*Review* **Pathophysiology of Cardiovascular Diseases: New Insights into Molecular Mechanisms of Atherosclerosis, Arterial Hypertension, and Coronary Artery Disease**

**Weronika Fr ˛ak, Armanda Wojtasi ´nska, Wiktoria Lisi ´nska, Ewelina Młynarska \*, Beata Franczyk and Jacek Rysz**

> Department of Nephrology, Hypertension and Family Medicine, Medical University of Lodz, ul. Zeromskiego 113, 90-549 Lodz, Poland

**\*** Correspondence: emmlynarska@gmail.com; Tel.: +48-(042)-6393750

**Abstract:** Cardiovascular diseases (CVDs) are disorders associated with the heart and circulatory system. Atherosclerosis is its major underlying cause. CVDs are chronic and can remain hidden for a long time. Moreover, CVDs are the leading cause of global morbidity and mortality, thus creating a major public health concern. This review summarizes the available information on the pathophysiological implications of CVDs, focusing on coronary artery disease along with atherosclerosis as its major cause and arterial hypertension. We discuss the endothelium dysfunction, inflammatory factors, and oxidation associated with atherosclerosis. Mechanisms such as dysfunction of the endothelium and inflammation, which have been identified as critical pathways for development of coronary artery disease, have become easier to diagnose in recent years. Relatively recently, evidence has been found indicating that interactions of the molecular and cellular elements such as matrix metalloproteinases, elements of the immune system, and oxidative stress are involved in the pathophysiology of arterial hypertension. Many studies have revealed several important inflammatory and genetic risk factors associated with CVDs. However, further investigation is crucial to improve our knowledge of CVDs progression and, more importantly, accelerate basic research to improve our understanding of the mechanism of pathophysiology.

**Keywords:** cardiovascular disease; atherosclerosis; arterial hypertension; coronary artery disease; inflammation; matrix metalloproteinases; oxidative stress; vascular endothelium dysfunction; genetic factor

#### **1. Introduction**

Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels [1]. They are a set of heterogeneous diseases whose underlying cause of development is most often atherosclerosis [2]. CVDs are chronic diseases that gradually evolve throughout life and remain asymptomatic for a long time [3]. Moreover, CVDs are the leading cause of morbidity and mortality in patients worldwide [4]. In Europe, CVDs are responsible for 45% of deaths [5], thus being particularly important for public health. Atherosclerosis, coronary artery disease (CAD), and arterial hypertension (AH) are the leading causes of CVDs [6].

Atherosclerosis is the main cause of cardiovascular-related death worldwide [7]. It is a thickening and hardening of the arterial wall, accompanies aging, and is related to major adverse impact on the cardiovascular system and various other diseases [8]. Elevated plasma cholesterol level (>150 mg/dL) is a major cause of the development of atherosclerosis [9].

CAD is a common heart condition in which we can observe the narrowing or blockage of major blood vessels—coronary arteries. CAD is caused primarily by plaque formation within the intima of the vessel wall [10], with plaque being defined as a fatty material

**Citation:** Fr ˛ak, W.; Wojtasi ´nska, A.; Lisi ´nska, W.; Młynarska, E.; Franczyk, B.; Rysz, J. Pathophysiology of Cardiovascular Diseases: New Insights into Molecular Mechanisms of Atherosclerosis, Arterial Hypertension, and Coronary Artery Disease. *Biomedicines* **2022**, *10*, 1938. https://doi.org/10.3390/ biomedicines10081938

Academic Editors: Tânia Martins-Marques, Gonçalo F. Coutinho and Attila Kiss

Received: 30 June 2022 Accepted: 6 August 2022 Published: 10 August 2022

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**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/).

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growing inside intima along with a severe inflammation, especially if the inflammation is chronic. This in turn causes difficulties in supplying the cardiomyocytes with enough blood, oxygen, and nutrients [11]. As a result, atherosclerotic plaque may erode or rupture, initially resulting in thrombosis and then a closure of the vessel, leading to myocardial infarction, stroke, limb ischemia, and death [12]. The other factors causing this condition are a diseased endothelium, low-grade inflammation, and lipid accumulation [13].

AH is one of the most common CVDs. AH causes few or no symptoms, but is an important risk factor for a myocardial infarction, stroke, renal failure, and peripheral vascular disease [14]. The diagnosis of AH, in accordance with the most significant guidelines, is diagnosed when a person's systolic blood pressure (SBP) in the office or clinic is ≥140 mm Hg and/or their diastolic blood pressure (DBP) is ≥90 mm Hg following a repeated examination [15]. CVDs are caused by multiple factors. Some of them are unvarying, such as age, gender, and genetic background, whereas others could be variable and, therefore, reduced (smoking, physical inactivity, poor dietary habits, elevated BP, type 2 diabetes, dyslipidemia, and obesity) [16].

