*Review* **The Diagnostic and Therapeutic Potential of Galectin-3 in Cardiovascular Diseases**

**Grazyna Sygitowicz \* ˙ , Agata Maciejak-Jastrz ˛ebska and Dariusz Sitkiewicz**

Department of Clinical Chemistry and Laboratory Diagnostics, Medical University of Warsaw, 02-097 Warsaw, Poland; agata.maciejak@wum.edu.pl (A.M.-J.); dariusz.sitkiewicz@gmail.com (D.S.) **\*** Correspondence: gsygitowicz@poczta.onet.pl

**Abstract:** Galectin-3 plays a prominent role in chronic inflammation and has been implicated in the development of many disease conditions, including heart disease. Galectin-3, a regulatory protein, is elevated in both acute and chronic heart failure and is involved in the inflammatory pathway after injury leading to myocardial tissue remodelling. We discussed the potential utility of galectin-3 as a diagnostic and disease severity/prognostic biomarker in different cardio/cerebrovascular diseases, such as acute ischemic stroke, acute coronary syndromes, heart failure and arrhythmogenic cardiomyopathy. Over the last decade there has been a marked increase in the understanding the role of galectin-3 in myocardial fibrosis and inflammation and as a therapeutic target for the treatment of heart failure and myocardial infarction.

**Keywords:** galectin-3; cardiac fibrosis; heart failure; atrial fibrillation; chronic inflammation; MMPs; microRNAs; lncRNAs

#### **1. Introduction**

Galectins are a family of glycan-binding proteins, which were named and classified in 1994 considering their affinity for β-galactosides and significant sequence similarity in the carbohydrate-binding domains (CRDs) [1,2]. To date, 16 protein-coding galectin genes have been identified including 12 members in human tissues encoding galectins 1, 2, 3, 4, 7, 8, 9, 10, 12, 13, 14 and 16 [2–4]. Galectins 5 and 6 are present in rodents, while galectins 11 and 15 are found in sheep and goats [2].

Galectins are synthesized in cytosol but they can be secreted from the cells in not yet fully elucidated mechanisms. Functioning both outside and inside the cell—galectins participate in various cell processes, including transport of glycoprotein vesicles, chemotaxis, proliferation, pre-RNA splicing and apoptosis. Extracellular galectins can act through binding to cell surface glycans, while intracellular effectors, mediating galectin function, still remain, to a great extent, unknown.

The members of the galectin family are classified into three types according to their molecular architecture: (1) prototype, which are usually homodimers containing one carbohydrate recognition domain in each subunit; (2) tandem, which are monomers containing two CRDs joined by a linker sequence; (3) chimeric, containing C-terminal CRD joined to a large repeating sequence and N-terminal domain [2].

#### **2. Galectin-3 Structure and Functions**

Galectin-3 (Gal-3) is widespread and present in various organs including: the lungs, heart, stomach, colon, adrenals, uterus and ovaries [5]. Galectin-3 is the only galectin of chimeric type in the galectin family. Galectin-3, also known as a binding protein for IgE, Mac2, CBP30 and CP35, is encoded by the *LGALS3* gene present on chromosome 14, locus q21-22. It consists of six exons and five introns involving about 17 kilobases. Galectin-3 expression is regulated by promotor methylation status of *LGALS3*, and such elements as: CRE motifs, region similar to nuclear factor kappa B (NF-κB), GC regions located in

**Citation:** Sygitowicz, G.; Maciejak-Jastrz ˛ebska, A.; Sitkiewicz, D. The Diagnostic and Therapeutic Potential of Galectin-3 in Cardiovascular Diseases. *Biomolecules* **2022**, *12*, 46. https://doi.org/10.3390/ biom12010046

Academic Editor: Alexander V. Timoshenko

Received: 30 November 2021 Accepted: 27 December 2021 Published: 29 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

galectin-3 promoter [6]. The galectin-3 gene contains also a special regulatory element called galig (galectin-3 internal gene) located in the second intron of the *LGALS3* gene [7].

κ

Galectin-3 consists of 251 amino acid residues of relative molecular mass 29–35 kDa and it has been identified for the first time in murine peritoneal macrophages [1]. It contains three domains: (1) short N-terminal constituting a unique region of 12 amino acids and containing a site of serine 6 phosphorylation for controlling its nuclear location and abolishing its affinity to ligand [8,9]; (2) a 100-amino acid sequence similar to collagen, containing proline, glycine and tyrosine tandem repetitions and containing a fissionable domain of collagenase H, in which histidine 64 is the site of action of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-2; (3) spherical C-terminal CRD containing an Asp-Trp-Gly-Arg motif (NWGR), similar to those described in the anti-apoptotic protein Bcl-2 [1].

Galectin-3 can form dimers or pentamers in specific circumstances, when galectin-3 concentration is high or when ligands are present [10]. Monomeric galectin-3 undergoes physicochemical modifications, which increase the range of its biological functionality, particularly extracellular activity. The most important mechanisms leading to galectin-3 "bioactivation" is multimerization and formation of galectin-3 lattice. Galectin-3 has various functions, depending on its cellular location (Table 1).

**Table 1.** Various functions of galectin-3 in relation to cellular location.


Galectin-3 participates in various pathophysiological processes, including apoptosis [6], adhesion [16], angiogenesis [17], cell migration [17], proliferation [16], and differentiation [18], but its main function is induction of inflammatory condition and fibrotic process [6] (Figure 1).

**Figure 1.** Biological functions of galectin-3.

#### **3. Galectin-3 in Cardiovascular Diseases**

In view of its multidirectional activity, galectin-3 plays an important role in many various clinical conditions and disease entities. Increased galectin-3 expression has been documented in cardiovascular diseases (CVDs), such as atherosclerosis, acute ischemic

stroke, acute coronary syndrome (ACS) and heart failure (HF), arterial hypertension, cardiomyopathies or atrial fibrillation (AF) [19] (Figure 2).

**Figure 2.** Galectin-3 and cardiovascular diseases.

#### *3.1. Atherosclerosis*

Atherosclerosis is a complex inflammatory process, initiated by changed permeability of arterial wall cells and focal subendothelial accumulation of LDL lipoproteins, leading to formation of atheromatous plaques characterised by inflammatory condition and increased oxidative stress [20,21]. The later inflammatory reaction includes a massive participation of monocytes and macrophages changing the structure of the vascular wall [10,22]. Endothelial cells become activated and vascular smooth muscle cells (VSMCs) actively proliferate to produce extracellular matrix. Over the years, the participation of many inflammatory condition markers in the atherosclerotic process has been studied [21–25] and then a potential role of galectin-3 as a mediator of atherosclerosis has been suggested. Many studies have shown that galectin-3 contributes to macrophage differentiation [26], formation of foam cells [27], endothelial dysfunction [27,28] and VSMC proliferation and migration in atherogenesis [29]. The amplification of cardiovascular system inflammatory condition and accumulation of lipids in macrophages caused by galectin-3 are the most important mechanisms of atherosclerosis development, which are stimulated by local or circulating galectin-3.

Study performed by Ou et al. [30], using human umbilical vein endothelial cells (HUVECs), showed that exogenous addition of galectin-3 and oxidised low-density lipoprotein (oxLDL) to cell cultures increased the expression of lectin-like oxLDL receptor 1 (LOX-1) and promoted endothelial dysfunction via LOX-1/ROS/p38/NF-kB-mediated signalling pathway. It has been suggested by authors that galectin-3 enhances LOX-1 expression and induced pro-inflammatory response [30].

#### *3.2. Acute Myocardial Infarction*

Galectin-3 has been described as a factor contributing to the development and destabilisation of atheromatous plaques through propagation of inflammatory condition, interaction with lipopolysaccharides (LPSs) and promotion of VSMC phenotypic transformation [31]. Therefore, galectin-3 can be involved in the pathogenesis of ACSs caused by atherosclerosis. A significantly increased galectin-3 expression has been demonstrated in the early phase of acute myocardial infarction (AMI) and ACS [32,33]. In in vivo studies, using recombinant galectin-3 in rat experimental model, it has been observed higher collagen deposition and

thick collagen content in the infarct region. This study presented potential application of exogenous galectin-3 to assess the myocardial remodelling process after MI [34]. Both experimental and clinical studies have demonstrated that galectin-3 is an independent predictor of mortality for any cause, death for cardiovascular causes and development of HF [35,36]. In the later phase of ACS, galectin-3 enhances the transition from acute to chronic inflammatory condition and causes myocardial fibrosis, leading to unfavourable ventricular remodelling [35].

#### *3.3. Heart Failure*

In recent decades, cardiac remodelling and myocardial fibrosis have been accepted as the main heart failure-inducing factors. Galectin-3, exogenously added, is closely related to myocardial fibrosis and is strongly expressed in cardiac myofibroblasts, which can be used as an independent predictor of myocardial fibrosis [37]. Sharma et al. [37] revealed that recombinant galectin-3, exogenously injected into the pericardial sac of healthy rats over a long term, led to LV dysfunction and deterioration of cardiac function. Galectin-3 induced collagen deposition and fibroblast proliferation. Liu et al. [38] demonstrated that when the transforming growth factor β (TGFβ)/Smad3 signalling protein pathway was inhibited by N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), the expression of profibrotic and inflammatory factors induced by intrapericardial infusion of galectin-3 was significantly reduced and cardiac remodelling and dysfunction were less pronounced. That fact suggests that galectin-3-induced myocardial remodelling may be associated with an activation of TGFβ/Smad3 signal transduction pathway. On the other hand, another experiment [39] has proved that excessive galectin-3 expression favours type I collagen synthesis, while galectin-3 secretion inhibition reduces the synthesis and deposition of that collagen.

#### *3.4. Hypertrophic Cardiomyopathy*

Hypertrophic cardiomyopathy (HCM), a primary myocardial disease, is characterised by myocardial hypertrophy. The development of cardiac fibrosis and irreversible change of ventricular structure significantly contribute to sudden cardiac death in patients with HCM [40]. In their case-control study, Yakar et al. [41] analysed and compared the relationship between serum galectin-3 concentration and left ventricular (LV) function in 40 patients with HCM and in 35 age-matched healthy volunteers. That study demonstrated that concentration of serum galectin-3 was significantly elevated in HCM patients compared with the control group. Furthermore, levels of serum galectin-3 were positively correlated with interventricular septum thickness and LV mass index. The concentration of serum galectin-3 was, however, not related to the degree of LV outflow tract obstruction. For that reason, it was accepted that serum galectin-3 concentration was related to the degree of LV hypertrophy but not to the LV diastolic and systolic dysfunction [41]. However, whether galectin-3 plays a major causative role in discussed process is not completely justified because there are studies showing that up-regulation of cardiac galectin-3 is not a critical disease modulator of cardiomyopathy induced by β2-adrenoceptor over-expression [42]. These findings raise a possibility that role of galectin-3 in the development of cardiomyopathy might not be universal but rather dependent to disease aetiology. On the other hand, another experiment [43] has revealed that galectin-3 is not a critical mediator of the fibrotic cardiomyopathy associated with pressure overload. Myocardial galectin-3 expression did not affect the survival, systolic and diastolic dysfunction, and cardiac fibrosis.

#### *3.5. Atrial Fibrillation*

Atrial fibrillation (AF) is a cardiac arrhythmia characterised by a rapid and irregular heart rate and is the most frequent and most serious arrhythmia. AF is closely related to high mortality rate, cerebral stroke and HF [44]. Particularly, atrial interstitial fibrosis seems to be the key factor contributing to AF development [45]. It is known that left atrial (LA) interstitial fibrosis plays a significant role in initiating and maintaining atrial fibrillation [45,46].

Galectin-3 enhances cardiac fibrosis and remodelling and is a well-documented cause of arrhythmia. Most studies have focused on the relationship between levels of serum galectin-3 and cardiac fibrosis in HF. The role of galectin-3 in the pathophysiology of atrial fibrillation has not been fully elucidated as yet. Sonmez et al. [47] were the first to study the levels of new biomarkers of inflammation in serum in patients with atrial fibrillation compared with those with sinus rhythm. The results of the study demonstrated that the levels of the new circulating markers of remodelling, such as galectin-3, MMP-9 and PIIINP (amino terminal peptide of type III procollagen) were significantly higher in patients with atrial fibrillation. Moreover, galectin-3, MMP-9 and PIIINP concentrations in serum were strongly positively correlated with LA volume and LA volume index [47]. The observational study by Gurses et al. [48] demonstrated also that serum galectin-3 concentration and LA volume index were significantly higher in patients with atrial fibrillation, which could have suggested that concentration of serum galectin-3 in AF can be correlated with atrial remodelling. The study also demonstrated that serum galectin-3 concentration was significantly higher in patients with persistent atrial fibrillation than in those with paroxysmal atrial fibrillation [48]. The study conducted by Yalcin et al. [49] revealed that the LA volume index and serum galectin-3 concentration were independently correlated with the range of LA fibrosis detected by means of delayed-enhancement magnetic resonance imaging (DE-MRI) in patients with paroxysmal atrial fibrillation with preserved LV function. These results suggest that concentration of serum galectin-3 is significantly correlated with atrial remodelling in patients with paroxysmal atrial fibrillation with preserved LV function [49].

