Role of Cathelicidins in Atherosclerosis and Associated Cardiovascular Diseases

Abstract

Cathelicidins (human LL-37 and rat CRAMP) are multifunctional peptides involved in various cardiovascular conditions. This review integrates the recent findings about the functional involvement of LL-37/CRAMP across atherosclerosis, acute coronary syndrome, myocardial infarction, heart failure, diabetic cardiomyopathy, and platelet aggregation/thrombosis. In atherosclerosis, LL-37 interacts with scavenger receptors to modulate lipid metabolism and binds with mitochondrial DNA and lipoproteins. In acute coronary syndrome, LL-37 influences T cell responses and mitigates calcification within atherosclerotic plaques. During myocardial infarction and ischaemia/reperfusion injury, LL-37/CRAMP exhibits dual roles: protecting against myocardial damage through the AKT and ERK1/2 signalling pathways, while exacerbating inflammation via TLR4 and NLRP3 inflammasome activation. In heart failure, LL-37/CRAMP attenuates hypertrophy and fibrosis via NF-κB inhibition and the activation of the IGFR1/PI3K/AKT and TLR9/AMPK pathways. Moreover, in diabetic cardiomyopathy, these peptides alleviate oxidative stress and fibrosis by inhibiting TGFβ/Smad and AMPK/mTOR signalling and provide anti-inflammatory effects by reducing NF-κB nuclear translocation and NLRP3 inflammasome formation. LL-37/CRAMP also modulates platelet aggregation and thrombosis through the FPR2 and GPVI receptors, impacting apoptosis, autophagy, and other critical cellular processes. This comprehensive overview underscores LL-37/CRAMP as a promising therapeutic target in cardiovascular diseases, necessitating further elucidation of its intricate signalling networks and biological effects for clinical translation.

1. Introduction

Host defence peptides (HDPs) are crucial players in non-specific immune response against infectious bacteria (both Gram-positive and Gram-negative, including bacterial biofilm), fungi, viruses, and other parasites. In recent decades, these peptides have been explored as immunomodulators because of their ability to induce an immune response [1,2]. HDPs are short bioactive polypeptides found in virtually all living beings as part of the first line of defence [3,4]. Cathelicidins and defensins are the two subgroups of HDPs produced by mammals as part of the innate immune system. Humans synthesise many classes of defensins, but only a single one of the cathelicidins [5]. Cathelicidins are one of the most important groups of mammalian HDPs produced by humans and other vertebrates as large 18 kDa precursors (94–114 amino acids), a highly conserved N-terminal region containing a signal peptide and a pro-domain called cathelin (cathepsin L inhibitor)-like domain (CLD), while the C-terminal region exhibits a highly variable antimicrobial domain (AMP) (Figure 1). For the further evolutionary analysis of protein sequences and comparison of physical, chemical, and biochemical parameters, we wish to redirect interested readers to recent specialised reviews [6,7,8]. Among the known mammalian HDPs, human and mice genes hCAP18 (or Cathelicidin Antimicrobial Peptide [CAMP]) and Cathelicidin-related antimicrobial protein (CRAMP), respectively, encode a precursor, which, under specific conditions, is cleaved by protease into an active 37-amino-acid long peptide (4.5 kDa) with dileucine at the N-terminus and called Cathelicidin, or LL-37. Human LL-37 is a positively charged peptide (+6), at neutral pH, with a high content of basic and hydrophobic amino acid residues [9].

LL-37 is primarily known as an important player in the human innate immune system, being produced against bacterial (both Gram-positive and Gram-negative) and viral infections [10,11,12,13]. During the last decades, the exploration of the LL-37 structure/function relationship facilitated the development of a wide range of new synthetic peptides capable of inducing an immune response while destroying bacteria [14]. The development of novel LL-37 analogues and derivatives was discussed in several excellent recent papers [8,15,16] and, therefore, will be omitted here. We wish to redirect readers interested in the molecular mechanisms underlying LL-37-induced receptor activation to another excellent review [17].