In this review, we summarize the available evidence of the pathophysiological implications of CVDs, focusing on atherosclerosis, CAD, and AH. It is particularly essential to explain the mechanisms of their formation and progression.

#### **2. Coronary Artery Disease and Atherosclerosis**

CAD and atherosclerosis are discussed first, due to their broad subject matter and mutual implication. The pathophysiological basis of these diseases is constantly being researched in order to finally find the reasons behind their formation and what factors additionally exacerbate the ongoing processes and may directly contribute to their induction. Understanding the exact course of the entire pathophysiological and pathogenic process will allow for accurate prevention and diagnosis of both diseases. Cardiovascular diseases are classified as civilization diseases [17]; hence, it is important to create accurate and effective algorithms that will help doctors during their work. Those that already exist require further improvement and improvement, due to the continuous discoveries and introduction of new pharmacotherapeutic solutions.

In our review, we focus on endothelial dysfunction as one of the first and most important causes of the processes leading to CAD and atherosclerosis [18]. In the case of both diseases, these processes constitute a starting point for further research and implications in the course of the disease development. This brings us to the remainder of the paper, i.e., inflammatory processes involving diseased tissue and oxidative factors.

We also do not forget about the genetic basis; dynamically developing research shows completely new faces of these diseases known to us and makes it possible to reflect on the real causes of their diversity and changeable forms, as well as create new possibilities for better diagnostics and considering whether the current judgement that the lifestyle and drugs mainly allow controlling the disease-causing process. They allow the use of new solutions and deepen our knowledge in this subject.

#### *2.1. Endothelial Dysfunction in Atherosclerosis*

In arterial vasculature there are areas (branch points, bifurcations, and major curvatures arterial geometry), which are much more prone to atherosclerotic lesions [19]. The mechanical forces such as a turbulent flow, which is related to the geometry and shape of vessels, also influence the endothelial cell [19].

Atherosclerosis occurs in these regions as a result of differences in flow, which is present at sites of low shear stress, turbulence, and oscillating flow. We do not maintain that these factors cause atherosclerosis but rather that they "prime the soil" in which lesions start to develop [20]. Endothelial cells are exposed to various degrees and types of shear stress, which have influence on their shape, intracellular signaling, and gene expression [21]. In the regular state, which is the quiescent state of the endothelium, nitric oxide (NO) is produced in order to bind to cysteine groups in NF-κB and the mitochondria, which inhibit cellular

processes. Moreover, the endothelial layer is covered by a glycocalyx, which is layer of proteoglycans and extracellular matrix components, involved in transendothelial transport, e.g., of lipoproteins, which can be lost or reduced in inflammation due to plasminogen activator inhibitor [21]. In homeostasis, endothelial cells prevent platelet activation, blood clotting, and leukocyte adherence by secreting substances such as NO, prostacyclin, t-PA, and antithrombin III [21]. When inflammation occurs there is an increase in the number of adhesion molecules (E-selectin, ICAM, and VCAM), which participate in the infiltration of leukocytes through the endothelial layer [22]. With leukocytes, lipoproteins penetrate the endothelium, and they are trapped in the subendothelial space and oxidatively modified. Endothelial dysfunction in modern cardiovascular medicine is described as changes in the production and availability of endothelial-derived NO, prostacyclin, and endothelin, as well as their impact on vascular reactivity. In this case, reactive oxygen species (ROS) such as H2O2 reach the regulatory molecules, which leads to the activation of the cells [23]. The endothelial membrane is permeable to compounds such as NO and H2O2, which causes the activation of the transcription factors and protease. Moreover, production of endothelial ROS may be triggered by inflammation and cells that participate in this process, such as leukocytes and growth factors. Other mechanisms that cause endothelial dysfunction are the formation of peroxynitrite, NO synthase uncoupling, prostacyclin formation inhibition, endothelin expression stimulation, and reduced NO signaling due to the inhibition of soluble guanylate cyclase activity [24]. All these mechanisms promote a vasoconstrictive and procoagulant milieu. Moreover, endothelial cells can make a transition to the mesenchymal cells [21]. Consequently, the extracellular matrix deposits between cells and dysregulates the junctional proteins (e.g., occludin and claudin-5), which lose cell–cell contact [21]. As a result, the endothelium loses its integrity with media, and this causes a higher activity in that field. Then, the changes extend beyond NO metabolism reactivity, including increased level of permeability for lipoprotein, oxydation, leukocyte adhesion and accumulation, and altered extracellular matrix metabolism, with all of these accumulating in the arterial wall [19]. In this way, macrophages, cholesterol, and inflammatory cells access the media, and atherosclerosis begins. Moreover, the mechanical forces such as a turbulent flow, which is related to the geometry and shape of vessels, also influence the endothelial cell [19].