#### *3.6. Arterial Hypertension*

Arterial hypertension is usually a chronic pathological condition characterised by elevated arterial pressure. Hypertensive cardiac remodelling starts with inflammation, increased deposition of extracellular matrix proteins leading then to myocardial fibrosis and, finally, to heart dysfunction [50]. It has been demonstrated that serum galectin-3 concentration increases in hypertensive patients, but that phenomenon is more pronounced in patients with LV hypertrophy. Therefore, galectin-3, as an important biomarker of early cardiac remodelling, is independently correlated with left ventricular remodelling (LVR). Moreover, LV mass was independently correlated with concentration of serum galectin-3 in hypertensive patients [51].

Primary aldosteronism is regarded as the most frequent cause of secondary arterial hypertension. Patients with primary aldosteronism show an increased susceptibility to heart muscle inflammation and fibrosis [52]. It has been demonstrated that aldosterone can induce galectin-3 secretion [50]. Galectin-3 is one of the most important mediators between macrophage activation and myocardial fibrosis. In a prospective pilot clinical study, serum galectin-3 concentration was significantly higher in the group of patients with aldosteroneproducing adenoma. Moreover, both the degree of myocardial fibrosis and serum galectin-3 concentration returned to normal after adrenalectomy [53]. Azibani et al. [54] observed for the first time that hyperaldosteronism increased the number of inflammatory factors, including galectin-3, and accelerated the hypertension-induced fibrosis. Many studies in this aspect were conducted. However, the role of galectin-3 in hyperaldosteronism-induced inflammation remains unclear [54].

#### *3.7. Acute Ischemic Stroke (AIS)*

AIS is a cerebral tissue infarction caused by occlusion of cerebral arteries, with injury of the neurons, astrocytes and oligodendrocytes. It is the most important vascular event in the central nervous system, leading to death or disability. Dong et al. [55] assessed the role of galectin-3 in patients with AIS. They demonstrated that serum galectin-3 concentration was significantly higher in AIS patients compared with healthy individuals, what increased the intensity of AIS and infarction volume. Besides that, higher levels of serum galectin-3 were independently correlated with increased risk of death, significant disability, recurrent stroke and vascular events [56,57]. In in vitro studies, performed using human cortical neuronal (HCN) cell lines culture, a knockdown of galectin-3 expression with siRNA dramatically increased neuronal cell viability and simultaneously reduced apoptosis and serum levels of proinflammatory cytokines, including interleukin-1, -6 (IL-1, -6) and NF-κB and also caspase-3, a protein associated with apoptosis [55]. As a result of ischaemia, galectin-3 is released by microglia in the injured cerebral tissue. Then, the released galectin-3 contributes to microglia activation through binding to toll-like receptor 4 (TLR4), what causes an exacerbation of the inflammatory response [58]. Furthermore, the effect of serum galectin-3 concentration of AIS patients may be associated with serum lipid concentration regulation. AIS is reversely proportionally related to HDL cholesterol concentration, which, in turn, is reversely related to mortality from ischemic stroke and vascular events [59]. Earlier studies have demonstrated a reverse relationship between galectin-3 and HDL-C in serum, what suggests that AIS regulation, in which galectin-3 participates, is possibly associated with dyslipidaemia and inflammatory condition [60,61]. In a prospective cohort study involving 2970 patients with AIS, increased galectin-3 and decreased HDL-C levels of serum were observed, what could have intensified inflammatory condition and oxidative stress after ischemic stroke [57]. Moreover, it seems that concentrations of serum galectin-3 and HDL-C exerta combined effect on AIS prognosis.

Furthermore, a neuroprotective effect in cerebral stroke has been described. Galectin-3 is of key importance for activation, migration and proliferation of microglia after ischemic stroke [62]. Galectin-3 deficiency is associated with a significant increase of the size of ischemic lesions and number of apoptotic neurons. Microglial activation and proliferation mediated by galectin-3 are associated with Gal-3/IGF-R1 (insulin-like growth factor receptor 1) interaction in response to ischemic injury [62]. The data suggest that galectin-3 plays a neuroprotective role in injured brain.

In summary, galectin-3 can simultaneously exert a negative effect and show a neuroprotective action in AIS. One of the possible causes is that circulating galectin-3 serum levels are associated with various stages of AIS and a coincidence of two unfavourable effects or the neuroprotective role are not necessarily associated with the circulating galectin-3.

#### **4. Diagnostic Usefulness of Serum Galectin-3 Concentration**

The inflammatory process and especially its acute phase revealed as a result of bacterial infection and in certain types of tumours, is closely related to the circulating neutrophils. The recruitment of the cells is a characteristic feature of acute inflammation and they are the first cells to migrate towards the inflammation site. Galectin-3 is strongly involved in the modulations of inflammatory processes and disorders underlying the inflammatory condition. The proinflammatory effect of galectin-3 is associated with activation of NF-kB transcription factor, induction of tumour necrosis factor α (TNF-α) and interleukin 6, regulation of cell adhesion, promotion of cell activation and chemotaxis and regulation of cell growth or apoptosis [63].

Inflammatory condition underlies many atherosclerosis-related diseases. Galectin-3 seems to be an important and, at the same time, useful biomarker of atherosclerosis and its special role has been observed in the process of atheromatous plaque destabilisation. A positive relationship has been demonstrated between serum galectin-3 concentration and the number and calcification area of atheromatous plaques [64]. Moreover, high concentrations of serum galectin-3 are a harbinger of clinical failures associated with higher risk of overall mortality or mortality due to cardiovascular reasons and HF [65]. The authors of another paper [66] have stressed that higher galectin-3 serum concentrations determined in patients on admission to hospital are associated with severe course of stroke and frequently with a poor prognosis at discharge from hospital. An unfavourable prognosis is frequently even poorer when a close relationship is present between levels of serum galectin-3 and markers of lipid metabolism [60], carbohydrate metabolism [67,68], renal function [69,70] or echocardiographic parameters serving for assessment of myocardial function and structure [71,72]. In individuals, in whom no metabolic disorders or excessive body mass or increased triglyceride and total cholesterol concentrations have been found, it has been

demonstrated that the age itself can distinguish subjects with higher and lower serum galectin-3 concentrations (individuals <40 years of age: 11.5 (9.5–13.60) ng/mL vs. those aged ≥40 years: 12.4 (10.6–14.4) ng/mL) [73].

In the assessment of the cardiovascular risk of primary importance are disorders of lipid–carbohydrate profile or renal function. It was found [60] that in individuals aged <40 years after myocardial infarction, serum galectin-3 concentration was strongly positively related to non-HDL-cholesterol concentration and negatively related to HDLcholesterol level. Patients with type 2 diabetes mellitus had higher serum galectin-3 concentrations than healthy individuals [67,68]. In patients with chronic and acute heart failure a relationship was observed between higher serum galectin-3 concentrations and renal failure parameters assessed by cystatin C or uric acid concentrations or by reduced estimated glomerular filtration rate (eGFR) [74]. The studies conducted by Mueller et al. [75] demonstrated that patients with eGFR >90 mL/min/1.73 m<sup>2</sup> had lower levels of serum galectin-3 (median: 10.7 (9.3–12.4) ng/mL) compared with patients with eGFR <90 mL/min/1.73 m<sup>2</sup> , in whom higher levels of serum galectin-3 were found (median: 12.1 (10.2–14.1) ng/mL). The diverse clinical involvement of galectin-3 in many diseases is due to its role as a regulator of acute and chronic inflammation that links inflammation-related macrophages and the promotion of fibrosis. This makes galectin-3 not an organ-specific marker, but a marker specific to the pathogenesis of inflammatory and/or fibrotic disorders. This is because the primary sources of circulating galectin-3 are not always identifiable. A patient with heart disease may have varying degrees of inflammation and progression of the fibrotic process. Thus, the serum galectin-3 concentration may reflect different stages of the pathophysiological state. This is because the level of circulating galectin-3 in patients with different stages of heart disease does not differentiate between myocarditis and fibrosis and therefore it does not specifically reflect these conditions. Moreover, there are gender-related differences in serum galectin-3 concentrations. In women, higher serum galectin-3 concentrations were observed compared to men, as well as a stronger correlation between concentration of serum galectin-3 and other cardiovascular disease risk factors [61].

An increased secretion of galectin-3 promotes a release of inflammatory mediators, including TGF-β or interleukin 1 and 2, and also intensifies cardiac fibroblast proliferation. Activated cardiac fibroblasts are the main source of extracellular matrix (ECM) proteins, particularly collagen. Galectin-3 affects in the first place synthesis of type I collagen, leading to an impairment of the homeostasis between type I/III collagen content and thus to impairment of the systolic/diastolic function of the myocardium. These disorders contribute to progression of myocardial failure [76].

Galectins, apart from modulating inflammatory processes, play the dominant role in fibrotic processes. In heart failure the pathophysiological element of key importance is the progressing fibrosis of the myocardial tissue. In the myocardium, the level of galectin-3 expression is almost undetectable in cardiomyocytes, but in cardiac fibroblasts that lectin reaches a significantly higher concentration. Chronic kidney disease (CKD) is one of the risk factors of cardiovascular diseases, hence cardiac biomarkers play a significant role in the development of kidney diseases. An elevated serum galectin-3 concentration is associated with myofibroblast proliferation or intense fibrogenesis and may be a harbinger of kidney fibrosis process or even of CKD development [70].

In heart failure galectin-3 is released into extracellular space, promoting fibrotic process through activation of fibroblasts. The fibroblast activation is characterised by increased expression of cytoskeletal protein—alpha smooth muscle actin (α-SMA)—an intracellular fibrosis marker, and collagen type 1 (COL1α1)—an extracellular fibrosis marker. Both α-SMA and COL1α1 regulation in tissues affected by fibrosis is mediated by galectin-3. That process is mediated by activation of cyclin-dependent kinase inhibitor 1A (CDKN1A), inhibin beta A, fibronectin 1, as well as extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). Thus, the central place in the regulation of fibrotic process development is taken by galectin-3, which participates in the regulation of expression of fibrotic matrix components (α-SMA and COL1α1) and in extracellular matrix

turnover through a number of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases. Galectin-3 influences the development of myocardial fibrosis through an effect on the important intermediates of that regulation: phosphatase and tensin homolog (PTEN) and protein tyrosine kinase 2 (PTK2). Exerting an effect on PTEN, it inhibits MMP-14 activity, contributing to the development of fibrosis, and inhibits MMP-9 activity, preventing the cardiac fibrosis process. On the other hand, the promoting of myocardial fibrosis with participation of MMP-9 is mediated by PTK2 [77].

The effect of galectin-3 is closely related to the heart failure markers useful in clinical practice, namely natriuretic peptides. The studies conducted by Felker et al. [78] have confirmed the relationship between increased galectin-3 serum concentration and the intensity of heart failure in individual NYHA classes, monitored by NT-proBNP concentration. In other studies a positive correlation between serum galectin-3 concentration and a negative correlation between left ventricular ejection fraction (LVEF%) and the degree of heart failure progression (max. NYHA IV class) have been demonstrated. A relationship has also been found between plasma galectin-3 concentration and the change of the left ventricular structure and function, what has confirmed that galectin-3 can participate in the left ventricular remodelling process in patients with HF [79,80]. Moreover, the studies have confirmed a higher specificity of galectin-3 in predicting the occurrence and development of HF than that in the case of determination of NT-proBNP concentration alone. However, this has not been shown with respect to galectin-3 sensitivity in the prognosis of the occurrence and development of HF [80].

Our studies have unequivocally stressed the importance of galectin-3 as a predictor of death in one-year follow-up [81]. The research has also shown a positive correlation between concentrations of serum NT-proBNP and galectin-3 (r = 0.565, *p* = 0.035). In the studies conducted in patients with HF in NYHA III class [82] it has been observed that galectin-3 is an important predictor of the risk of death, taking into account the age and gender and also HF intensity (based on NT-proBNP concentration) and renal function disorders (acc. to eGFR value). In the studies by Tang et al. [83], increased plasma galectin-3 concentration was associated with advanced age and poor renal function. That correlation, revealed in a group of patients with chronic systolic heart failure, demonstrated that high plasma galectin-3 concentration was associated with renal failure and shorter survival of the patients.

On the other hand, Fermann et al. [84] demonstrated in their study higher serum galectin-3 concentrations in patients with myocardial failure and with confirmed renal dysfunction. It is worth to stress here, that in the case of heart or kidney failure it is recommended to analyse in serum galectin-3 concentration together with natriuretic peptide concentration values. A joint analysis of changes of these markers offers a possibility of a more accurate prognostication and, at the same time, confirmation of organ failure development.

A group of researchers [85] made an attempt to find a correlation between levels of serum galectin-3 and NT-proBNP and the inflammatory condition assessed by serum hsCRP concentration in patients with the first myocardial infarction, treated by percutaneous coronary intervention (PCI). In the paper by Szadkowska et al. [85] a detailed analysis of the above-mentioned markers in serum, depending on galectin-3 level, demonstrated three times higher NT-proBNP concentrations and two times higher hsCRP levels in patients presenting higher galectin-3 concentrations (>16 ng/mL), than in those with galectin-3 concentrations <16 ng/mL. In the paper, a positive correlation was also demonstrated between NT-proBNP and hsCRP concentrations (r = 0.45, *p* < 0.001) and galectin-3 and hsCRP concentrations (r = 0.20, *p* < 0.05).