Initially, LL-37 was described in 1995 as a lipopolysaccharide (LPS)-binding antimicrobial protein isolated from neutrophils. The ability of LL-37 to bind LPS protected mice from lethal quantities of injected LPS, thus preventing lethal endotoxemia [18]. Also, LL-37 is known to control inflammation, acting as both a pro- and anti-inflammatory factor in different cell types and tissues. Therefore, LL-37 negatively regulates anti-inflammatory interleukin 10 (IL-10) and positively regulates pro-inflammatory ILs (1β, 12 and 18) and type I interferons (IFNs), while inhibiting Absent in melanoma 2 (AIM2) inflammasome formation, as well as suppressing IFNγ, Tumour necrosis factor (TNFα), IL-4, and IL-12 [19]. LL-37 immunomodulatory properties have been attributed to neutrophils, monocytes, macrophages, dendritic cells, lymphocytes (thymocytes [T] cells, bone marrow [B] cells, and natural killer [NK] cells), mast cells, and mesenchymal stromal cells [20]. Furthermore, LL-37 regulates the production of reactive oxygen (ROS) and nitrogen (NOS) species, antigen presentation stimulus, and leukocyte differentiation [21]. LL-37 also prevents the translocation of Nuclear factor kappa B1 (NF-kB) subunits p50 and p65, which are factors regulating the expression of pro-inflammatory genes [22]. LL-37 can promote, directly or indirectly, the attraction of a variety of leukocytes, including neutrophils, eosinophils, monocytes, and CD4+ and CD8+ T cells through the Formyl Peptide Receptor-Like 1 (FPRL1) receptor [23,24]. Additionally, LL-37 was shown to promote cell migration, proliferation, and differentiation [25,26], to regulate angiogenic [27,28] and wound-healing processes [29,30], to maintain skin barrier homeostasis [31], and to contribute to cancer [32] and auto-immune disease progression [33]. Furthermore, recent research demonstrated that LL-37 has anti-amyloidogenic properties [8], which are mediated through interaction with the amyloid self-assembly of islet amyloid polypeptide, thus suppressing amyloid self-assembly and decreasing damage to the pancreatic β-cell, thus protecting from type 2 diabetes pathogenesis [34]. Finally, the serum LL-37 levels were associated with psychotic disorders. In particular, elderly women with major depressive disorder and patients with bipolar disorder showed increased levels of LL-37, while no association was found with other parameters (such as age, C-reactive protein, white blood cells, body weight, smoking status, hypertension, diabetes, and dyslipidaemia) [35,36].

Atherosclerosis is the main risk factor for other cardiovascular diseases, often referred to as atherosclerotic cardiovascular diseases (ASCVDs). Growing plaque increases the risk of vessel obstruction, gradually reducing blood flow in the coronary arteries, generating ischaemic cardiopathies, such as cardiac insufficiency and angina pectoris, and, eventually, causing myocardial infarction or stroke. Thrombus, detached from the arterial wall, can produce a clot circulating within the cardiovascular system. At some point, the clot can settle in the distal arteries and cause local ischaemia, organ dysfunction, or potential infarction [37]. Further in this section, we review recent publications deciphering the role of LL-37 in atherosclerosis and associated complications, such as coronary artery disease, heart failure, diabetic cardiomyopathy, and others.

2. Atherosclerosis

Atherosclerosis is a leading cause of various cardiovascular diseases, including peripheral artery disease (PAD), coronary artery disease (CAD), heart failure, cardiomyopathy, ischaemic stroke, and others. This chronic inflammatory condition is characterised by the pathological lipid accumulation and remodelling of the arterial wall. The development and progression of atherosclerosis result from the interaction between numerous genetic, environmental, and behavioural risk factors, with inflammation and hyperlipidaemia playing pivotal roles in its pathogenesis [38]. Globally, advanced atherogenesis and its complications account for 17.9 million deaths annually, representing 32% of all deaths [39].

Atherosclerosis begins with the influx of cholesterol-rich, low-density lipoprotein (LDL) particles coated with Apolipoprotein B (ApoB) protein into the intimal layer of the arterial walls, particularly in susceptible regions such as arterial bifurcations (Figure 2) [40]. Within the intima, LDL particles undergo modification by various proteolytic and lipolytic enzymes and oxidative agents, resulting in the formation of multiple modified low-density lipoprotein (mmLDL) particles. The accumulation of mmLDL in the intima leads to endothelial cell (EC) dysfunction, marked by increased expression of inflammatory mediators (such as IL-1β, IL-6, TNFα, and INFγ) and adhesion molecules (such as Intercellular adhesion molecule 1 [ICAM1], Vascular cell adhesion protein 1 [VCAM1], Monocyte chemoattractant protein-1 [MCP-1], and P- and E-selectins), thereby promoting inflammation and leukocyte adhesion. Macrophages then uptake mmLDL to form foam cells, which further facilitate inflammation and stimulate humoral and adaptive immunity [41,42].

Oxidative stress is another critical factor in the initiation and progression of endothelial dysfunction and atherosclerosis [43]. An increase in ROS and insufficient antioxidant production promote lipid deposition, inflammation, and endothelial injury [44]. Several other processes contribute to the disease’s progression, including the transition of epithelial cells to mesenchymal cells and the differentiation of fibroblasts to myofibroblasts under the influence of inflammatory cytokines and pro-apoptotic regulators. These processes exacerbate endothelial dysfunction and plaque formation. Advanced plaques can lead to the necrosis of macrophages and smooth muscle cells, resulting in a necrotic core covered by a fibrous cap that is prone to rupture, causing thrombosis and vessel occlusion [45]. Additionally, the dysregulation of calcium homeostasis in the plaque initiates a mineralisation process similar to bone formation, leading to plaque calcification [46]. While extensive calcifications can stabilise the plaque, spotty calcifications increase its vulnerability and the risk of rupture [47]. Consequently, effective anti-atherosclerotic treatments typically focus on lipid-lowering, antioxidant, and/or anti-inflammatory strategies [48,49].