#### *2.2. Inflammatory and Oxidising Factors in Atherosclerosis*

Following the mechanisms of endothelial dysfunction, we can see that inflammatory factors also play a huge role in the development of this pathology. Atherosclerosis is characterized by the retention of lipids and inflammatory cells such as macrophages, T lymphocytes, and mast cells in damaged arterial wall, the intima [25]. Modified lipids activate inflammatory cells in the intima, producing chemokines and cytokines such as tumor necrosis factor (TNF-alpha), interleukin -1, -4, and -6, and interferon-gamma, which activate other leukocytes, endothelial cells, and adhesion molecules, especially vascular cell adhesion molecule-1 (VCAM), intercellular adhesion molecule-1 (ICAM), and E-selectin, on the endothelial surface. These, in turn, recruit other inflammatory cells. As a result, the monocyte-derived macrophages release enzymes in order to modify the lipoproteins. These modified lipoproteins become atherosclerotic plaques. Then, macrophages absorb and build in the cholesterol-rich lipoproteins from LDL, as well as secrete pro-oxidant substances, which contribute to the process of atherosclerosis: ROS and RNS (reactive nitrogen species). These are the same compounds that participate in the endothelial dysfunction, which aggravates the condition of the endothelium, indicating that the process is self-perpetuating. This is not desirable, because this damage of the cellular functions of biomolecules (such as proteins, carbohydrates, and lipids) can result in lipid peroxidation and LDL oxidation. As we know, oxidized phospholipids trigger inflammation, because of the extensive binding to the Toll-like receptors, which can activate the transcription factors nuclear factor-κB (NF-κB) cytokines which trigger proinflammation; hence, oxLDL is called a clinical marker of plaque inflammation [26]. OxLDL irritates endothelial cells, increasing

the production of adhesion molecules. ROS and RNS convert the LDL-C to the OX-LDL, which are built into the intimal layer. The inner layer also migrates the muscle cells from the media and proliferates itself. When all these processes combine, the atherosclerotic plaque is created, featuring fiber tissue, muscle cells, and many inflammatory cells. Accelerated cell turnover is likely to lead to an enhanced macromolecular permeability, increasing lipid uptake in the regions with a disturbed flow. This in turn would lead to the atherosclerotic phenotype expression of VEGF increasing in response to low shear stress, leading to greater endothelial permeability [27]. In addition, in the vessels, hyperglycemia promotes the overproduction of ROS by the mitochondrial electron transport chain. Excess superoxide leads to DNA strand breakage and activation of nuclear poly ADP ribose polymerase (PARP). These processes inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), shunting the early glycolytic intermediates into the pathogenic signaling pathways during inflammation [28].

#### *2.3. Epigenetic Factors in Atherosclerosis*

The first major epigenetic mechanism that contributes to the complexity of atherosclerosis is DNA methylation, which is catalyzed by DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) DNA methylation [27]. Moreover, DNA demethylation is also an important mechanism explaining the pathogenesis of atherosclerosis. During studies on mice, the *TET2* overexpression significantly reduced atherosclerotic lesion formation, likely by oxidatively demethylating 5meC to 5hmC in the endothelial vessel wall. This *TET2*-induced rescue occurs via the upregulation of autophagy, as *TET2* overexpression decreases the methylation level of the promoters of autophagic flux-related genes [29]. Moreover, the loss of *TET2* functions in hematopoietic cells and myeloid cells enhanced atherosclerosis in mice, as shown in [30]. Bone marrow was transplanted from control mice into an atherosclerosis-prone *Ldlr*−*/*− recipient mice, and a diet high in cholesterol was introduced. After 5, 9, and 13 weeks on the diet, the recipients of *Tet2*−*/*− marrow had 2.0-fold, 1.7-fold, and 1.4-fold larger lesions in the arteries. DNA methylation and histone acetylation are key processes in regulating the expression of inflammatory cytokines and chemokines in atherosclerosis. This involves methylation at the C5 position of cytosine residues in a *CpG* dinucleotide context, exerted by DNA methyltransferases (DNMTs). DNMTs are capable of both methylation and demethylation, making the modification reversible, but those modifications are reserved for the Tet methylcytosine dioxygenases (TET1, 2, and 3) [29]. In addition, studies have shown that other genes, *DNMT3A*, *JAK2*, and *ASXL1*, which mutated, increase the risk of incident coronary heart disease 12-fold in *JAK2* V617F and 1.7-fold to 2.0-fold in other genes mentioned above [30]. Mutations in the *DNMT3A* and *TET2* influence DNA methylation, whereas those in the *ASXL1* alter histone modifications, thereby influencing clonal expansion of hematopoietic stem cells [31]. This phenomenon was found to influence risk of CAD [32].