In clinical practice, apart from commonly used heart failure markers—natriuretic peptides, increasingly frequently new tools are tested for the assessment of the degree of heart failure—and that concerns not only myocardial failure. They include, among other markers: GDF-15 (growth and differentiation factor-15) and ST2 (suppression of tumourigenicity 2), which belongs to the interleukin 1 family. GDF-15 is regarded as a prognostic marker in cardiovascular diseases and is frequently determined in combination with other

prognostic factors, such as NT-proBNP or hsTnT. High GDF-15 concentrations were noted in hypertrophic and dilated cardiomyopathies, after volume overload, ischaemia and heart failure [86]. The ST2 receptor has two isoforms: membrane-bound receptor type 2 (ST2L) and soluble form (sST2), present in serum and most frequently used in diagnostic procedures [70,87]. The form sST2 is a "bait" for IL-33 and thus it counteracts its interaction with the membrane-bound ST2L, blocking the paracrine fibroblast-cardiomyocyte communication system and reducing the cardioprotective effect of IL-33 [87].

A good clinical practice is, however, a joint assessment of the changes of concentrations of several parameters. An assessment of the cardiovascular risk is possible with a higher sensitivity with simultaneous measurements of the concentrations of: sST2, hsTnI, hsCRP or GDF-15 [70]. The addition of serum galectin-3 concentration to that group is extremely useful in the case of disorders associated with myocardial dysfunction. It should be also mentioned that the biological variability of galectin-3 is low in comparison to other established and novel cardiovascular biomarkers (e.g., NT-proBNP, GDF-15, ST2) [19,88]. Apart from this, galectin-3 had 8.1% within-individual coefficient of variation in healthy controls and chronic HF patients [88]. Schindler et al. [89] presented that galectin-3 shown relatively low biological variability in healthy individuals and stable HF patients. Furthermore, among healthy subjects, galectin-3 had minimal biological variation in both the shortand long-term without sex differences.

Galectin-3 is a biomarker of ventricular remodelling and myocardial fibrosis [90]. It is also an important regulator of chronic and acute inflammatory condition and inflammation leading to fibrosis in various tissues [91]. The study by Tuegel et al. [92] demonstrates that simultaneously determined high concentrations of three biomarkers in serum: galectin-3, sST2 and GDF-15 in patients with chronic kidney disease are associated with a higher mortality of such patients. At the same time, it has been observed that only GDF-15 serum concentration is associated with development of heart failure. Serum galectin-3 concentration shows a low tissue specificity and, therefore, a multimarker strategy of cardiovascular risk assessment is needed [70]. Two studies involving patients with HF (CORONA and COACH trials) analysed the kinetics of changes in levels of serum galectin-3 after 0, 3 and 6 months [19,93]. The increase in serum galectin-3 concentration by ≥15% was found to indicate a 50% higher risk of mortality and hospitalization due to HF compared to patients with stable values of galectin-3 in the same time range, regardless of age, sex, diabetes, renal function, ejection fraction (LVEF) and NT-proBNP concentration. Moreover, the results of our study [81] demonstrated that patients with acute heart failure who died within 1 year of follow-up had significantly higher levels of serum galectin-3 at baseline compared to those who survived (55.6 ± 37.6 ng/mL vs. 15.0 ± 7.04 ng/mL; *p* = 0.005), which is in agreement with some previous reports. Our study was only a preliminary pilot research and the size of the study group is small.

In summary, it should be stressed that the proinflammatory effect of galectin-3 is not only limited to its participation in the pathogenesis of cardiovascular diseases. Recently, a significant importance of that marker has been demonstrated in the first place in SARS-CoV-2 infections. In patients with a severe COVID-19 course a systemic inflammatory condition develops, with intense cytokine storm, being the cause of respiratory failure and other multiple organ injuries [94,95]. It seems that serum galectin-3 concentration reflects the severity of COVID-19 course. In patients infected with SARS-CoV-2, in whom an unfavourable course of the disease forced administration of intensive therapy in view of respiratory failure, an almost three times higher serum galectin-3 concentrations were found, compared with patients not requiring treatment at intensive care units (ICUs) (23.46 (15.51–27.80) ng/mL vs. 8.93 (7.58–12.97) ng/mL, respectively) [63].

The study conducted by Ku´snierz-Cabala et al. [63] also called attention to the use of galectin-3 in the diagnosis of pneumonia, particularly in situations of severe COVID-19 course. In patients with pneumonia, an almost twice higher serum galectin-3 concentrations were found compared with those, in whom no pneumonia developed (13.30 (8.93–17.38) ng/mL vs. 8.55 (6.73–10.98) ng/mL, respectively). In that group of patients also higher concentrations

were revealed of other proinflammatory markers: IL-6, pentraxin-3 (PTX-3), endothelial damage marker–soluble fms-like tyrosine kinase-1 (sFlt-1) and a number of tissue damage markers [63].

#### **5. Therapeutic Potential of Galectin-3**

A galectin-3-targeted therapy requires an understanding of the biology and pathologies associated with that molecule. Galectin-3 plays an important role in chronic inflammatory condition and is involved in the development of many diseases, including diseases of the heart [96–98], kidneys [99,100], viral infections [101], autoimmune diseases [102], neurodegenerative disorders [103–105] and many neoplastic diseases. In recent years a significant intensification has been observed of the studies on the role of galectin-3 as a central regulator of fibrosis and on therapeutic strategies aimed at inhibition of galectin-3 function in profibrotic diseases. The strategy of galectin-3 expression or function inhibition may be effective, not disturbing at the same time its normal main function.

Preclinical studies, in which a deletion of galectin-3-encoding gene was used, demonstrated the role of galectin-3 on the long list of cardiovascular diseases, including those focused on heart remodelling [106,107], hyperaldosteronism and arterial hypertension [108,109], acute myocardial infarction [110], myocardial ischemic/reperfusion injuries [111] and dilated cardiomyopathy [112]. That led to putting forward the hypothesis that galectins are potential targets for new anti-tumour and anti-inflammatory compounds. This hypothesis has been supported in several in vivo studies with the use of amino acid-derived lactuloseamine [113] and modified citrus pectin (MCP) [114]. The mentioned derivatives inhibit the expression of galectin-3. A development of small and large molecules blocking galectin-3 function may pave the way for a novel and exciting therapy with inhibitors, the action of which is, however, limited in healthy tissues.

#### *5.1. Galectin-3 Inhibitors*

#### 5.1.1. Monosaccharides

Pharmacological inhibition of galectin-3 with N-acetyllactosamine (N-Lac) prevented left ventricular dysfunction in heart-failure-susceptible REN2 rats [107] and also demonstrated a protective effect against hypertensive nephropathy in REN2 rats [115]. Low molecular mass saccharides, such as lactose or N-Lac, cannot be, however, used as "drugs" since they are rapidly absorbed and metabolised. Low molecular mass organic compounds, galactose derivatives, have also been synthesised and tested in respect of binding to the carbohydrate-binding domain of various galectins [116]. An inhibitor, 3,3′ ditriazolylthiodigalactoside exerted the strongest effect on galectin-3, with low values of the inhibition constant Kd = 29 nM [116].

#### 5.1.2. Galactomannans and Modified Citrus Pectins

Galactomannans (GMs) are plant-derived galectin-3 antagonists. GM-CT-01, known under proprietary name Davanat® is a galactomannan with molecular mass 50 kDa and half-life from 12 h to 18 h [117,118]. Various types of modified citrus pectins with masses over 1000 kDa, e.g., GCS-100 [118] and PectaSol-C® are already available on the market. The galectin-3-inhibiting effect of MCP was tested in various cell and animal models. The tests included: inhibition of haemaglutination by galectin-3, reduction of heart inflammatory condition, suppression of organ fibrosis and reduction of atherosclerosis in mice with apolipoprotein E deficiency [25,108,119]. The inhibiting effect of MCP on *LGALS3* gene expression was tested in cardiac fibroblasts in a rat experimental model of heart failure [120]. It was demonstrated [120] that MCP alleviated heart dysfunction, decreased the degree of myocardial damage and reduced collagen deposition. A reduction of *LGALS3*, TLR4, MyD88 gene expression and NF-κB-p65 factor inhibition were also described. A reduction was also observed of expression of IL-1β, IL-18, TNF-α– the proinflammatory cytokines involved in the pathogenesis of heart failure. The use of MCP as a galectin-3 antagonist exerted a favourable effect on myocardial dysfunction process through inhibition

of inflammation and fibrosis. The use of galectin-3 as a potential therapeutic target in the treatment of heart fibrosis after infarction may bring many benefits [120]. It should also be said that in many in vivo studies no specificity of MCPs has been established, and it is possible that their inhibitory activity is caused by an effect also on other therapeutic targets.

#### 5.1.3. Thiodigalactosides

Recently, thiodigalactoside derivatives have been developed, with action targeted at new CRD sites, other than the canonical binding site. TD-139 is a thiodigalactoside analogue approved by the FDA for treatment of idiopathic pulmonary fibrosis, in the form of inhalation powder, and it has been speculated that it shows an effect antagonistic to galectin-3 through binding to B and E subsites [118,121]. TD-139 is a small molecule (C28H30F2N6O8S) of about 648 g/mol molecular mass and it can bind with a high affinity to both galectin-3 and galectin-1. Although the mechanism of action still remains unclear, it is supposed that the molecule allosterically modulates the CRD of galectin-3. Some research groups have also reported that thiodigalactosides can be preferentially adjusted to form more specific galectin-3 inhibitors [122].

#### 5.1.4. Heparin-Based Inhibitors

Heparin-based inhibitors are a relatively new and attractive group of galectin-3 inhibitors, which are sulphated or acetylated heparin derivatives. The results of in vitro studies have demonstrated that they are non-cytotoxic and selective for galectin-3 (i.e., they do not inhibit galectin-1, -4 and -8). The experimental in vivo studies with nude mice demonstrated that compounds induced by galectin-3 significantly inhibited the metastasizing of human melanoma and colonic cancer cells to the lungs. Moreover, the compounds showed no detectable anti-thrombotic activity and they seemed to be promising therapeutic agents [123]. They were, however, only tested in vivo models of metastases and, in the future, studies should be conducted in order to estimate their potential as anti-fibrotic agents.

#### 5.1.5. Neoglycoconjugates

Galectin-3 binds to branched saccharides with increased binding power, and large dendrimers connected with lactose as a functional group, provide an "excess of ligands" for galectin-3 binding. Michel et al. [124] studied the effect of various types of dendrimers, functionally bound to lactose, on tumour cell aggregation. They found that smaller dendrimers inhibited cell aggregation, possibly through competitive inhibition, while larger dendrimers containing several terminal lactose groups increased aggregation, providing thus many sites for galectin-3 binding [124]. Recently, yet other chemically modified glycoproteins (neoglycoproteins) have been developed, which show a potential to be used as new therapeutic molecules against fibrosis, through effective targeting at galectin-3. They serve not only as ligands with a high affinity, but can be also modulated in order to achieve selectivity for galectin-3, compared with other galectins. Their use in clinical context has not been assessed yet [125].

#### 5.1.6. Peptide Based Compounds

Amino-terminally truncated galectin-3 (Gal-3C) has been studied in the therapy of galectin-3-related tumours and seems to be a promising therapeutic target, showing a low toxicity profile [126,127]. There have been, however, no sufficient studies conducted, assessing its potential in the treatment of other galectin-3-associated disorders. Recently, Sun et al. [128] used the galectin-3-binding peptide i.e., G3-C12 for inhibition of intracellular galectin-3 in tumour cells. Since G3-C12 is highly selective for galectin-3 compared with other galectins, it acts as a selective galectin-3 target ligand. Then, when that peptide is coupled with the drug by means of a universal drug carrier, such as copolymer of N- (2-hydroxypropyl) methacrylamide (HPMA), the created G3-C12-HPMA-drug conjugate can easily penetrate the cells with galectin-3 over-expression [128]. The above-described

concept of selective intracellular galectin-3 supplying can also find use in other scenarios of galectin-3-related diseases, such as organ fibrosis and HF.

Galectin-3 inhibitors are promising therapeutic agents, but still not much is known about their critical features, such as: in vivo power of action, absorption, metabolism, pharmacokinetics and toxicology. Although it has been demonstrated that they are active in some disease models, further studies are needed on the mechanism of their action, to establish whether they are active in a therapeutically acceptable model of supplying and dosing of the drugs for patients.

#### **6. NcRNAs as the Modulators of** *LGALS3* **Gene Expression and a New Potential Therapeutic Target**

The learning of the role of galectin-3 in the processes of myocardial fibrosis, inflammation and postinfarction dysfunction has also started studies, the aim of which was the search, at molecular level, for post-transcriptional mechanisms regulating *LGALS3* gene expression. The effect has been studied of short, non-coding RNAs, microRNAs (miRNAs), which participate in the regulation of many genes involved in such processes as: cell differentiation, division, proliferation, apoptosis and angiogenesis [129]. The mechanism of miRNAs action is based on the inhibition of protein translation process or direct degradation of mRNA of the target genes through binding of miRNAs to the complementary region of the target mRNA molecule [130]. Much data is available confirming the participation of miRNAs in the process of physiological regulation of the heart function and also in the progression of cardiovascular diseases [131–134].