2.1. The Role of LL-37 in Atherosclerosis Progression

Several early reports demonstrated atherosclerotic plaques produced a surplus amount of LL-37, which triggered vascular smooth muscle cell (VSMC) apoptosis and induced the expression of the adhesion molecule (ICAM1 and MCP-1), thus promoting atherosclerosis development [50,51]. Later experiments on LL-37 cathelicidin-related, antimicrobial peptide (mCRAMP)-deficient atherosclerotic (ApoE^–/– and ApoE^–/– Cramp^–/–) mice fed a high-fat diet (HFD) confirmed a positive effect of CRAMP on atherosclerosis progression. CRAMP was localised in the interstitium of the aortic root sections of ApoE^–/– mice fed HFD, while a normal diet reduced CRAMP production. Furthermore, ApoE^–/– Cramp^–/– mice fed HFD showed decreased lesion sizes and reduced macrophage accumulation in early atherosclerotic lesions [52] (Figure 2).

Interestingly, CAMP serum levels in healthy volunteers were positively correlated with LDL cholesterol, while negatively correlated with HDL cholesterol. The oral lipid ingestion (a mixture of saturated, mono-unsaturated, and poly-unsaturated fatty acids free of proteins and carbohydrates) decreased CAMP serum concentration in healthy volunteers. The molecular mechanism underlying this effect is currently unknown and requires further investigation [53]. CRAMP blood serum levels in atherosclerotic Low-density lipoprotein receptor-deficient (Ldlr^−/−) mice fed HFD were higher compared to mice fed a standard diet. Moreover, Cramp expression in Ldlr^−/− mice fed HFD was increased only in the spleen and liver, while no expression was detected in aortic tissue, and expression in the adipose tissue, kidney, colon, and ileum was not affected by HFD. Furthermore, the experimental induction of myocardial infarction (MI) reduced CRAMP serum levels in wild-type mice. On the contrary, the LL-37 blood serum levels in aged CAD patients were not different from healthy controls. However, the LL-37 levels correlated positively with triglycerides (TG) and blood pressure (both systolic and diastolic) and negatively correlated with left-ventricle ejection fraction in CAD patients. Therefore, further studies are required to elucidate the connection between LL-37 and diet in atherosclerosis progression [54].

A recent study established the connection between LL-37 and autophagy, a lysosome-dependent, cellular stress-dependent process of the bulk degradation of malfunctional and damaged cellular components. Thus, in human umbilical vein endothelial cells (HUVECs), LL-37 promoted autophagy by up-regulating autophagosomal membrane marker Microtubule-associated protein 1 Light Chain 3 Alpha (LC3-II). Furthermore, LL-37 co-localised with LC3 at the autophagosomal membrane and formed a complex with Autophagy receptor ubiquitin-binding protein p62, suggesting that LL-37 is ubiquitinated, recognised by p62, and transferred to the autophagosomes for degradation [55].

However, another study demonstrated that LL-37 could form a complex with mitochondrial DNA (mtDNA), which released dying cells in atherosclerotic plaques to escape from autophagy and degradation by DNase II, while activating Toll-like receptor 9 (TLR9)-mediated inflammatory responses (Figure 2). The mtDNA concentration in the plasma of patients with atherosclerosis was 3–7 times higher compared to healthy controls, and atherosclerotic plaques showed a 15-fold higher amount of LL-37-mtDNA complex compared with the normal tissue. Accordingly, the injection of LL-37-mtDNA complex increased the atherosclerotic lesion area in the aortic root in ApoE^–/– mice. Interestingly, the treatment of HFD-fed ApoE^–/– mice with anti-Cramp-mtDNA antibody decreased plaque size and reduced the levels of Cramp-mtDNA complex, TNFα, IL-6, and IFNα. These results proposed a crucial role of the LL-37-mtDNA complex in inflammatory response and atherosclerosis progression [56]. Interestingly, Apolipoprotein A-I (ApoA-I), the main component of high-density lipoprotein (HDL) particles, which is also known as “good cholesterol” for its atheroprotective properties, protected HUVEC against LL-37-induced cytotoxicity. Mechanically, ApoA-I effectively and specifically bound LL-37 and caused its structural rearrangement, thereby reducing LL-37-induced cytotoxicity. Accordingly, ApoA-I knockdown in HepG2 cells increased sensitivity to LL-37-mediated cytotoxicity. However, further in vivo studies are required to evaluate what role this mechanism plays in the HDL-related repertoire of atheroprotective properties [57].