#### *2.4. Endothelial Dysfunction in CAD*

The vascular endothelium is the layer of cells lying under the epithelium lining the inside of the vessel and the muscular layer, which is a boundary between the circulating blood and the vascular wall. Its cells are specialized in maintaining vascular homeostasis, which is crucial for the proper functioning of organs, especially the heart. Through its role in signal transduction and as a source of many vasoactive substances, it is their key regulator. The vascular endothelium reacts to physical and chemical stimuli through the release of autocrine and paracrine vasoactive agents. Factors of endothelial origin regulate surface tension and cell adhesion, including platelet activation and leukocyte adhesion, smooth muscle cell proliferation, and vascular wall inflammation. The endothelium is considered to be a strong indicator of cardiovascular function and fitness. Its dysfunction is considered to be the earliest marker of atherosclerosis and, in effect, CAD.

First, we focus on the critical process of the NO signaling pathway [33]. Nitric oxide is a gas with relatively small particles that strongly dilates blood vessels and has additional anti-inflammatory and antioxidant properties [34]. It is synthesized by three distinct subtypes of the NO synthase (NOS) enzyme, each with unique expression patterns and functional properties: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). Broadly, these proteins catalyze the production of NO and l-citrulline from L-arginine and O2, using electrons donated from dihydronicotinamide adenine dinucleotide phosphate (NADPH). Finally, in the presence of heme and tetrahydrobiopterin (BH4), NOS monomers form homodimers, which are capable of using the donated NADPH electrons to catalyze the two-step oxidation of L-arginine to L-citrulline and NO. The expression of the nicotinamide adenine dinucleotide phosphate oxidase in the vessel wall with the consequent overproduction of NO has been proposed as an initial step in the chronic dysregulation of normal NO production by eNOS, which is characteristic of the monomeric forms of eNOS. In effect, eNOS produces a superoxide, rather than NO. The superoxide reacts with NO to form peroxynitrite, which in turn increases the uncoupling of eNOS and further superoxide production [35,36]. This again promotes eNOS uncoupling and is itself a mediator of effects [37]. When uncoupled, eNOS switches from its oxygenase-induced oxidant excess, and then exerts a deleterious effect on the endothelial and vascular function.

In the second step, NOS catalyzes the oxidation of *N*ω-hydroxy-L-arginine to Lcitrulline, thereby releasing NO [38,39]. For example, nNOS and eNOS are highly dependent on Ca2+-activated CaM for homodimerization and activity, whereas iNOS is minimally dependent on calcium concentration. These nuances have critical functional effects. NADPH oxidase (NOX) is another element that has received much attention as a key element in the development of vascular dysfunction. NOX has the primary function of producing reactive oxygen species (ROS) and is considered the main source of the ROS production in endothelial cells. The enzymatic production of NO by eNOS is critical in mediating the endothelial function, and oxidative stress can cause eNOS dysregulation and endothelial dysfunction [40].

Taking this into account, the endothelial dysfunction is directly related to a decreased production and sensitivity of cells to NO. As a result, we have an effective disturbance in the functioning of the entire vessel and its homeostasis, which leads to an observation of prothrombotic and proinflammatory phenomena, along with lower susceptibility of the blood vessel wall.

Another factor of interest is phospholipase A2 and its influence on the endothelial dysfunction. Lp-PLA2 (lipoprotein-bound phospholipase A2), also known as plateletactivating acetylhydrolase, is a vascular-specific inflammatory enzyme mainly produced by macrophages, lymphocytes, and foam cells in the atherosclerotic plaques. The circulation of Lp-PLA2 is mainly associated with apolipoprotein B-containing lipoproteins and, therefore, closely related to low-density lipoproteins (LDLs). Lp-PLA2 can trigger proinflammatory and proatherogenic properties in the vascular wall. The enzyme hydrolyzes oxidized phospholipids on the LDL particles in the intima of the artery, producing two highly inflammatory mediators with proinflammatory and atherosclerotic effects. Elevated levels of Lp-PLA2 correlate with arterial stiffness in patients with a stable CAD (Figure 1), regardless of the risk factors and pharmacotherapy [41].