The increased interest in circulating miRNAs as potential biomarkers of cardiovascular diseases has caused that successive studies started to appear, concerning the question whether miRNAs molecules can reflect the changes occurring at various stages of a pathological process. The correlation was studied between miRNAs specific to acute heart failure and the serum concentration of well-documented biochemical biomarkers including galectin-3 [135]. A negative correlation was demonstrated between miR-199a-3p expression and galectin-3 serum concentration in the 48th hour of hospitalisation in patients with impaired heart function and poor prognosis (r = −0.73; *p <* 0.001). The relationship observed between microRNA and established biomarkers, including galectin-3, can contribute to better elucidation of the role of unfavourable heart remodelling processes and fibrosis in the pathogenesis of acute heart failure. In study by Song et al. [136] *LGALS3* gene expression is increased in cardiac fibrosis process and over-expression of miR-199a expression has also been observed with hypertrophy heart failure. The biochemical and molecular biomarkers analysed can serve for predicting and better identification of patients with unfavourable prognosis in the case of impaired heart function [136,137].

The study by Zhang et al. [138] presented, in vivo and in vitro, using a murine experimental model, the role of miR-27b and explained some mechanisms underlying myocardial hypertrophy. In the case of miR-27b over-expression the heart function in the course of hypertrophy was restored, what suggested a protective role of miR-27b against the pathological process. The study demonstrated and experimentally confirmed that *LGALS3* was the target gene for miR-27b. Experimental *LGALS3* gene inactivation significantly suppressed the myocardial hypertrophy process. Both *LGALS3* and miR-27b have a potential as therapeutic and diagnostic targets in the treatment of cardiovascular diseases, including myocardial hypertrophy [138].

Increased *LGALS3* expression was also noted in the experimental murine model, in damaged cardiac tissue subjected to myocardial ischemic reperfusion (I/R) procedure [139]. On the other hand, an experimental silencing of *LGAL3* gene expression alleviated myocardial damage resulting from the I/R procedure. A bioinformatic prediction of the interactions between mRNA and miRNA demonstrated that the *LGALS3* was regulated by miR-204-5p. A reduced miR-204-5p expression level was observed in the cardiac tissue subjected to the I/R procedure. Moreover, a negative correlation was demonstrated between miR-204-5p expression and *LGALS* expression in the studied experimental system. Another important observation from the cited study [139] concerns the interaction between miR-204-5p and long non-coding RNA (lncRNA) *KCNQ1OT1*. A reduced *KCNQ1OT1* expression can provide protection against heart injury in the course of I/R after myocardial infarction, and *KCNQ1OT1* can modulate the expression of genes through a network of lncRNA/miRNA/mRNA interactions. *KCNQ1OT1*, binding to miR-204-5p, modulates the process of heart injury in the course of I/R through interaction with *LGALS3.* An increased *KCNQ1OT1* expression was noted in the heart tissue subjected to I/R, and a negative correlation was demonstrated between *KCNQ1OT1* expression and miR-204-5p expression in the studied experimental model. From the therapeutic point of view, the observation seems important, that experimental reduction of *KCNQ1OT1* expression and increase of miR-204-5p expression suppress cardiac injury in the course of I/R through a reduction of *LGALS3* expression. The presence of the network of *KCNQ1OT1*/miR-204-5p/*LGALS3* interactions may constitute a potential therapeutic target in the case of myocardial damage after I/R procedure [139].

*LGALS3* expression and influence of the lncRNA *SNHG20* and miR-335 regulatory factors were assessed in the process of myocardial fibrosis and hypertrophy induced by angiotensin II (Ang II) in a murine experimental model [140]. An increased *SNHG20* expression was noted, which was reflected in an increase of *LGALS3* expression, but a decreased miR-335 expression was observed in the cardiac tissue. An experimental loss of *SNHG20* function, which resulted in *SNHG20* expression reduction, caused a reduction of expression of the proteins involved in heart fibrosis and apoptosis processes and also increased the viability of the cells. An experimental over-expression or silencing of *SNHG20* through interaction with the miR-335/*LGALS3* system can modulate the cardiac fibrosis process induced by Ang II. *SNHG20* lncRNA can be another therapeutic target in the process of myocardial fibrosis and hypertrophy [140].

The diagnostic usefulness of the miR-1 and miR-21, known in the pathogenesis of heart failure, and of galectin-3 protein was analysed in a group of patients with acute heart failure and coexistent asymptomatic type 2 diabetes mellitus [141]. A negative correlation was found between miR-1 expression and galectin-3 serum concentration and a positive correlation was observed between miR-21 expression and serum galectin-3 concentration in the studied group of patients. An application of a panel of determinations including miRNAs and biochemical biomarkers can lengthen the odds on an early identification of patients with acute heart failure among patients with asymptomatic type 2 diabetes mellitus [141].

The participation of the mentioned miR-1, miR-21 and serum galectin-3 concentration was also studied in patients with symptomatic heart failure and left ventricular hypertrophy and history-confirmed arterial hypertension [142]. It was shown that in patients with heart failure a reduction of miR-1 and miR-21 expression occurred, and these results were significantly correlated with the concentration of serum galectin-3, the important factor playing the key role in the fibrotic process. The changes of miR-1 and miR-21 expression and serum galectin-3 concentration were analysed also in patients with systolic heart failure with various degrees of intensity of left ventricular dilatation [143]. A relationship was observed between reduced miR-1 expression with increased serum galectin-3 concentration and the progression of unfavourable heart remodelling, assessed as left ventricle dilatation. An increased miR-21 expression was also found in patients with decompensated systolic heart failure. In the study, it has been demonstrated that both up-regulation of miR-1 and miR-21 as well as galectin-3 lead to myocardial hypertrophy and unfavourable heart remodelling.

In the study by Han et al. [144], the clinical importance was assessed of miR-214 and serum galectin-3 concentration in whole blood of patients with chronic heart failure. A statistically significant increase was noted of both miR-214 expression and galectin-3 serum concentration in the group with chronic heart failure. A positive correlation was demonstrated between miR-214 expression and serum galectin-3 concentration, what suggested the participation of both factors in the development of chronic heart failure. Galectin-3 exerts an effect promoting the myocardial fibrosis process, while miR-214 can regulate fibroblast proliferation [144].

Galectin-3 can be not only a clinically important biomarker of fibrotic process in cardiovascular diseases, but also an interesting therapeutic target that can possibly decelerate the progression of heart fibrosis. lncRNAs and miRNAs are involved in the regulation of signalling pathways in cardiac fibroblasts. Expression silencing or over-expression of lncRNAs or miRNAs in vivo can prevent the fibrotic process and improve the diastolic heart function. The interrelations in the lncRNA/miRNA/Gal-3 axis can constitute a susceptible target for therapeutic interventions, but further studies on this topic are required in order to increase the knowledge of their complex roles in the pathogenesis of cardiac fibrosis (Figure 3).

**Figure 3.** ncRNAs modulation of *LGALS3* gene expression in cardiovascular diseases. miR microRNA; SNHG20—Small Nuclear RNA Host Gene 20; KCNQ1OT1—KCNQ1 Opposite Strand/Antisense Transcript 1. ↓—down-regulation; ↑—up-regulation; . . . .—interrelation between lncRNA and miRNA.

#### **7. Conclusions and Perspectives**

Based on the increasing literature data, galectin-3 emerges as a structurally unique and functionally extremely important galectin, expressed in various tissues and cell types and present not only inside but also outside cells, and also bound to cell membrane surfaces. The biological role of galectin-3 was initially ascribed to its carbohydrate-binding activity, but in the last decade a completely new spectrum of its functions was proved, not directly associated with the activity of that lectin. It has been found that galectin-3 participates in many pathological processes, in the first place in cardiovascular diseases but also in viral infections and many tumours.

Galectin-3 is an important factor in the pathophysiology of HF, mainly in view of its role in cardiac ventricular remodelling. Galectin-3 initially plays a protective role in the heart through its anti-apoptotic and anti-necrotic functions, while a prolonged expression of that protein leads to fibrosis and unfavourable remodelling of the damaged tissue. The sites of galectin-3 binding are mainly located in the extracellular matrix of the myocardium, fibroblasts and macrophages. Galectin-3 is released at the site of damage and activates the resting fibroblasts to become matrix-producing fibroblasts. The role of galectin-3 in fibroblast activation includes increased synthesis of cytoskeleton proteins, such as collagen type I, and inhibition of the activity of matrix metalloproteinases, what suggests that galectin-3 is involved in the initiation and development of the process of myocardial fibrosis. Usually, the expression of galectin-3 in a healthy heart is low, while

its synthesis and release increase in fibrotic diseases, such as HF and AF. That creates wide possibilities of galectin-3 use in the diagnosis of cardiovascular diseases. Galectin-3 can provide additional information for the prognostication and stratification of HF risk. It seems, however, that the combination of biomarkers Galectin-3 and NT-proBNP or galectin-3 and cardiac troponins can provide a more precise clinical information than serum galectin-3 concentration alone. Apart from that use, galectin-3 seems to be a promising biomarker in cardiovascular diseases initiated and stimulated by inflammatory condition. It remains controversial whether this factor mediates unfavourable heart remodelling, or if it is merely a marker of heart failure. Some studies have provided evidence for a causal role of endogenous galectin-3 in the pathogenesis of myocardial fibrosis, hypertrophy and dysfunction in patients with heart failure. Based on these studies, it has been suggested that galectin-3 is not only a biomarker but also a mediator of the disease and it can be used as a therapeutic target [145].

Yu et al. [107] reported that the inhibition of galectin-3 was beneficial in mice that underwent transverse aortic construction (TAC) and in Ren2 hypertensive transgenic rats since it reduced fibrosis and improved function. Furthermore, in both murine and rat models of aldosterone-induced cardiac fibrosis, the loss of galectin-3 attenuated fibrotic changes and decreased cardiac dysfunction [108,109].

On the other hand, pharmacological and genetic inhibition of galectin-3 did not bring beneficial effects in a murine model of cardiomyopathy induced by transgenic activation of β2-adrenoceptors [42]. Both pharmacological and genetic inhibition occurred before the development of cardiomyopathy, and the obtained results suggest that the loss of galectin-3 cannot prevent, let alone, reverse the dysfunction in this experimental model. The sympathetic nervous system is activated in the course of cardiovascular diseases, which leads to enhanced and sustained stimulation of cardiac β-adrenoreceptors [146]. The activation of β-adrenoreceptors modulates the role of galectin-3 in heart disease, both as a biomarker and the mediator of the disease, as well as increases the level of circulating galectin-3 and directly regulates galectin-3 expression in the heart [147]. This could be the reason for obtaining different results depending on the experimental model and for attempts to undermine the role of galectin-3 as both an important mediator in the pathogenesis of CVD and a therapeutic target. In our opinion, galectin-3 plays a significant role in the pathogenesis of fibrotic heart disease as a factor stimulating the development of inflammation and worsening the prognosis. Thus, it seems that galectin-3 may be recognized not only as a biomarker but also as a promising therapeutic target. However, this thesis should be evaluated in further experimental and clinical research.

The role of galectin-3 in physiological and pathophysiological processes has inspired the development of its inhibitors as not only new therapeutic methods, but also as experimental tools for the basic sciences. These inhibitors may be useful in studying of the role of galectin-3 both in vitro (cell and tissue cultures) and in animal models, therefore they can contribute to the extension of the knowledge and better understanding of the intra- and extracellular functions of galectin-3.

The molecules of non-coding RNA (lncRNA and miRNA) play an important role in the regulation of *LGALS3* expression in various pathological conditions of the myocardium. These molecules can silence *LGALS3* expression at post-transcription level and thus can have an influence on the disease development. Moreover, an experimental over-expression of lncRNA and miRNA can prevent or alleviate the process of myocardial fibrosis. The learning of the lncRNA/miRNA/Gal-3 interrelations is very important, since they can constitute a target for therapeutic interventions. To achieve that goal an extension is needed of our knowledge of their complex role in the pathogenesis of heart diseases.

**Author Contributions:** Conceptualization, G.S.; writing—original draft preparation, G.S., A.M.-J. and D.S.; writing—review and editing, G.S., A.M.-J. and D.S.; visualization, G.S.; supervision, G.S. and D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Lucas de Freitas Pedrosa 1 , Avraham Raz <sup>2</sup> and João Paulo Fabi 1,3,4, \***


**Abstract:** Galectin-3 is the only chimeric representative of the galectin family. Although galectin-3 has ubiquitous regulatory and physiological effects, there is a great number of pathological environments where galectin-3 cooperatively participates. Pectin is composed of different chemical structures, such as homogalacturonans, rhamnogalacturonans, and side chains. The study of pectin's major structural aspects is fundamental to predicting the impact of pectin on human health, especially regarding distinct molecular modulation. One of the explored pectin's biological activities is the possible galectin-3 protein regulation. The present review focuses on revealing the structure/function relationship of pectins, their fragments, and their biological effects. The discussion highlighted by this review shows different effects described within in vitro and in vivo experimental models, with interesting and sometimes contradictory results, especially regarding galectin-3 interaction. The review demonstrates that pectins are promissory food-derived molecules for different bioactive functions. However, galectin-3 inhibition by pectin had been stated in literature before, although it is not a fully understood, experimentally convincing, and commonly agreed issue. It is demonstrated that more studies focusing on structural analysis and its relation to the observed beneficial effects, as well as substantial propositions of cause and effect alongside robust data, are needed for different pectin molecules' interactions with galectin-3.