Scavenger receptors (SRs) are integral membrane proteins that typically bind and/or internalise multiple ligands. The initiation and progression of atherosclerosis is linked to the SRs of five SR classes: SR-A, B, E, G, and J, with Cluster of differentiation 36 (CD36), lectin-like oxidised LDL receptor-1 (LOX-1), and receptor for advanced glycation end-products (RAGEs) as the most important members of SR-B, SR-E, and SR-J classes, respectively. Lipoprotein particles, phospholipids, carbohydrates, and cholesterol esters are the common ligands for these SRs, which explains their significance in ASCVD diagnosis and therapy [58,59]. Recent reports suggested a functional association between LL-37 and SRs in the context of ASCVD. Experiments on HFD-fed, streptozotocin-induced diabetic mice demonstrated that cathelicidin overexpression reduced fat mass without affecting glucose metabolism. Mechanically, this effect was achieved through the LL-37-mediated inhibition of extracellular signal-regulated kinase (ERK)/CD36 signalling pathway, which prevented fat accumulation in hepatocytes and adipocytes and manifested in phenotype by reduced fat mass [60].

Furthermore, the unique ability to bind LDL and enhance LDL uptake was found in cathelicidins from humans and some primates, but not in mice or rabbits. Further, LL-37–LDL complex uptake in the macrophages was mediated partly by the low-density lipoprotein receptor (LDLR), SR-B1, and CD36 (Figure 2). In addition to increased LDL uptake, LL-37 treatment modulated the macrophages’ transcriptional response by downregulating the expression genes associated with lipid metabolism (such as LDLR, Fatty acid desaturase 2 [FADS2], Methylsterol monooxygenase 1 [MSMO1], 7-Dehydrocholesterol reductase [DHCR7], and others). Accordingly, ApoE^–/– HFD-fed mice expressing human LL-37 developed larger atheroma plaques compared to control mice, while showing no difference in body weight and circulating total cholesterol (TC), LDL, HDL, and TG. Finally, LL-37 co-precipitated mainly with ApoB-containing lipoproteins, including VLDL/chylomicron, intermediate-density lipoprotein (IDL), and LDL, while smaller amounts were identified in HDL fractions. In total, these results provide evidence that LL-37 interaction with atherogenic lipoproteins may modulate lipid metabolism in macrophages and increase atherosclerosis development [61].

Recent research showed the involvement of immune auto-reactivity to CRAMP in atherosclerosis progression. Thus, the immunisation of HFD-fed ApoE^–/– mice with 20 μg of Cramp reduced the aortic atherosclerosis burden, while a 100 μg dose increased the aortic sinus plaque size, increased neutrophil infiltration, and was accompanied by increased CD4+ and CD8+ T cells with Effector Memory (EM) markers and increased CD11b+ cDC subpopulation (Figure 2). These results suggested that the therapeutic manipulation of the Cramp amount can modulate the immune response to the self-antigen and affect atherosclerotic plaque development [62].

In total, the provided results suggested high LL-37/CRAMP levels as an active player stimulating atherosclerosis progression. These effects were mediated through interaction with mitochondrial DNA, LDL, scavenger receptors, and autophagy. Additionally, a high-fat diet was shown to increase the cathelicidin serum level through an unknown mechanism. Also, different Cramp doses modulated the immune response to the self-antigen and affected atherosclerosis progression.

2.2. Immunomodulatory Role of LL-37/CRAMP in the Acute Coronary Syndrome

LL-37 treatment induced differential T cell immune responses in peripheral blood mononuclear cells (PBMCs) from patients with acute coronary syndrome (ACS) and stable coronary artery disease (CAD). In particular, LL-37 treatment reduced CD8+ effector T cell responses in CAD patients and controls, but not in ACS patients, in whom it was associated with the reduced expression of programmed cell death protein 1 (PDCD1). T cells isolated from CRAMP-immunised donor ApoE^–/– mice were transferred into recipient HFD-fed ApoE^–/– mice. CRAMP T cell-recipient mice showed a 28% reduction in aortic plaque area and increased IL-10 and IFNγ expression in CD8+ T cells compared to the control mice (Figure 2). Furthermore, 56% of adjuvant T cell-recipient mice (controls) demonstrated calcification in atherosclerotic plaques, while CRAMP T cell-recipient mice showed no calcification. These results suggested that LL-37/CRAMP-reactive T cells are active players in atherosclerosis development and may be involved in the plaque calcification process [63].