#### *2.5. Inflammation in CAD*

Systemic vasculitis is a term referring to a group of diseases characterized by inflammation and fibrinoid necrosis of blood vessel walls. The underlying pathogenesis involves many mechanisms such as cell-mediated inflammation, immune complex (IC)-mediated inflammation, and ANCA-mediated inflammation, For example, inflammation in GCA is mostly a T-cell-driven process in which dendritic cells present antigens in blood vessel walls. These T-cells activate other inflammatory cells such as monocytes and macrophages, which, as a result, release proinflammatory cytokines (interleukin-1, interleukin-6, and interferon-γ). Inflammation in diseases such as polyarteritis nodosa and cryoglobulinemia is driven by IC deposition (antibody-mediated IC formation, microaneurysms) [42], whereas Wegener's granulomatosis, Churg–Strauss syndrome, microscopic polyangiitis, and necrotizing glomerulonephritis result from interactions between antibodies and enzymes within inflammatory cells, which is typical for ANCA-related vasculitis [43]. As a consequence of these processes, manufactured antibodies and immune complexes attach to the inner layer of blood vessels. This is a cause of aggravated ET-1 release, which, in a positive feedback loop, recruits more monocytes and macrophages. Vasculitis, as a very diverse group of diseases, has many cellular mechanisms that we mentioned above and affects different arteries and veins, in terms of both distribution and size [44]. The size of vessels is also crucial in terms of factors sustaining inflammation. Vasculitis of small veins is related to involvement of the endothelium, necrose (which is associated with ANCA), damage of the arterial walls, aneurysm formation, and hemorrhage. It is also characterized by leukocytoclasia, which is the excessive accumulation of neutrophils. Moreover, in vasculitis of large systemic vessels, the response to the inflammation leads to thickening of the intima, restenosis, and overall remodeling. These changes are manifested in the malfunctioning of blood vessels and subsequent events such as infarction and hemorrhage [44].

**Figure 1.** Lp-PLA2-dependent activation cycle [40]. The macrophages, lymphocytes, and foam cells present in the atherosclerotic plaques have an influence on the increased level of Lp-PLA2, which in turn catalyzes a reaction that, in the presence of oxidized phospholipids on LDL, is a direct contribution to the secretion of an increased amount of inflammatory mediators, which in turn leads to endothelial dysfunction and further CAD.

#### *2.6. Genetic Background in CAD*

In recent years, there has been substantial research showing correlations between genetic factors and endothelial function and dysfunction, which in turn are associated with an increased risk of developing CAD. In our work, we selected genes that, in our opinion, have gained attention in recent years and have shown a significant impact on the development of CAD. However, it should be remembered that, since 2007, in addition to all the genes known so far that have a confirmed impact on CAD, new research has resulted in nearly 60 distinct genetic loci [45].

As mentioned before, atherosclerosis underlies CAD. Previous studies have shown that it has an important but poorly understood and defined genetic component [41,46]. An earlier genome-wide association study (GWAS) identified many loci associated with an increased risk of CAD. A recent study by Redouane Aherrahrou, Liang Guo, and others accurately described the characteristics of atherosclerosis that are associated with the migration and proliferation processes in the vascular smooth muscle (VSMC) cells. The phenotypic variability shown was of great importance here; more specifically, four loci

directly related to the atherosclerosis in VSMC were identified. Moreover, as many as 79 out of 163 loci associated with CAD are associated with one of the VSMC phenotypes [47].

One of them is the chromosome 1q41 locus, which harbors *MIA3* protein. The G allele of the lead risk SNP rs67180937 is associated with a lower VSMC *MIA3* expression and a lower proliferation. The *MIA3* protein, which plays a role in the secretion of collagen, is the likely cause of the genetic basis of CAD at its 1q141 locus. Silencing this protein resulted in a reduction in the VSMC profiling, with a lentivirus used for this purpose. This protein also significantly impacts the thickness of the fibrous cap of the atherosclerotic plaque, both in humans and in mice [48].

Next one, *JCAD* (junctional cadherin 5-associated, also known as *KIAA1462*, encoding a junctional protein associated with CAD) is one of more than 160 GWAS-identified genes [49]. *JCAD* is a protein that binds cells in the endothelium and is responsible for the regulation of pathological angiogenesis, less frequently than its development [50]. CAD depletion also increased EC apoptosis and reduced EC proliferation, migration, and angiogenesis [51]. *JCAD* depletion inhibits the activation of the YAP/TAZ pathway and the expression of further proatherogenic genes including CTGF and Cyr61. Proteomic studies suggest that *JCAD* regulates the YAP/TAZ activation by interacting with the actin-binding protein TRIOBP, thus stabilizing stress fiber formation. In addition, endothelial *JCAD* expression was increased in murine and human atherosclerotic plaques [52].