**Keywords:** galectin-3; pectin; structure and function; bioactive polysaccharides; galectin-3 inhibition

#### **1. Introduction**

Pectins are complex polysaccharides and versatile hydrocolloids, vastly available in plant cell walls and the middle lamella of higher plants. These polysaccharides are major components for maintaining rigidity and integrity of the plant tissues, positively impacting plant growth and health [1]. Every source of pectin has a variable amount of sub-structures, such as homogalacturonans (HG), rhamnogalacturonans (RG-I and II), and xylogalacturonan [2]. The HG region is primarily composed of a homopolymer of partially esterified 1-4-α-D-galactopyranuronic acid (Gal*p*A). RG-I regions are composed of repeated and intercalated α-D-Gal*p*A with α-L-rhamnopyranose (Rha*p*) [→2)-α-L-Rha*p*- (1,4)-α-D-Gal*p*A-(1→]. This region also has variable side chains of galactans (β-1,4-Gal*p* residues with varying degree of polymerization), arabinans (α-1,5-L-Ara*f* with 2- and 3 linked arabinose/arabinan branches), and arabinogalactans (Type I: β-1,4-D-galactans with *O*-3 linked L-arabinose or arabinan; Type II: β-1,3-linked D-galactans with β-*O*-6-linked galactans or arabinogalactan), attached to the Rha*p* residues (side chains are linked at Rha*p O*-4, as demonstrated in Figures 1 and 2) [2–4]. In diverse fruits, these structures undergo a series of chemical and enzymatic alterations during the ripening process, resulting in a

**Citation:** Pedrosa, L.d.F.; Raz, A.; Fabi, J.P. The Complex Biological Effects of Pectin: Galectin-3 Targeting as Potential Human Health Improvement? *Biomolecules* **2022**, *12*, 289. https://doi.org/10.3390/ biom12020289

Academic Editor: Alexander V. Timoshenko

Received: 14 December 2021 Accepted: 7 February 2022 Published: 10 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

wide variety of intramolecular changes inside the pectic chain, as illustrated before for the papaya pulp, a fruit that rapidly undergoes the ripening process [5–8].

α α α α β α β α α **Figure 1.** Schematic representation of major pectin components in chair conformation. HG homogalacturonan, composed of linear α-1,4-D-galactopyranuronic acids; RG-I—intercalated α-Dgalactopyranuronic acids and α-L-rhamnopyranose through α-1,4 and 1,2 glycosidic bindings; AG-I β-1,4-D-galactopyranose with occasional *O*-3 α-L-arabinofuranose; AG-II—β-1,3-D-galactopyranose with *O*-6 α-L-arabinofuranose/arabinogalactans. Arabinans and galactans consist of linear α-1,5-Larabinofuranoses and β-1,4-D-galactopyranoses, respectively.

β

**Figure 2.** Schematic representation of major pectin components in Symbol Nomenclature for Glycans (SNFG-B) [9] model. Different glycosidic bounds common to pectins are illustrated.

As part of the dietary fiber group, pectins are not digested by the human tract, although the fermentation process by the human microbiota is greatly important for the maintenance of the colonic and systemic health through local signaling. The fermentation products and molecular fragments can improve metabolic syndrome and attenuate hypercholesterolemia, hypertriglyceridemia, and hyperglycemia, markers related to heart disease risks in mice, rats, and humans [10–14]. The pectic products and fragments after colonic fermentation also possess anti-oncogenic attributions in the colon for mitigating cancer-related risks in the colon and even in other types of neoplasia in humans [15].

β β Galectin-3 (Gal-3) is the chimeric representative member of the galectin protein family, located ubiquitously in the nucleus, cytoplasm, outer cell surface, and extracellular space in mammals. Gal-3 is classified as a β-galactoside binding protein, although—as it is discussed further ahead—its binding range cannot be restrained only to β-galactoside ligands. Gal-3 is composed of a flexible N-terminal domain, containing up to 150 amino acid residues with sequences rich in proline, tyrosine, glycine, and glutamine. This collagenlike region rich in proline, glycine, and tyrosine ends up in a C-terminal domain with a carbohydrate recognition domain (CRD) containing about 135 amino acids. The CRD region is responsible for the signature pattern of the galectins family [16–18]. Although the ubiquitous expression, the main biological source is derived from immunological and collagen-producing cells, where it helps to establish cell recognition and communication through protein–protein or glycan–protein interactions [19,20]. For example, one important and recently elucidated Gal-3 effect in physiological conditions is the recruitment of endosomal sorting complexes required for transport to damaged lysosomes (ESCRTs), so they can be effectively repaired [21]. However, alterations of Gal-3 expression are strongly related to tumor growth, cancer cell proliferation, cell-to-cell adhesion properties, fibrosis stimulation, T-lymphocytes apoptosis, macrophage differentiation into infiltrative forms that stabilize

tumor environment, and other features [22–28]. Those attributions are shown in Figure 3, where the intestinal model was chosen, as it is simple to explain the interface between endogenous and exogenous stimuli that differently activate Gal-3 functions. Moreover, pectin is considered a bioactive compound when ingested as a food component or as a dietary supplement, a topic that is explained in the next sections. It can be noted in Figure 3 the different Gal-3 forms available at biological environments, such as the monomeric unit (intra and extracellular) recently secreted present at initial interactions with natural ligands and pentameric (chimeric appearance) after associations of other monomeric units through their N-terminal domain due to ligand stimulus [18,29].

**Figure 3.** Schematic representation of (patho)physiological effects of galectin-3. As it is demon-**Figure 3.** Schematic representation of (patho)physiological effects of galectin-3. As it is demonstrated, the physiology is separated by a thin line from the pathological scenario, such as the extracellular matrix (ECM) secretion stimuli or the chemotactic signaling for infiltrative immune cells. Bcl-2— B-cell lymphoma 2; ERK—extracellular signal-regulated kinases; NT—N-terminal tail/domain; PKC—protein kinase-C; ROS—reactive oxygen species.

While there are extensive high-quality data regarding Gal-3 binding sites modeled to small glycoconjugates and an important level of binding/inhibition by those [30,31], the present review looks forward to establishing ways described in the literature regarding pectin and its fragments and different molecular interactions with Gal-3 protein, as it is highly controversial due to structural variability and reproducibility of experimental conditions. This review also covers the Gal-3 independent pectin interactions. The gathering of these data is fundamental for a better understanding of the overall perspectives regarding this promising—but not yet fully understood—area to be explored. Likewise, all the structural parameters, such as distinct molecular sizes, a higher number of galactan/arabinan side chains or galacturonic acid contents, methyl esterification, and other

properties important to pectin biological effects are also discussed by in vitro and in vivo perspectives [7,32,33].

#### **2. Basic Pectin Molecular Aspects**

#### *2.1. Pectin Molecular Weight*

Although having extremely variable structures, pectins are composed of large polysaccharide chains translated to solutions with high viscosity, a factor which would likely be an impairing factor for possible absorption through oral administration [34]. There are several approaches for extracting and, at the same time, lowering pectic molecular weight. The most commonly utilized method, especially in industrial extraction, is the chemical modification with mineral acids [35], such as hydrochloric and nitric acids, and organic acids [36–39], such as citric and malic acids. Enzymatic digestion is a more subtle and precise approach, using different cleavage agents, mostly arabinanases and galactanases, to remove excess side chains and polygalacturonases (exo- and endo-) to break down the larger HG backbones/pectic domain, aiming to obtain pre-planned molecular patterns [33,40]. Another category, thermic modifications, is less specific but far more practical and inexpensive. These modifications can be achieved by different methods, such as high temperature and pressure [41,42], ultrasound-assisted heating [43–45], and more underused but promising approaches, such as electromagnetic induction heating [46]. There are described extraction methods capable of conserving more of the native pectin structure, such as the use of low temperatures (40 ◦C) but with low extraction yields, and the decision which technique should be used depends on the final objective of the work [47], as it is discussed further that high molecular weight polysaccharides are not well suited for potential biological applications. Therefore, breaking down and/or manipulating the large polysaccharide chains into smaller portions while bringing better malleability to the molecule itself, improving viscosity and rheological properties, can set more biologically available binding sites, facilitating the potential interaction with proteins such as lectins or other cell-surface receptors.

#### *2.2. Monosaccharides, Backbone, and Side Chains*

The monomeric structure of polysaccharides is also extremely variable and inconsistent depending on the food source, extraction method, and modification strategy. These monomeric sugars are naturally presented as either pyranose (*p*, saccharides with chemical structure including six-member ring, composed of five carbon and one oxygen atoms) or furanose (*f*, saccharides with chemical structure including five-member ring, composed of four carbon and one oxygen atoms). This high variability represents a problem regarding standardization of recommended ratios between monosaccharides, such as the Gal*p*/Ara*f*, Ara*f* + Gal*p*/Gal*p*A, associated with the proportions of molecular side chains (e.g., arabinans and galactans) or main structure sequences of α-D-Gal*p*A. For example, important works regarding molecular modeling of Gal-3 inhibitors that have standardized to low molecular weight molecules (<1000 Da) as higher protein-inhibitor ligands [48–50] did not consider polysaccharides. Galactan and galactosyl residues were the main focus as contributors to biological effects observed between pectin and Gal-3 interaction and inhibition since this protein has a preference for binding β-D-galactopyranoside [40,51]. However, while still important targets of interest, other monomeric compounds such as the Gal*p*A, Ara*f*, and their association have been described more consistently, as found for polysaccharides with Ara*f* residues in higher quantities, mostly linked in α-(1→3,5)-L-arabinan side chains, demonstrating higher inhibition of Gal-3 through techniques such as Gal-3 hemagglutination assays (G3H) or binding through biolayer interferometry assays (BLI) and surface plasmon resonance assays (SPR) [3,33,52,53]. Therefore, this focus on specific monosaccharides that are not only galactosyl could help to understand the biological action of pectin and the possible Gal-3 interaction and inhibition.

#### *2.3. Esterification Degree*

Pectin can also be classified by the esterification degree throughout its molecule, where the common approach is the division in a lower degree of esterification (DE) (<50% DE—low-methylation or LMP) or higher DE (>50%—high-methylation or HMP), as well as how the esterification is distributed on pectin molecule (degree of blockiness). The determination of this parameter as a characterization step is crucial to establish the best application of the referred polysaccharide, such as the gelling property and potential where HMP achieves through hydrophobic interactions and hydrogen bonding, while LMP forms gels through the salt-bridge connecting adjacent or opposite carboxyl groups from divalent ions [54,55], emulsion property for protein complexation or drug delivering [56–58], in addition to the direct biologic relationship that is further discussed in the following topics. The RG-I backbone region's side chains are mostly a mixture of arabinans, galactans, and arabinogalactans attached to the rhamnose residue. RG-II is highly methyl-esterified and much more diverse in sugar composition side chains than RG-I. The main pectin backbone is composed of HG, also known as the "smooth" region. This main portion of pectic molecules can also exhibit varying degrees of methylation or acetylation (DM and DA, respectively) depending on the food source and extraction process [2]. Alkali treatment is the most common chemical method to achieve lower DM for pectic samples through direct saponification but may result in chemical waste or even slight alterations to the main pectin chain by β-elimination. Enzymatic treatment is less practical, requiring up to 25 h, depending on the expected DM. An alternative to this inconvenience is the high hydrostatic pressure-assisted enzymatic process, which is a promising operation to help attenuate this problem and facilitate the industrial application of this method [59].

#### *2.4. Rheological Properties*

When studying pectin rheology parameters, they are especially useful to determine potential applications of the characterized samples towards large-scale applications, both at food and non-food products, also helping at the definition of a better biologic destination [60]. The previously mentioned structural parameters directly impact the food product incorporation, such as viscoelasticity and thickness, which can be interpreted as gelling capacity, stabilizing potential in acidified milk beverages or fruit juices, emulsion capability with protein-rich solutions, and many others [61–64]. Therefore, the stratification generated from this type of analysis can lead to the best type of use for each pectin sample [65].

#### *2.5. Food Source*

The food source—exclusive from plants—together with the pectin extraction methods, and in some cases, the ripening parameters of fruits, are important factors in obtaining functional pectic molecules, as already mentioned. Usually, there is an additional ecologically sustainable status, as many of the possible sources are residues and byproducts from the juice and food processing industries. Some residues of apple [66], *Prunus domestica* and *Prunus mume* [67,68], jaboticaba [69], citrus [70,71], and papaya [7,8,72,73] have been explored for pectin extraction and biological activity studies.