Further analysis demonstrated that the T cell response to LL-37 stimulation in PBMC was different in ACS patients compared to healthy controls. Specifically, CD8+CD137+, CD8+CD69+CD137+, CD8+CD25+, CD8+CD69+, CD8+CD25+CD69+, and CD4+CD69+CD134+ T cells were significantly increased by LL-37 stimulation in ACS PBMC. Interestingly, platelets derived from healthy controls down-modulated the CD8+CD69+CD137+ T cell response to LL-37 in autologous PBMC. The CD8+CD69+CD137+ T cell activation-induced marker (AIM) profile negatively correlated with the platelet count in ACS patients. These results provide novel insight into important mechanics underlying T cell activation and the generation of immunologic memory in ACS [64].

2.3. Role of LL-37/CRAMP in Myocardial Infarction and Myocardial Ischaemia/Reperfusion INJURY

The immediate reperfusion of an occluded coronary artery is a prerequisite for salvaging ischaemic myocardium; however, rapid blood flow restoration to the ischaemic region paradoxically causes further tissue damage, known as myocardial ischaemia/reperfusion (MI/R) injury, and may account for up to 50% of final infarct size. Despite paramount clinical interests, no effective treatments are currently available to reduce MI/R injury in MI patients, demonstrating the understanding of the pathogenesis of MI/R injury is incomplete and further research in this area is required [65]. Some recent research evidence demonstrated the involvement of LL-37 in MI pathogenesis.

The α-chemokine Stromal derived factor 1 (SDF-1) is one of the major chemotactic factors responsible for the recruitment of the bone marrow-derived stem/progenitor cells (BMSPCs), which are required for post-MI regeneration. Despite enhanced SDF-1 expression during the MI events, the following inflammatory response creates a proteolytic environment that quickly degrades SDF-1, thus limiting the clinical efficacy of this type of MI treatment [66]. Recent research demonstrated that CRAMP pre-treatment enhanced the BMSPC response to the physiological levels of SDF-1 in vitro and increased BMSPC recruitment and retention in vivo after intracoronary transplantation to mice with induced MI (Figure 3). Accordingly, MI model mice administrated with Bone Marrow-Derived Mononuclear Cells (BMMNC) pre-incubated with CRAMP showed smaller scars, higher capillary density, less adverse remodelling and fibrosis, and enhanced cardiac recovery. However, the underlying mechanism is currently unknown and requires further investigation [67].

Reduced CRAMP levels were found in heart and serum samples from MI/R mice and neonatal mouse cardiomyocytes treated with oxygen glucose deprivation/reperfusion (OGDR). Similarly, reduced LL-37 levels were found in MI patients compared to healthy controls, and low serum LL-37/neutrophil ratio was linked with the readmission and/or death of MI patients during one-year follow-up (Figure 3). Further in vitro and in vivo experiments demonstrated that CRAMP knockdown enhanced cardiomyocyte apoptosis and CRAMP KO mice showed increased infarct size, while CRAMP peptide supplementation reduced cardiomyocyte apoptosis and MI/R injury. Mechanically, CRAMP inhibited apoptosis by activating the AKT and extracellular signal-regulated kinase (ERK1/2) signalling pathways and increasing Forkhead box O3a (FoxO3a) phosphorylation and nuclear export. Proto-oncogene c-Jun (c-Jun) was identified as a negative regulator of the CRAMP gene [68].

The high-plasma LL-37 level is associated with lower risks of ischaemic cardiovascular events in patients after acute ST-elevation myocardial infarction (STEMI). Analysis of peripheral plasma samples and clinical and laboratory data from STEMI patients at three-year follow-up showed that the number of major adverse cardiovascular events (MACEs) (causing mortality, reinfarction, unscheduled revascularisation, or ischaemic stroke) was higher in STEMI patients with low LL-37 levels. Interestingly, while patients with higher LL-37 levels were associated with reduced risks of reinfarction, all-cause death, and unscheduled revascularisation, they were not associated with the risk of ischaemic stroke [69]. Additionally, among acute MI patients, a high LL-37 level was associated with a lower risk of MACE in patients with elevated lipoprotein(a) and Proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels [70].

However, another study found that neutrophil-derived cathelicidin played a negative role at the early stage of MI/R. Therefore, mice subjected to MI/R injury showed increased levels of cathelicidin, which was mostly produced by heart-infiltrating neutrophils. Furthermore, Cramp knockout mice reduced MI/R-induced myocardial inflammation, infarct size, and heart damage. On the other side, CRAMP administration to WT mice before MI/R exacerbated injury by activating the Toll-Like Receptor 4 (TLR4), Purinergic receptor P2X, and ligand-gated ion channel, 7 (P2X₇R)/NLR family pyrin domain-containing 3 (NLRP3) inflammasome signalling pathways (Figure 3). However, these effects were reversed by the application of TLR4, P2X₇R, and NLRP3 inflammasome inhibitors and neutrophils depletion before MI/R in CRAMP-treated WT mice. Moreover, in vitro experiments showed that LL-37 stimulated the processing and secretion of IL-1β from heart-infiltrating neutrophils by activating the TLR4 and P2X₇R/NLRP3 inflammasome signalling pathways [71].