Moreover, the SIRT1 protein (Sirtuin 1) plays an important role in regulating the physiological mechanisms taking place in the cell, consequently influencing the mechanisms against CAD. SIRT1 is a cardioprotective molecule due to it regulating the expression of eNOS. It regulates angiogenesis, and it protects the endothelium against dysfunctional changes and damage to the heart muscle resulting from a reduced perfusion and ischemia. Suppression of the SIRT1 causes monocyte affinity due to endothelial dysfunction [53]. The gene encoding transcription factor *TCF21* has been linked to CAD risk by GWAS in multiple racial/ethnic groups. *TCF21* antagonizes the MYOCD/SRF pathway through multiple mechanisms, further establishing a role for this CAD-associated gene in smooth muscle cells [54]. Two genes associated with high susceptibility of atherosclerotic plaque were also found. These two genes changed at different timepoints after myocardial infarction, and both had the lowest prognosis of heart failure when expressed at low levels. *TLR2* and *CD14* are closely associated with the worsening of CAD, the instability of atherosclerotic plaques, and the prognosis of heart failure after myocardial infarction [55]. Other studies revealed an atheroprotective role of *SVEP1*. The deficiency of wildtype *SVEP1* increased the endothelial *CXCL1* expression, leading to an enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in an enhanced plaque progression in the *SVEP1* deficiency (Figure 2) [56]. An intronic region of the disintegrin and metalloproteinase with thrombospondin motifs-7 (*ADAMTS7*) is one of the two crucial factors that can reduce the wildtype *SVEP1* [57]. *ADAMTS7* is one of the many proteins involved in the remodeling of the blood vessel walls due to the properties that dissolve their substrate proteins [57]. Increased expression of this gene is associated with an increased proteolytic activity and a migration of smooth muscle cells, which favors the abovementioned remodeling properties, as demonstrated on the smooth muscle cells in mice [58].

Two more genes have attracted our attention: *NOS3* and *GUCY1A3* and their common variants. Their presence significantly contributes to a reduction in the level of BP, which is known to be one of the important factors in reducing the occurrence of CAD [59]. Lossof-function mutations in GUCY1A3 and CCT7 result in a reduced level of α1 subunit of soluble guanylyl cyclase (α1-sGC), as well as β1-sGC protein content, and they impair the soluble guanylyl cyclase activity, which was correlated with risk of myocardial infarction in a large family enriched for premature CAD2 [60].

**Figure 2.** The role of *SVEP1* in atherosclerosis and CAD [56]. *CXCL1* expression is dependent on the presence of *SVEP1*. Only in the presence of *SVEP1* is the *CXCL1* expression silenced. Two factors have a significant influence on the expression of *CXCL1*: MMPs and ADAMTS- 7 (specific metalloproteinases), which can reduce the wildtype *SVEP1*; mutant *SVEP1* (*SVEP1\_p.D2702G* missense variant). Consequently, the secretion and recruitment of inflammatory cells are increased.

The following genes, when inactivated, show increased risk of CAD: *LDRL* (lowdensity lipoprotein receptor), *APOA5* (apolipoprotein A-V) [61], and *LPL* (lipoprotein lipase) [45]. Abnormal mutations in *APOA5* increase the risk of CAD due to this gene coding a protein responsible for the increased activity of *LPL* [61]. The opposite effect is shown by mutations in the *APOC3* and *ANGPTL4* genes, which are responsible for the inhibition of lipoprotein lipase [62,63].

#### **3. Arterial Hypertension**

#### *3.1. Matrix Metalloproteinases in AH*

The remodeling of the vascular extracellular matrix (ECM) during hypertensive impairment has been associated with the involvement of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Several MMPs and TIMPs might participate in the vascular remodeling associated with the AH. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, involved in many physiological and pathological processes, particularly tissue repair and modulation, cellular differentiation, cell mobility, angiogenesis, and cell proliferation, migration, and apoptosis. The dysfunction of MMP activity leads to a progression of various pathologies, tissue destruction, fibrosis, and matrix weakening [64]. It is worth mentioning that MMPs are also involved in the development of hypertension-mediated damage to the vascularity, heart, and kidneys, leading to organ failure and cardiovascular complications (Figure 3) [65]. Endogenous tissue inhibitors of MMPs (TIMPs) are crucial in restraining the degradation of ECM. Furthermore, the pathological processes in the vessel wall might be provoked by an excessive amount of MMPs, due to an imbalance between MMPs and TIMPs [66].

**Figure 3.** Summary of physiological and pathological functions of MMPs. MMPs, matrix metalloproteinases.

The results of a meta-analysis conducted by Marchesi et al. showed increased plasma levels and activities of MMP-2, MMP-9, and TIMP-1 among hypertensive patients [67]. Furthermore, Bisogni et al. found that a high BP along with the development and progression of CVDs correlated with increased levels of MMP-2 and MMP-9. It is also worth mentioning that MMP-9 is involved in the vascular remodeling, thus leading to the perpetuation of the elevated BP [65]. Additionally, Kostov et al. reported elevated MMP-1 levels and increased collagen degradation in patients with AH [68]. Thus, the impaired vascular remodeling and aggravated proteolytic activity might result from an MMP/TIMP imbalance in vascularity, principally in the intima and media of the vessel wall [16]. On the contrary, a study by Basu et al. attempted to demonstrate a crucial role of the TIMP-3, which is the protection of the arterial ECM in response to Ang II [69]. Furthermore, Pushpakumar et al. observed worsening of kidney functions and renovascular remodeling due to an increased activity of MMP-9 among hypertensive mice with TIMP2 deficiency [70]. These alterations in clinical AH suggest an important role for MMPs in AH. What is interesting, the determination of the MMPs and TIMPs in the serum can be used as a noninvasive approach to diagnosing and monitoring the structural changes in the cardiovascular system in AH [71].