#### **3. Gal-3 Binding Sites and Pectin Interactions**

Structurally, Gal-3 N-terminal tail (NT) transiently interacts with its CRD F-face and is linked to the glycine/proline-rich sequence, conferring its uniqueness in the galectin family, allowing self-oligomerization [74–76]. Gal-3 CRD is composed of 11 β-sheets, whereas five belong to the F-face and six belong to the conventional β-galactosides binding S-face, one opposed to the other, forming a β-sheet-sandwich (Figure 4A–C). In the S-face CRD, there is the NWGR conserved motif (Asparagine–Tryptophan–Glycine–Arginine), which is similarly encountered in the BH1 domain from B-cell Lymphoma-2 (Bcl-2) anti-apoptotic molecule, and suggested as the possible interaction that enables apoptosis evasion in tumoral cells (Figure 4B) [19,77,78]. Within the CRD, specifically the S-face, there are some subsites that can be named for easier comprehension of binding interactions. There are

two conserved subsites (C and D), two non-conserved (A and B), and one not well-defined subsite (E) [30], where their interfaces are mainly constituted of hydrogen bonds. The CDassociated subsites are a target of natural ligands such as the N-acetyllactosamine (LacNAc) Gal-3 inhibitor or the β-D-galactopyranoside residue located in the Lactose molecule, where it remains tightly bonded mainly to C subsite amino acids (Figure 4D) [79]. β

β

β

β

β β β β β β β **Figure 4.** Galectin-3 crystalized tertiary structure X-ray diffraction, PDB ID 49RB [75,80,81]. (**A**) Gal-3 complete CRD, with the anti-parallel β-sheet sandwich; (**B**) CRD S-face β-sheets (S1–S6), which holds the ABCDE subsites. The NWGR motif is highlighted because of their biochemical importance; (**C**) CRD F-face β-sheets (F1–F5, also numbered as B-9, -8, -7, -2, and -11, respectively); (**D**) Schematic representation of β-D-galactopyranosyl-1,4- β-D glucopyranose (β-Lactose) binding at the canonical S-face. The hydrogens atoms are colored as red, the hydrogen bonds as blue dotted lines, and the lactose chain as orange. The binding is stronger at the C subsite (between S4 and S5) amino acids and the β-D-galactopyranosyl residue.

However, there is extensive literature regarding other synthetic and sugar-derived molecules (especially tetrasaccharides and thiosaccharides) that utilize the whole ABCD region (also mentioned as Gal-3 binding groove, as its morphological 3D structure conformation due to the interactions between the groups AB/CD and each one of them individually) for a better affinity performance and inhibition potential [30,49,74,82]. In addition to this, the interaction of pectic poly- and oligosaccharides with Gal-3 is suggested through N-tail epitope recognition by pectin side-chain residues (e.g., galactans). The F-face interacts with β-galactosides (but also other portions), disrupting the CRD F-face binding with NT. Additionally, the same pectic structure could show multiple interactions involving both the S face region (disturbance of amino acids residues from the canonical binding site, e.g., 154–176 sequence observed with Heteronuclear single quantum coherence spectroscopy

β

(HSQC spectra)) and F-face region (amino acids residues of β-sheets 7, 8, and 9 mostly, e.g., 210–225 sequence) (Figure 5) [76,83,84]. β

β α **Figure 5.** Hypothetical interaction of RG-I fragments with both F and S-face of Galectin-3 simultaneously. Here, the main protagonists would be the β-D-galactopyranose and α-D-galactopyranuronic acid residues and would not act like specific pharmacological inhibitors, but maybe as potential Gal-3-ligand blockers through multivalency or allosteric occupation. Hydrogen bonds are represented by the blue dotted lines. PDB ID 49RB [75,80,81].

β Other Gal-3 inhibitors that have been thoroughly and increasingly studied are pectin and its fragments. Some of the possible Gal-3 inhibition effects by pectin and fragments might include protecting pancreatic β-cells against oxidative and inflammatory stress [85]. Xu et al. [86] observed modified citrus pectin (MCP) downregulated pathways involved in myocardial fibrosis, but the authors did not study the Gal-3 inhibition by MCP in vivo, although Gal-3 was downregulated in the treated group [86]. Some other biological effects from Gal-3 inhibition are related to cancer proliferation control [7,32], with a strong positive relationship between overexpression of Gal-3 and carcinogenic processes, such as apoptosis evasion, higher cell invasion, and metastatic progression, which are key signatures in tumor and metastatic types of cells [87]. The structural relationship between the pectic chain and Gal-3 binding sites is also very important for the expected positive functional effects [33,88]. Extracellular Gal-3 biological functions are exerted mostly by interacting with glycoconjugates in cell surfaces, such as laminin and adhesin, signaling and activating specific pathways. Some studies regarding binding interactions between pectin and its fragments with Gal-3 are listed below (Table 1).


#### **Table 1.** Polysaccharide-Gal-3 binding articles summary.


The Gal-3 binding range is considerably wide, including oligolactosamines, sulfated and sialylated glycans, and α-binding glycans, such as Fucα1-2, Galα1-3, and Galα1-4 added to the core of LacNAc molecule (as terminations) [89,90,93,94], which could indicate why non-β-Gal pectic fragments and other sources can still perform positively in some binding experiments. The β-1-4-galactan side chains inside the pectic RG-I domains have been highly attributed as the molecular factor involved in direct inhibition of Gal-3 (recombinant proteins and native proteins from cells), mainly discussed as a motif varying reaction, such as galactose residues in the middle or at terminal parts of those linear molecules. The longer galactan side chains in RG-I molecules identified in MCP and ginseng pectin were also associated with a stronger interaction with Gal-3 in vitro [40,95]. Similar positive binding affinity results with Gal-3, higher than observed with potato galactan, were obtained when isolating a 22.6 kDa RG-I polysaccharide from pumpkin [91]. Meanwhile, the RG-II enriched fractions extracted from *Panax ginseng* flower buds, with a high methylesterification and backbone substitution, as well as with lower content of galactose residues and low molecular weight, were associated with an absence of Gal-3 binding [3]. Highly esterified HG samples also did not inhibit Gal-3 [3]. However, other ginseng samples with an equivalent ratio between HG and RG-II regions had a positive binding affinity to Gal-3 in a similar way to the synergistic effect of HG and RG-I of citrus pectin [34,83]. In the latter case, the authors also suggested that the unesterified characteristic of pectin, alongside non-substituted GalA segments, were crucial structural elements for the observed biological effects [83]. Following this trend, commercial lemon pectin samples (from CP Kelco) with a low degree of methyl-esterification and low molecular weight were consistently and extremely potent in preventing negative outcomes in human islets with β-cell apoptosis (diabetes model) induced stress in vitro [85], in a dose–response manner. Gal-3 inflammatory stress induction was analyzed by evaluating oxygen consumption rate (OCR) in the presence and absence of a Gal-3 known inhibitor, α-lactose. After the previous incubation with α-lactose, the pectin sample effects that reversed OCR reduction induced by streptozocin and minimized inflammation induced by cytokine incubation were greatly compromised. The author's suggestions were based on the observed effects derived from pectin binding to Gal-3 [85]. However, specific data regarding the connection between the in vitro effects and Gal-3 inhibition by pectins were not proposed.

As already mentioned before in this topic, Gal-3 does not have only one direct binding site toward polysaccharides, known as the canonical S-face (sugar-binding) region inside the CRD. This can help to explain the different results in distinct binding experiments based upon competitive inhibition and chain length [88]. Detailed data suggest that the Gal-3 Nterminal binds to a 60 kDa RG-I-rich pectin portion of ginseng through galactans located in RG-I ramifications [92]. This same molecule with removed ramifications (Rha*p* and Gal*p*A intercalated residues only) did not bind with Gal-3 [92]. Another molecule rich in galactose but not derived from pectin—is a galactomannan isolated from *Cyamopsis tetragonoloba* guar gum flour (1-4-β-D-mannopyranose backbone with 1-6-α-D-galactose ramifications) also did not bind with Gal-3 [92]. However, experiments performed with potato galactans oligosaccharides derived from pectin had similar results as observed for the ginseng RG-I regarding Gal-3 binding [92], demonstrating the probable uniqueness structural features of pectin-derived molecules that can result in Gal-3 binding.

Gal-3 F-face has shown strong binding signals with pectin molecules, mainly at lower polysaccharide concentrations, opening more space to conventional S-face binding in higher concentrations. Like S-face, the F-face non-orthodox site is enriched by hydrophilic and charged amino acids residues. In addition to not having the Trp key-residue, other hydrophobic side chains could have similar functionality, as well as similar concave shape conformation [84,92] (Figure 4A–D). It is specified that resonance broadening was attributed as the primary capability of binding between Gal-3 and the RG-I-4 (a ginseng-derived rhamnogalacturonan), but also that broadening would be directly correlated to higher noise and lower sensitivity in analytical techniques [84,92], a factor which has to be taken into account. The presence of those non-conventional sites was suggested when lactose (CRD S-face inhibitor) did not interfere in RG-I binding to Gal-3 [40]. Furthermore, the binding of a determined ligand to the F-face of Gal-3 CRD could allosterically modify S-face residues. These shifts influence the conformation of the opposing face affecting the affinity between ligand (peptide, glycoconjugate, or polysaccharide) and the Gal-3, improving or attenuating the inhibitory/activity effects [76]. Moreover, Gal-3 N-terminal tail phosphorylation, although having little impact in CRD F-face, may be related to allosterically influencing carbohydrate-binding to the canonical CRD S-face site [76]. This structure-related information and respective identification method improvement contributed to more profoundly describing how the synergistic effects between pectin fragments work, as will be thoroughly discussed below.

Unusual non-galactoside poly-oligomeric ligands could act through multivalence interaction with Gal-3. The synthesis or identification of molecules that represent potential inhibitors of multimeric (chimeric) Gal-3 to cell-membrane glycoconjugates could be critical for minimizing known observed effects, such as the glycoclustering of receptors resulting in apoptosis induction of T cells [96]. Chelation, subsite binding, and reassociation of the binding site towards different monomeric structures within a molecule are also candidates for multivalent interactions with Gal-3 [97]. Although pectins and other natural ligands are harder to validate in comparison to synthetic compounds, parameters such as molecular flexibility and occupation of binding sites can be less manipulated/predicted [98]; the study of monosaccharide and consequently substructures ratios influencing Gal-3 binding is a promising area of investigation.

A recent in vitro study analyzed Gal-3 inhibition and MCF cell viability after specific enzymatic modification of citrus water-soluble fraction (WSF) rich in pectin. It was demonstrated that although β-1,4-galactan side chains in RG-I are still considered the most accountable molecular part for the observed effects, the partial removal of the side chains composed of other monosaccharide residues, such as α-1,5-arabinan, also contributed to inferior results both at cancer cell proliferation and direct gal-3 binding [33]. Other complex structures, such as the pectic acidic fractions extracted from *Camellia japonica* pollen, described as an RG-I-like polysaccharide, had their branched α-1-3,5 arabinan and type II arabinogalactans attributed to their strong Gal-3 inhibition effects [53]. It is suggested that, despite not having a significant relationship to the ratio of RG-I/HG, pectin-rich WSF bioactivity was dependent on cooperation between RG-I and HG regions [33]. In addition to this pectin fragment ratio, the monosaccharide residue composition ratio had an impactful performance. The Gal*p* amount is necessary for the best Gal-3 inhibitory results

since pectic fragments with lower Gal*p*/*Araf* ratio resulted in lower Gal-3 inhibition even with similar molecular sizes than other studies (50 to 60 kDa) [52].

There are also in vitro data for anti-proliferative characteristics for sugar-beet pectin, in which both the RGI/HG backbone and the galactans/arabinan side chains exhibited those positive effects in HT-29 (human colorectal adenocarcinoma) cell populations [99]. The alkali treatment not only increased the RG-I/HG ratio (which translates to more neutral sugar side chains) but also enhanced the anti-proliferative effects. The removal of almost all side chains in the sample did not completely abolish the effects, denoting the importance of the cooperative effects between different pectic structures [99]. Similar results were obtained in our lab, in which papaya uronic fraction, a Gal*p*A fraction enriched with galactans, had the best in vitro results in inhibiting Gal-3 hemagglutination than neutral sugar-enriched fractions [7]. The difference of the samples was the degree of methylation, in which the former had low DM, and the latter had high DM.

Zhang and colleagues [83] indicated that a combination of RG and HG polysaccharides enhances the Gal-3 binding in vitro through HG interaction with RG, opening more Gal-3 binding epitopes in the RG molecule. At a particular RG/HG molar ratio, the interaction between polysaccharide and the F-face of Gal-3 could establish the new activated binding epitopes due to the higher prevalence of galactose residues, facilitating the S-face CRD interaction. This interaction between HG and RG could perform alterations in its conformation and increase or enhance the synergistic functional effects [83]. Each 130 kDa molecule of the isolated pectin fraction could bind up to 16 Gal-3 molecules. Overall, the combination of both structures showed biologically better activity than separated molecules [83]. Additional ginseng HG-rich fractions were responsible for inducing apoptotic process in vitro at higher doses and cell cycle arrest at lower doses in HT-29 cells, and these biological effects were increased after heat treatment of the polysaccharide fractions [100].