The major finding of the discussed studies suggested both positive and negative roles for LL-37/CRAMP in MI. Several studies showed a high LL-37 level as a crucial factor necessary for cardiac recovery, post-MI regeneration, and the reduction of MI/R injury that is associated with a lower risk of MACE in the future. These effects were achieved through the recruitment of the bone marrow-derived stem cells, activation of the AKT and ERK1/2 signalling pathways, and increased FoxO3a phosphorylation and nuclear export. The discussed adverse effects of high cathelicidin levels were mediated through the activation of the TLR4 and P2X₇R/NLRP3 inflammasome signalling pathways, which resulted in increased infarct size and heart damage. While, in general, LL-37 may be considered as a novel treatment for cardiac ischaemic injury, future studies should also address and characterise, in detail, the adverse effects of MI/R injury.

2.4. The Role of LL-37/CRAMP in Heart Failure

Myocardial ischaemia (partial or complete blockage of blood flow to the heart because of atherosclerotic plaques in a coronary artery) may cause left-ventricular remodelling, myocardium thinning, and decreased systolic function, eventually resulting in heart failure. Shockwave treatment (SWT) involves waves of mechanical pressure that were originally used for kidney stone lithotripsy and later applied to regenerative purposes in the treatment of other diseases and conditions. SWT application was beneficial for chronic ischaemic heart disease, where it induced angiogenesis and stimulated regeneration in a TLR3-dependent way [72,73,74]. Recent experiments on mice with induced myocardial infarction that were subjected to SWT demonstrated the involvement of LL-37 in SWT-mediated therapeutic effects. Thus, SWT increased the number of capillaries and arterioles and reduced left-ventricular fibrosis, increased RNA release and cellular RNA uptake, and increased LL-37 levels. Interestingly, pre-treatment with RNase had no effect on SWT, while sequential digestion with proteinase and RNase abolished the SWT effect. These results suggested SWT protects from left-ventricular remodelling and cardiac dysfunction by increasing LL-37 levels and RNA release, which further forms the RNA/protein complex and induced angiogenesis [75].

The serum levels of LL-37/CRAMP were reduced in both acute HF patients and experimentally induced HF and hypertrophic mice models, respectively. Moreover, CRAMP knockout manifested with a further decline of cardiac function and exacerbated HF in model mice (increased cell size, fibrosis, and apoptosis parameters). On the other side, CRAMP supplementation attenuated HF (decreased cell size, interstitial cardiac fibrosis, and reduced expression of Collagen 1 and Bax/BCl2 and cleaved caspase 3/caspase 3 ratios) in both in vivo HF mice models and in vitro cell cultures. Mechanistically, the anti-hypertrophy effects of CRAMP were mediated through the inhibition of Nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signalling [76].

Cardiac hypertrophy (alterations in the shape, size, and function of the heart after injury or stress) is the pathological process of HF, which initially is beneficial for the maintenance of cardiac function, but later leads to maladaptation [77]. Recent research has found that CRAMP suppressed the cardiac hypertrophic response by directly binding Insulin-like growth factor1 receptor 1 (IGFR1), thus activating the IGFR1/Phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. Additionally, CRAMP ameliorated cardiac oxidative stress by activating the TLR9/AMP-activated protein kinase (AMPK) pathway. Accordingly, the CRAMP anti-hypertrophic and anti-oxidative effects were reversed in AKT Serine/Threonine Kinase 1 (AKT) and TLR9 knockout mice, respectively [78].

In summary, LL-37/CRAMP protected against heart failure and pressure overload-induced cardiac hypertrophy by inhibiting NF-κB signalling and activating both the IGFR1/PI3K/AKT and TLR9/AMPK pathways in cardiomyocytes.

2.5. The Role of LL-37/CRAMP in Diabetic Cardiomyopathy

Similarly, in the Streptozotocin (STZ)-induced mice model of diabetic cardiomyopathy (DCM), CRAMP injection improved cardiac function, inhibited endothelial-to-mesenchymal transition, and reduced fibrosis levels. The molecular mechanism, identified in high glucose-treated mouse heart endothelial cells (MHECs), suggested that the CRAMP effect was mediated through the inhibition of the Transforming growth factor β(TGFβ)/Smad and AMPK/mammalian target of rapamycin (mTOR) signalling pathways [79].