Alterations in the structure, along with the degradation of several components of the vascular wall, including ECM, are critical during AH. The ECM is critical for maintaining the homeostasis in vasculature. It is thought to support stability and vascular cell behaviors [72]. A study by Cui et al. indicated that MMPs cause a degradation of the ECM proteins such as collagen or elastin; therefore, MMPs disturb the structural integrity of the vascular wall and lead to a decreased elasticity of the vascular wall [73].

Vascular ECM remodeling during AH applies effects on the functional and structural alterations in the vascular smooth muscle cells (VSMCs). Wang et al. demonstrated that MMPs promote VSMC growth and proliferation by activating growth factors such as insulin growth factor-1, transforming growth factor-h, and heparin-bound epidermal growth factor, in addition to promoting interactions between VSMC and these growth factors [16].

Martínez et al. found that, in rats, MMP-2 may regulate BP by destroying the vasodilator peptides adrenomedullin (AM) 1–52, 8–52, and 11–52 (Figure 4). AM plays a role in the regulation of BP. MMP-2 cleaves the vasodilator peptide AM (1–52, 8–52, and 11–52) into smaller peptides AM (11–22), which act as the vasoconstrictors. MMP-2 activity may be related to the development of AH, both by reducing the levels of the potent vasodilator AM (1–52, 8–52, and 11–52) and by generating hypertensive molecules [74].

**Figure 4.** MMP-2 cleavage of AM. Adrenomedullin (AM 11–52) is a peptide that affects vessels and leads to its vasodilation. MMP-2 regulates BP by reducing the AM (1–52, 8–52, and 11–52) into smaller peptides AM (11–22), which act as vasoconstrictors. Therefore, MMP-2 may worsen vascular function and increase BP in hypertensive patients. Numbers in brackets are the peptide length before and after the MMP2 cleavage. MMP-2, matrix metalloproteinase 2; AM, adrenomedullin; BP, blood pressure.

In addition, the MMPs may play a role in hypertensive complications such as intracranial hemorrhage. A study by Wakisaka et al. found increased levels of MMP-9 in the endothelial cells and ECM of cerebral vessels that are considered to be associated with a spontaneous intracranial hemorrhage in the hypertensive rat models [75].

MMPs/TIMPs are involved in the regulation of the ECM metabolism, which plays a significant role with regard to the maintenance of tissue integrity [72].

#### *3.2. Immune System in AH*

Both innate and adaptive immune cells have been implicated in the development of AH. The majority of studies correlated the presence of AH with elevated levels of circulating inflammatory markers, cytokines, and antibodies. The findings by Mirhafez et al. showed an association between the concentrations of several cytokines and AH (an increased level of IL-6, IL-1β, IL-1α, IL-18, IL-2, IL-8, TNF-α, IFN-γ, C-reactive protein (CRP), and MCP-1, and a decreased level of IL-10) among patients with AH [76].

Sesso et al. evaluated the relationship between IL-6 and CRP and the risk of developing AH in a nested case–control study of 400 women. The results showed that IL-6 was indifferently associated with while CRP was firmly related to the AH risk [77]. Furthermore, CRP has been found to be a mediator in the development of endothelial dysfunction, vascular stiffness, and elevated BP [78]. Kong et al. investigated CRP gene polymorphisms. The results showed that plasma levels of CRP could predict the development of AH. On the contrary, the relationship between genotype and CRP levels was not associated with a change in AH risk [79]. Lima et al. demonstrated that IL-10 inhibits the pressor activity of Ang II and vascular dysfunction associated with AH, while also modulating the RhoA/Rho kinase pathway. Strategies to increase IL-10 levels during AH may enhance the benefits provided by regular treatments [80]. Peng et al. established in mice models that elevated levels of IL-4 are linked to the development of with cardiomyopathy, as a result of an angiotensin II-induced cardiac damage [81].

Macrophages are the main effector cells of the innate immune system, and numerous studies indicate their role in the pathogenesis of AH. Monocyte levels are elevated in hypertensives in comparison to normotensives [82]. Interestingly Ang II-preactivated circulating monocytes in hypertensive patients might lead to subendothelial infiltration and thereafter augment the risk of arteriosclerotic complications [83].