Another ginseng polysaccharide fraction had 91 kDa, an Ara*f*/Rha*p* ratio of 2:1, and a Gal*p*/Xyl*p* ratio of 1:1 and was characterized as xylo/rhamnogalacturonan I with arabinan/galactan side chains. This fraction was evaluated for in vitro Gal-3 inhibition, anticancer effect, and in vivo gut microbiota modulation [101]. The authors indicated that the xylans were mostly responsible for the microbiota's healthy recovery and protection. Meanwhile, arabinogalactan side chains can interact with Gal-3 down-regulating tyrosinase through its N-glycan binding site [101]. Overall, the polysaccharide was effective in restoring normal levels of the important interleukin for tumor rejection and T cell activation, such as IL-10. The polysaccharide also modulated IL-17, a mediating molecule over-produced by the tumor cell microenvironment, revealing a multi-targeted functionality of the ginseng polysaccharide [101]. Although studied for many years, this pectic polysaccharide immunomodulatory property is still a trending topic, especially when the structural differences influence the interaction between the pectin and immune receptors, such as the toll-like receptor family (TLR) and interleukins at the macrophage cell surface [73,102], and will be more profoundly discussed later.

After determining the direct inhibition potential by competing with Gal-3 ligands in ECM, it was demonstrated that MCP can also downregulate Gal-3 expression. The in vitro induction of cell cycle arrest at the G2/M phase, through Cyclin B1 decrease and cyclin-dependent kinase 1 (Cdc2) phosphorylation, disrupts the Gal-3 pathological function of maintaining cell cycle arrest at the late G1 phase that leads to evasion from apoptosis induction [103]. Gal-3 can also induce phosphorylation in the signal transducer and activator of transcription 3 (STAT-3) in ovarian cancer cell spheroids. Although demonstrating slight decreases in cell viability after treating with paclitaxel, a strong synergistic effect between this drug and MCP was observed. The IC50 values decreased when both compounds were together, alongside a 70% increase in caspase-3 activity and a 75% Cyclin D1 expression level decrease against the values obtained using only paclitaxel [104].

Regarding fibrosis induction, MCP alleviated liver fibrosis and stress-induced secretions, such as decreased malondialdehyde (MDA), TIMP metallopeptidase inhibitor 1 (TIMP-1), collagen-1 α-1 (Col1A1), and Gal-3 expressions, improving HSC apoptosis

rate and the upregulation of glutathione and superoxide dismutase in vivo [105]. Renal fibrosis-related biomarkers in an adult male Wistar murine model were also attenuated by treatment with MCP in drinking water. Albuminuria, proinflammatory cytokines, such as small inducible cytokine A2, osteopontin, epithelial transforming growth factor-β1, and other epithelial to mesenchymal transition factors, were all controlled or restored to normal levels after treatment in normotensive experimental models of renal damage [106].

Gal-3 is up-regulated by aldosterone, mediating inflammatory and fibrotic response in vascular muscle cells in vitro and in vivo [107]. The mechanism was demonstrated as inducing Gal-3 secretion by macrophages via phosphatidylinositol 3-kinase inhibitor/AKT and nuclear factor κB transcription signaling pathways in vitro and in vivo [108]. Furthermore, in aldosterone-induced cardiac and renal injuries, MCP subcutaneous injection in mice—different from the major in vivo experiments when MCP was diluted in drinking water—downregulated Gal-3 at protein and mRNA levels while also minimizing cardiac adverse effects induced by aldosterone salt [109]. In addition, for the renal injuries, MCP inhibited Gal-3 at the tubular level but not in the glomeruli, which is highlighted by the authors since Gal-3 is not expressed at the glomerular level [109]. Nephrotoxicity is a major side-effect of cisplatin chemotherapy, which contributes to the number of acute kidney injury (AKI) hospitalizations. Mice that received 1% MCP in drinking water during the same period after cisplatin injection had better morphometric preservation and prognostic regarding renal fibrosis, such as lower serum creatinine levels, Gal-3, fibronectin, and collagen-1 expression, which could point to a protective effect of MCP in this treatment [110]. Specific mechanisms and routes of Gal-3 downregulation, however, were not successfully addressed by those studies. Therefore, those points should be considered for better knowledge of the possible link between treatment and the biological effect.

AKI-induced mice through the ischemia/reperfusion (IR) model led to Gal-3 expression, cardiac injury, and systemic inflammation. The authors indicate that the induction of inflammation alongside cardiac fibrosis was Gal-3 dependent, demonstrated through significant reduced deleterious outcomes in genetically Gal-3-KO mice that received orally MCP (100 mg/kg/day), which was also seen at WT-MCP-treated mice group [111], but the expression of other markers such as MCP-1 and ICAM-1 mRNA were also decreased by MCP treatment. Rats submitted to myocardial IR also had better prognostics when treated with MCP (in drinking water) one day before and eight days after the procedure, such as improved perfusion, serum brain natriuretic peptide normalization, IL-1b and C-reactive protein (CRP) reduction, lower Gal-3 expression levels at the ischemic tissue, and other parameters. The Gal-3 specific blockade suggested was measured by the authors through the expression of two proteins that are down-regulated by Gal-3, fumarase, and reticulocalbin-3, which were restored after MCP treatment [112]. Perindopril and MCP (in drinking water) were similarly effective as treatments for ischemic heart failures in rabbit models, lowering Collagen-I, III, and Gal-3 mRNA and protein expression, alongside slight reversion of histological remodeling (an important level of fibrosis was still maintained, visible by Masson staining of myocardial tissues). However, the exact mechanisms by which MCP or perindopril could exert their observed effects are still unclear and were not directly addressed [113]. Both the effects of Gal-3 and isoproterenol-induced left ventricular systolic dysfunction in the mice model with selective hyperaldosteronism, alongside myocardial fibrosis installation. Combined therapies with MCP in drinking water and canrenoate as an aldosterone blocker enhanced the anti-inflammatory and anti-fibrotic effects [114]. Rats in a pressure overload model that were treated with MCP in drinking water had lower Gal-3 mRNA expression and Gal-3 immunostaining-confirmed presence than control. Other fibrosis-related proteins such as α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor (TGF)β-1 and fibronectin, and also inflammation factors such as IL-6, IL-1β, and TNF-α were at lower levels than control [115]. The studies described herein regarding oral MCP supplementation, either isolated or in combination with other molecules: (i) did not only show effects influencing Gal-3, but also other pathways, receptors, and protein expressions; (ii) authors did not explain, or at

least brought up for the discussion, if orally consumed MCP could reach the target organs, such as kidney, heart or liver. These factors should be considered whenever reading these interesting but overly biased results, denoting not-so exclusive observations that are also promising but opening many questions regarding the systemic distribution of pectin's preor post- colonic fermentation.

As mentioned before, there are physiological activation pathways that could involve Gal-3 action, important data to account for whenever testing new possible therapeutic agents that could selectively induce activation/apoptosis depending on the target [116]. This in vitro work has demonstrated that recombinant human Gal-3 initiates three distinct pathways, one for T cell activation (PIK3) and two hybrids (reactive oxygen species and protein kinase C—ROS and PKC, respectively). Furthermore, MCP and acidic fraction from ginseng roots inhibited T cell apoptosis in vitro by caspase-3 cleavage, and ginseng-derived fractions did not interfere in IL-2 secretion [116]. One of the Gal-3 connections established by the authors to the observed effects is proposed as upregulation of PI3K/Akt phosphorylation, in which the presence of an inhibitor for each molecule also inhibited IL-2 secretion. However, no similar effects regarding the effects of MCP in cardiac protection, fibrosis regulation, and normalized hypertension were observed in a recently published clinical trial from Lau et al. [117] in patients with high Gal-3 levels and established hypertension. The results raise many doubts and counterpoints regarding the MCP effects in in vitro and in vivo studies to be replicated in humans, which is discussed in the next chapter.

Recently, Gal-3 has been treated as a treatment target and prognostic marker for patients with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Higher serum levels of Gal-3 were related to a tendency of severe acute respiratory distress syndrome (ARDS) development, and alongside IL-6 and CRP, Gal-3 was demonstrated to be the best predictive power for mortality outcome [118]. There are also positive correlations between Gal-3 and other inflammatory markers such as PTX-3, ferritin, and the marker of endothelial dysfunction, sFlt-1 [119], which could be utilized for intensive care unit (ICU) admission biomarker. A phase 2a study, the first clinical trial using an inhalator treatment targeting Gal-3-GB0139—associated with the standard of care procedure (dexamethasone) identified lower Gal-3 serum levels, higher mean downward of CRP levels (although higher CRP was identified at first in the treatment group) and other inflammatory agents and better fibrosis marker levels than the control group. However, there was no statistical difference between patient mortality rates between groups [120]. This enhances and augments the discussion to further explore alternative treatments regarding Gal-3 inhibition.

#### **4. Pectin and Gal-3 Controversies**

As it has been widely studied and known, pectins are extensively fermented through the local intestinal microbiota, which could systematically impair "direct" mechanisms of action. In the above-discussed works showing systemic effects after oral ingestion of MCP, it is often ignored the thought process and viability on how those molecules could reach the systemic circulation. In counterpoint, a work that used antibody recognition of RG-I fragments from *Bupleurum falcatum* L.—demonstrating reactivity at mice bloodstream and liver—could indicate a partial small-intestine related absorption [2,121]. Similar studies based on β-glucan uptake mechanisms should be more explored for pectins in general, as both are non-digestible carbohydrates that suffer fermentation to an extensive degree [2]. Modified pectin from broccoli (*Brassica oleracea* L. Italica) was suggested to be absorbed after an increased number of activated macrophages and lymphocyte proliferation when administered through oral treatment in mice, but with no same effect in vitro [122]. One possible suggestion of pectin "absorption" is related to asialoglycoprotein receptors that could play a role in absorbing modified pectin fragments throughout the intestine, as they are notable galactoside-terminal glycoproteins transporters [123,124]; however, much more pieces of evidence need to be described to confirm this hypothesis. The oral consumption of MCP reduced liver metastasis on a mouse colon cancer model [125], while MCP-derived galactans ([-4-β-D-Gal*p*-1-]n) and arabinans ([-5-α-L-Ara*f*-1-]n) with a low

degree of polymerization were absorbed through paracellular transport, and in lower rates through transcellular transport, similar to what was observed at in vitro culture of Caco-2 monolayer models [126]. Pectin-derived oligosaccharides with 1 kDa and rich in galactosyl residues were absorbed in BALB/c mice and human tumor cells while also changing membrane permeability in different human cancer cells, such as HepG2 and Colo 205 (hepatic and colon carcinoma, respectively) [127]. Microfold cells and gut-associated lymphoid tissue (GALT) are also proposed as explanations for the bloodstream presence of modified pectin fragments, where the former theoretically would serve as a facilitator to GALT macrophages to act internalizing these portions [122,128]. Once again, these assumptions of absorption models are still in germinative steps; therefore, they cannot be taken as unreasoning facts, but they can open new potential transcription elucidation of in vivo observed effects towards clinical significance in the future.

The above described highly selective effects, although not completely mechanistically understood, could be related to the mentioned diverse binding sites and different chemical conformations between those polysaccharides and Gal-3, but the range of varied mechanisms unrelated to Gal-3 specific inhibition cannot be excluded or ignored, as it could also play a role or even be the major protagonists regarding polysaccharides action.

In a related but slightly different context, it is highly important to clarify that a binding molecule is not the same as an inhibitor by itself. Additionally, this capability of pectins to exert both instances, binding to or inhibiting Gal-3, has been contested. As demonstrated by Stegmayr et al. [50], the use of a range of plant polysaccharides to study the capacity of interacting with representatives of the galectin family (including Gal-3) resulted in a very low or even absent binding and agglutination inhibition [50]. It is further discussed that the discrepancy observed in similar studies, such as the one from Gao et al. [95], could be due to a fine-tuning difference (concentration and temperature) or even multivalence interaction factors [50,95]. Furthermore, noteworthy, immobilized surface techniques may be prone to developing suitable conditions to the multivalent aspect, therefore potentially overestimating affinity results [88]. Nevertheless, the same authors also found a potential indirect effect. Re-incubating the JIMT-1 cells with the pectin samples led to Gal-3 accumulation around intracellular vesicles, feasibly inclining towards a "directional" change of location inside the cell, although more experiments regarding this property need to be performed to detect plausible applications of this observed scenario in vivo [50]. Other studies demonstrated biological effects in different in vitro cell cultures were totally or highly independent of Gal-3 inhibition; as demonstrated through Gal-3 hemagglutination (G3H) assay, cell lines with low expression of Gal-3 and/or usage of lactose (Gal-3 inhibitor) did not influence HG or RG-I activities, suggesting that cell migration inhibited by those polysaccharides did not rely upon human/mouse Gal-3 binding [8,129]. The main problem observed in literature is the overused statement of plant polysaccharides acting as specific pharmacological direct inhibitors of Gal-3, without specific data of inhibition shown, or even proposed study models to evaluate the integrity of those polysaccharides in reaching potential target organs. The facts were described in the letters written by Hakon Leffler, MD, Ph.D., and Anwen Shao, MD, Ph.D. [130,131], in response to pectin attribution of Gal-3 inhibition at blood–brain barrier disruption, relying on the lower expression detected by immunoblotting [132], but this is only one of the examples. Specific human Gal-3 inhibition of the S-face CRD region, mostly between C and D subsites, is achieved mainly by small glycoconjugates, such as lactose, N-acetyllactosamine, and many synthetized neo-glycoproteins [30,49,133–136].