These results were confirmed in another study that investigated the ability of Cathelicidin to protect the heart from DCM. Thus, Cathelicidin injection ameliorated cardiac dysfunction and reduced cardiac fibrosis (lowered both the transcriptional and protein levels of collagen I, collagen III, TGFβ, and Connective tissue growth factor [CTGF]), inflammation (lowered the levels of IL-1, IL-6, IL-18, and TNFα, and decreased the nuclear translocation of NF-κB), oxidative stress (enhanced the activities of MnSOD and Gpx, and lowered the activity of NADPH oxidase and MDA levels), and apoptosis in DCM mice. Further experiments on neonatal rat cardiomyocytes stimulated with high glucose demonstrated that the cardioprotective effects of Cathelicidin were mediated through the inhibition of Thioredoxin-interacting protein (TXNIP), p65, and NLRP3 expression, thus reducing NLRP3 inflammasome formation. Accordingly, NLRP3 overexpression averted the beneficial effects of cathelicidin on the heart during DCM and on high glucose-induced myocyte injury [80].

In summary, CRAMP protected against diabetic cardiomyopathy by reducing apoptosis, inflammation, fibrosis, and oxidative stress levels, and by inhibiting the endothelial-to-mesenchymal transition. These effects were mediated through the inhibition of the TGFβ/Smad and AMPK/mTOR signalling pathways and reduction of NF-κB nuclear translocation and NLRP3 inflammasome formation.

2.6. The Role of LL-37/CRAMP in Platelet Aggregation and Thrombosis

Platelets are crucial players in regulating haemostasis, while their unwarranted activation under pathological conditions (such as acute lung injury, ischaemic stroke, and organ ischaemia–reperfusion injury) may cause the formation of blood clots (thrombosis) within the circulation, which is a frequent cause of hospitalisation and cardiovascular mortality [81]. Recently, the effect of cathelicidins on platelets was investigated in several research papers.

Thus, in vitro experiments demonstrated that LL-37 selectively inhibited platelet aggregation induced by ADP, Collagen, and U46619 in a dose-dependent manner. Also, LL-37 inhibited human platelets spreading on an immobilised fibrinogen in a dose-dependent manner. Furthermore, the LL-37 anti-thrombotic effect was exerted in vivo in an arterio-venous shunt thrombosis model in rats, where an LL-37 dose (15 mg/kg) provided an effect comparable to a 50 mg/kg dose of aspirin. Mechanically, the LL-37 effects were mediated through the inhibition secretion of a major platelet alpha-granule protein P-selectin, inhibition of Proto-Oncogene C-Src (SRC), and AKT kinase phosphorylation (Figure 2) [82].

However, the adverse role of LL-37 was defined in other reports. Cathelicidins were abundant in arterial thrombi taken from acute MI patients, in whom they were associated with areas of the platelet-rich thrombus in which leukocytes were mostly absent. Similarly, CRAMP was present in arterial thrombi leukocyte-poor areas of mice subjected to ligation injury of the carotid artery, suggesting that cathelicidins associate with platelets. Further experiments with the transplantation of bone marrow derived from wild-type and Cramp^−/− mice demonstrated that haematopoietic CRAMP was an active contributor to arterial thrombus formation and stability. In particular, CRAMP absence reduced platelet activation and arterial thrombosis, while having no effect on bleeding time, plasma clotting time, blood cell counts, or baseline platelet activation markers. Also, high doses of LL-37/CRAMP induced IL-1β, P-selectin, and CD40L expression and secretion in human/mouse platelets. Mechanically, these effects were mediated through glycoprotein VI receptor (GPVI) with the activation of the downstream signalling SRC and phospholipase C (PLC) pathways (Figure 2). Finally, CRAMP deficiency was associated with reduced formation of platelet–neutrophil aggregates in the systemic circulation and survival benefit in mice models of acute lung injury [83].

Another identified mechanism of adverse LL-37 action in platelets was mediated through formyl peptide receptor 2 (FPR2), a surface G-protein coupled receptor (GPCR). In particular, LL-37 induced platelet activation, increased the thrombus formation, and reduced bleeding time in a concentration-dependent manner (Figure 2). Experiments with Fpr2/3^−/− mice and FPR2 antagonist WRW4 confirmed the functional dependence of LL-37 on the FPR2 receptor. Thus, these models showed reduced effects of LL-37 on platelet aggregation, fibrinogen binding, and P-selectin exposure. Also, Fpr2/3^−/− mice showed increased bleeding time and diminished platelet function in general [84].

In total, the presented results suggested that LL-37/CRAMP was a crucial player in platelet activation and mediator of thrombo-inflammation. Interestingly, both LL-37/CRAMP-mediated effects (positive—anti-platelet/anti-thrombotic and negative—platelet/thrombo-inflammation activation) were mediated through FPR2 and glycoprotein VI receptors and their downstream targets (SRC and PLC). Apparently, LL-37 acted in a dose-dependent way and involved multiple signalling pathways and unknown proteins, which can affect and modulate LL-37-mediated physiological effects.

3. Conclusions

The multifaceted role of LL-37/CRAMP in cardiovascular and inflammatory diseases highlights its sophisticated involvement in diverse physiological processes. Across different experimental models and clinical studies, LL-37/CRAMP demonstrates both potentially beneficial and adverse effects, depending on the specific disease context and cellular environment. For example, LL-37 containing creams was successfully tested in trials to enhance the healing of venous leg ulcer and diabetic foot ulcer [30,85]. On the other side, other trials proved that LL-37 inhibition with azelaic acid and Heparan Sulfate Analog was effective in facial rosacea treatment [86,87]. So far, the application of LL-37/CRAMP for the treatment of ASCVD has not been tested in any clinical trials or observational studies. In atherosclerosis and acute coronary syndrome, LL-37/CRAMP exhibits immunomodulatory properties by influencing T cell responses and possibly contributing to plaque calcification, implicating its role in disease progression. Notably, discrepancies exist regarding the activation of the NLRP3 inflammasome by LL-37/CRAMP, which may either exacerbate inflammation or contribute to protective immune responses, depending on the cellular milieu and disease state.

In myocardial infarction and ischaemia–reperfusion injury, LL-37/CRAMP displays dual effects: promoting myocardial regeneration and reducing infarct size through mechanisms involving the AKT and ERK1/2 signalling pathways and the inhibition of apoptosis, while paradoxically exacerbating inflammation through TLR4 and P2X7R/NLRP3 inflammasome activation. These findings underscore the complexity of LL-37/CRAMP actions in cardiovascular pathophysiology, warranting further investigation into its precise mechanisms and therapeutic potential.

Moreover, in heart failure and diabetic cardiomyopathy, LL-37/CRAMP demonstrates protective effects by inhibiting NF-κB signalling and NLRP3 inflammasome formation, reducing oxidative stress, apoptosis, and fibrosis, and improving cardiac function. However, the exact signalling pathways mediating these effects remain to be fully elucidated, particularly in the context of endothelial-to-mesenchymal transition and the TGFβ/Smad and AMPK/mTOR pathways. Furthermore, LL-37/CRAMP influences platelet aggregation and thrombosis, exhibiting both anti-thrombotic properties through the inhibition of platelet activation pathways and the pro-thrombotic effects mediated by FPR2 activation. These contrasting effects underscore the complexity of LL-37/CRAMP in haemostasis and thrombo-inflammation, requiring cautious consideration in therapeutic applications.

Moreover, the divergent effects of LL-37/CRAMP across various diseases may stem from the complex interplay between its concentration, the microenvironmental context, and the differential expression of its receptors and interacting proteins. For instance, while high levels of LL-37/CRAMP appear harmful in promoting immune modulation and plaque growth and altering macrophage lipid metabolism in atherosclerosis and acute coronary syndrome, high concentrations may also enhance cardiac recovery and reduce scars, adverse remodelling, and fibrosis in conditions like myocardial infarction and ischaemia–reperfusion injury. Additionally, variations in the effects on inflammatory signalling pathways, such as NLRP3 inflammasome and NF-κB, likely contribute to these disparate outcomes. Furthermore, differences in tissue-specific responses and the presence of co-morbidities, such as diabetes mellitus, obesity, and the practising of an unhealthy, fat-rich diet, may further influence LL-37/CRAMP’s therapeutic efficacy and safety profile. Therefore, careful consideration of disease-specific factors and mechanistic insights into LL-37/CRAMP signalling pathways is essential for optimising its therapeutic potential in clinical settings.

Future research should focus on elucidating the precise mechanisms underlying the dualistic effects of LL-37/CRAMP in different disease contexts. Detailed investigation into the concentration-dependent effects, tissue-specific responses, and interplay with disease-associated pathways, such as NLRP3 inflammasome activation and NF-κB signalling, will be crucial. Additionally, the interplay between different affected pathways, most importantly associated with inflammation, apoptosis, autophagy, and thrombosis (TLR4, NLRP3, NF-κB, AKT, ERK1/2, TGFβ/Smad, AMPK/mTOR, GPVI/SRC, and PLC), should be elucidated in future research in different model systems and disease contexts. Moreover, exploring LL-37/CRAMP in various model systems, including genetically modified animal models, various cell cultures, and patient-derived samples, will provide valuable insights into its therapeutic potential and safety profile across different clinical conditions. Additionally, comprehensive clinical studies are warranted to validate its efficacy, optimise dosing regimens, and identify potential biomarkers for patient stratification. Addressing these research gaps will be instrumental in advancing LL-37/CRAMP towards practical applications in clinical settings, potentially paving the way for novel therapeutic strategies in cardiovascular diseases, diabetic complications, and inflammatory disorders.

In conclusion, while LL-37/CRAMP holds promise as a therapeutic target in cardiovascular diseases and inflammatory conditions, its dual nature and context-dependent effects underscore the need for further mechanistic studies. Understanding the precise signalling pathways activated or inhibited by LL-37/CRAMP across different disease states will be crucial for harnessing its therapeutic potential effectively.