To address those controversial perspectives, more studies regarding alternative mechanisms independent of Gal-3 interaction should be performed with polysaccharides while also exploring more structure-relation models. In the following chapters, alternative perspectives are further analyzed.

#### **5. Pectin as Dietary Fiber: Some of the Gal-3 Independent Beneficial Effects to Human Health**

Regarding pectin fermentation, it generates products that are essential for colonic and systemic health in general. One example is the inhibition of cholesterol intestinal absorption in an apoE−/<sup>−</sup> mice model through regulating mRNA levels of its transporters, resulting in controlled blood lipid levels in vivo [10]. This protection is also due to the physicochemical properties of the soluble fibers, where the bile acid excretion and cholesterol mobilization in the intestinal tract is compromised by the fiber viscosity [137]. Similar results were obtained in mildly hypercholesteremic humans, in which pectin with high DE and high molecular weight resulted in a cholesterol-lowering effect [12]. A rodent model of high-fat diets to induce non-alcoholic fatty liver disease also had positive results after receiving 8% citrus peel pectin. The diet had attenuated liver damage and lipid accumulation while also reducing some biomarkers such as carbohydrate-responsive element-binding protein (ChREBP) and reducing serum total triglyceride in vivo [11].

Regarding glucose metabolism, pectin (from apple and citrus) added to high fat/high sugar diets, even at low doses, were successful at ameliorating glucose serum levels [13,14], glucose tolerance, and insulin resistance biomarkers such as HOMA-IR and fasting insulin serum levels [13]. Those effects are suggested to be derived from a pectin capacity of lowering mucosal disaccharidase activities, specifically sucrase and maltase [14], and also through a potential pectin p-AKT upregulation, being beneficial to directing the insulin signaling [13].

Short-chain fatty acids (SCFA) are the most common by-product of pectin and other types of fibers fermentation, and there is extensive literature related to biological and health effects [138–141]. The SCFA help to maintain intestinal health through G-protein-coupled receptors (GPR) interaction, such as T regulatory cell homeostasis, epithelium integrity, and maintenance of an acute immune response and normal cytokine/chemokine expression of key modulators [142–145]. Different types of pectins and fragments can modulate microbiota and have different fermentation profiles. Sugar beet and soy pectin lowered *Akkermansia* relative abundance, while soy pectin showed high levels of propionate, butyrate, and branched SCFA concentrations in the colon of rats [146]. Supplementation of citrus pectin (CP) in piglets diets was also attributed to a higher relative abundance of Bacteroidetes members in colonic digesta and feces, and this pectin-enriched fraction also slowed the fermentation process, changing microbiota interaction [147]. In a dynamic digestion/fermentation simulator, CP could induce *Bifidobacterium* spp. growth, but not *Lactobacillus* spp. Both genera presence are considered beneficial to colonic health [148]. During in vitro fermentation, authors had similar results with high DM (70%) apple pectin [149], with in vivo observations also supporting the mentioned in vitro data [11]. The CP was capable of inducing growth of *Faecalibacterium prausnitzii*, a bacteria that has been pointed as a modulator in dysbiosis of Crohn's disease patients and a major agent of pectin utilization, with their lower levels correlated to inflammatory bowel disease [150–152]. Those data support the application of pectins in health investigations, even before going deeper into specific binding/modulation features, and they are summarized in Table 2 (for the diverse discussed biological effects of pectin).


**Table 2.** Summary of observed experimental effects in manuscripts studying pectin and its fragments.






#### **6. Should Gal-3 Inhibition Be the Main Biological Effect Expected from Pectin?**

Although the potential Gal-3 inhibition achieved by pectin could be a resourceful knowledge area, there is a great extent of literature showing parallel ways. Wild-type and Gal-3 knockout (Gal-3−/−) HCT-116 (human) cells were exposed to different papaya pectin fractions in vitro. Specifically, the most acidic fraction (uronic fraction from fourth day after harvest), with a mean molecular weight of 128 kDa and high antibody reactivity to LM5 and LM16 (1,4-β-galactan and type-1-rhamnogalacturonan, respectively), kept a slightly lower efficacy at the Gal-3−/<sup>−</sup> cells, suggesting that even though the cancer cell viability decrease could be in part due to Gal-3 inhibition, it was not the only molecular modulated pathway [7]. Commercially available CP and MCP demonstrated to have a pro-inflammatory action independent of Gal-3 inhibition, upregulating cytokine secretion in the spleen of BALB/c mice, IL-17, IFN-γ, and TNF-α through IL-4 [153]. Modified pectin obtained from *Theobroma cacao* pod husks, highly composed of uronic acids, galactose, and rhamnose, with a low degree of methylation and amidation, was also attributed with a pro-inflammatory profile, similar to LPS stimulation in isolated macrophages from mice, upregulating secretion of IL-12 and TNF-α, although stimulating the anti-inflammatory IL-10 simultaneously. The pectins, however, did not enhance the phagocytic activity of the peritoneal macrophages [154]. Similar effects were detected in differentiated macrophage (THP-1) cell cultures that were in contact with native sweet pepper pectin. The polysaccharide was characterized as pectin composed of uronic acids, galactose, and arabinose,

also confirmed through NMR with signals of methyl and acetyl groups linked to α-D-Gal*p*A with a high degree of methylation (85%) and low degree of acetylation (5%). There were also signals of (1→4)-linked-β-D-Gal*p* units, which the authors attributed to type-1-arabinogalactans. The sweet pepper pectin induced in vitro TNF-α, IL-1β, and IL-10 secretion at the highest concentration used [155]. After modifying the native structure and removing its side chains by partial acid hydrolysis, the composition was uronic acids (91%) and rhamnose (9%). The respective signals identified in the native molecule related to galactan core residues had disappeared, with also a great reduction in DM (down to 17%). This modified sweet pepper pectin was still able to induce—at a lower rate than native pectin—the TNF-α and IL-10 secretion but at a higher rate the IL-1β [155]. Here, it is again highlighted the importance of the structure-dependence of pectic fractions with different potential targets. In earlier studies, sweet pepper pectin (1,4-α-D-galacturonan partially substituted with methyl and *O*-acetyl backbone) and low methoxyl CP performed similarly by lowering TNF-α and enhancing IL-10 secretion, which resulted in ameliorated survival rate in endotoxin-shock induced mice models [102,156]. Elsewhere, CP was also capable of reducing LPS-induced hypothermia and inflammatory cytokine gene expression, suppressing IL-6 production and TLR-4 signaling in vivo [157].

Toll-like receptors (TLR) have been thoroughly explored, especially the dichotomy of weighing between agonists or antagonists for cancer treatment, where the same TLR can exhibit anti or pro-tumor immune responses [164–166]. Lemon pectin ranging from 40 to 100 kDa and from 30 to 74% of DM, activated in vitro THP-1 phagocytic cells depending on *MyD88* in a TLR-mediated manner [158]. Additionally, the pectin DM and its structural backbone were correlated to NF-κB/AP-1 activation through TLR, where highly esterified polymers were strong activators, and their oligomers (produced after extensive hydrolytic processing) did not perform equally [158]. Similarly, ginseng polysaccharide extract composed of RG-II was a TLR-4 up-regulator and MyD88 activator in a structure-dependent manner in vivo [116]. A polysaccharide extracted from a European berry (*Hipphoides rhamnoides* L.) with 85% DM and consisting of repeating units of (1→4)-β-D-galactopyranosyluronic acid residues strongly stimulated TLR-4 [159,167]. The blockage of TLR4/MyD88 was used by authors to explore the interaction between the pectic structures and macrophages, where it inhibited the increase observed in nitric oxide and other cytokines induced by treatment with the polysaccharides [167]. Ginseng RG-II stimulated TLR-4, increasing dendritic cell maturation and activation through induced cytokine and mitogen-induced protein kinases (MAPKs) production. This cytotoxic T cell response inhibited tumor growth of EG7 lymphoma cells [160]. Interestingly, the ripening process for some sources of pectin, such as papaya, can be of utmost importance regarding main structural changes to effectively interact with diverse TLR receptors [73]. The authors demonstrated that although TLR-2 and -4 were activated by pectins isolated from unripe and ripe papayas, TLR-3, -5, and -9 were not activated by the pectins isolated from unripe fruits in two different time points (pectins with 580 and 610 kDa, higher galactose and glucose contents, alongside with a proportionally lower composition of GalA residues; the second unripe point had 15% DM). Specifically, TLR-3 and -9 could be inhibited by pectins isolated from unripe papayas due to high molecular weight structures [73], similar to what was previously observed with lemon pectins that inhibited the TLR-2 heterodimer formation with TLR-1, but not with TLR-6 [161]. Citrus pectin ranging from 18 to 69% DM (but without further structural details or suggestions) was able to inhibit TLR/MyD88 by oral administration in mice and decreased TLR-2 mediated immune response in rats when administered both in alginate microcapsules and directly at drinking water [162]. Administration of pectin samples and usage of checkpoint inhibitors could be powerful measures to assure immune responses in certain types of cancer [168]. Neohesperidin nanoliposomes incorporated with citrus pectin (65% DM) and chitosan (50 kDa, degree of deacetylation of 85%) had higher cellular uptake rates in comparison to chitosan or neohesperidin single treatments [169]. A nephropathy murine model with MCP added in drinking water found protective effects of the pectin sample independent of Gal-3 inhibition at early proliferation, but with Gal-3 downregulation later on [163]. An overall scheme of the possible biological effects of pectins that were discussed throughout the paper are depicted in Figure 6 using an intestine model for easier comprehension, and the different biological effects observed in in vitro, in vivo, and cohort/clinical studies are summarized as a table (Table 2).

**Figure 6.** Schematic representation of the intestinal environment. Pectin molecules can interact in different ways with epithelial and immune components of intestinal tissues, regulating different responses directly and through fermentation by-products. SCFA—short-chain fatty acid; TLR—tolllike receptor.

Even though extensive human model studies are needed to confirm the positive outcomes of pectin ingestion regarding specific targets, the data obtained until now can help to demonstrate a variety of positive health-regulating effects of pectins. The pectin binding and inhibition of Gal-3 is one of them; however, this relationship is still highly controversial and contested. Human Gal-3 inhibition and the consequent beneficial effects on human health must be covered, from in vitro experiments to human clinical trials, passing through the bioavailability assays of pectin-derived fragments in the human body. The plausible wider range of usage for the polysaccharides is highlighted in this revision, enhancing the discussion of the inclusion of pectin molecules for synergistic effects with different molecules and drugs. Moreover, pectin could exert direct and indirect immunomodulatory effects, and their fragments could interact with different types of receptors. The fermentation products of pectins also help to sustain the intestinal and systemic environment, with all these possible beneficial effects of pectins being potentially achieved through supplementation by oral intake.

#### **7. Conclusions**

Targeted galectin-3 binding sites for therapeutic approaches are diverse. Pectin is an important food component classified as soluble dietary fiber. Its biological effects on human health go from colonic fermentation and microbiota modulation to potential direct interaction with intestinal cells and proteins, such as TLRs and Gal-3. Many studies can effectively suggest the multi-way interaction between pectin molecules and these ligands. The Gal-3 binding is suggested in the CRD motif, both through its F-face and S-face sites. Furthermore, the binding can occur in the N-terminal tail and even in a twostep interaction method, promoting subtle motif variations in the protein molecule and therefore enhancing an adequate interconnection with the pectin molecules. However, the biological effects of pectin transcend Gal-3 interaction and/or inhibition, which is far from being an established point, with several challenges to be overcome, and has undergone valid confrontations. All the literature and methodology improvements converge on the pectin diversity for enhancing human health. Moreover, pectin molecules exerting distinct regulatory/inhibitory effects, low side effects, and natural sources are interesting bioactive components to be added into dietary supplements. These pectin molecules could be used continuously to increase the natural intake of bioactive polysaccharides, e.g., in postcardiac arrest and renal fibrosis pathologies, as well as to a great extent as an auxiliary factor in chemotherapy and possibly as immunomodulatory molecules. Extensive additional research is needed before confirming any of those promising illustrated scenarios.

**Author Contributions:** L.d.F.P.: Conceptualization, Data curation, and Writing (Original draft and Review and Editing). A.R.: Writing (Review and Editing). J.P.F.: Conceptualization, Supervision, and Writing (Review and Editing). All authors have read and agreed to the published version of the manuscript.

**Funding:** We thank São Paulo Research Foundation (FAPESP—Brazil; #2016/19657-8 and #2020/08063-5) and the Brazilian National Council for Scientific and Technological Development (CNPq; #142112/2019-4) for Lucas de Freitas Pedrosa scholarships. The study was financially supported by grants #2012/23970– 2, #2013/07914–8, #2019/11816–8, and #2021/06419-0 from the São Paulo Research Foundation (FAPESP).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