Macrophages have a role in mediating hypertensive end-organ damage. In one study, perivascular macrophages were associated with the neurovascular and cognitive dysfunction induced by AH [84]. Shen et al. demonstrated that microglia, which are the resident immune cells in the brain, are the main molecular factors in mediating a neuroinflammation and modulating neuronal excitation, which contributes to an increased BP [85].

Barbaro et al. identified a novel mechanism via which excess sodium contributes to inflammation and AH. The result showed a pathway of sodium entrance into dendritic cells. Therefore, high-salt-treated DCs produce the prohypertensive cytokines IL-17 and INF-γ [86].

Antigen-presenting cells (APCs) are involved in the evolution of the inflammation associated with AH. Hevia et al. showed that APCs are essential for the development of AH, as the deletion of APCs produces rapid changes in the BP in mice with angiotensin II plus a high-salt diet. Additionally, the APCs activate the intrarenal renin/angiotensin system components and take part in the modulation of the natriuresis and tubular sodium transporters [87].

The NOD-like receptor protein 3 (NLRP3) inflammasome participates in the development of AH [88]. Krishnan et al. investigated MCC950—a recently-identified inhibitor of NLRP3 activity. The result showed that, in mice with established AH, MCC950 lowered BP and decreased renal inflammation, together with reduced fibrosis and kidney dysfunction [89].

Some studies investigated the neuro-immune axis associated with AH. Abboud et al. indicated that excessive sympathetic activity and reduced parasympathetic activity have a major role in pathological processes [90]. Furthermore, Harwani et al. demonstrated anti-inflammatory nicotinic/cholinergic modulation of the innate immune system in rats. Interestingly, they revealed proinflammatory innate immune responses in the hypertensive rats, prior to the development of AH [91].

Evidence from human and animal studies strongly suggests an association between inflammation and AH. Most recent findings in this field add to the growing body of evidence suggesting that AH is an inflammatory disease, while also drawing attention to the new possibilities for treating AH.

#### *3.3. Oxidative Stress in AH*

Disruption of redox signaling is a common pathophysiological mechanism observed in AH. Oxidative stress, resulting in either enhanced ROS production or decreases in antioxidant defense, is associated with an elevated BP, endothelial dysfunction, and vascular remodeling [92]. Oxidative stress and inflammatory responses act cooperatively in the pathogenesis of AH [93]. In the vascular system, the major source of ROS production is NOX, whose expression is increased in hypertensive conditions [94]. Dysregulation of enzymes such as NADPHNOX, nitric oxide synthase (NOS), xanthine oxidase, mitochondrial enzymes, or superoxide dismutase (SOD), which generate ·O2 <sup>−</sup>, H2O2, and ·OH together with reduced levels of antioxidants, results in increased formation of ROS within the vasculature. ROS contribute to vascular injury by promoting VSMC growth, extracellular matrix protein deposition, activation of matrix MMPs, inflammation, endothelial dysfunction, and increased vascular tone [95]. Furthermore, Crowley et al. indicated a novel mechanism via which oxidative stress promotes an inflammation in the vascular wall. The chaperone protein cyclophilin A (CypA), secreted from VSMCs due to ROS stimulation, leads to the recruitment of the inflammatory cells within the vasculature. Additionally, CypA triggers activation of MMPs, thus exaggerating vascular injury [93].

A variety of studies have indicated the role of NOX enzymes in the vascular remodeling during AH. Dikalova et al. indicated that an overexpression in the vascular smooth muscle of NOX1 exacerbates the hypertrophic and hypertensive responses to Ang II and increases the superoxide production in mice. Therefore, NOX1 participates in the development of cardiovascular pathologies [96]. Interestingly, Nosalski et al. investigated the pharmacological inhibition of NOX1/NOX4 in rats. The result showed that pharmacological inhibition of NOX4, elevated BP, increased accumulation of immune cell accumulation, and increased perivascular collagen deposition led to an accelerated vascular aging among both normotensive and hypertensive rats. Interestingly, the NOX1 inhibition did not affect the development of AH [97]. Murdoch et al. evaluated the overexpression of Nox2 and its association with AH. Thus, NOX2 contributes to the vascular remodeling and endothelial

dysfunction, in addition to being involved in the pathophysiology of AH [98]. On the contrary, NOX4 promotes the protection of the vascular system in situations of increased stress induced by ischemia or angiotensin II [99]. NOX4 enhances the H2O2 production; thus, it has valuable effect on vasodilator function and BP [100].

A large body of evidence has shown that the *nuclear factor erythroid factor 2-related factor 2*(*Nrf2*) is involved in the AH pathophysiology. Tanase et al. investigated *Nrf2* in oxidative stress and its role in AH. *Nrf2* is a critical redox-sensitive transcription factor, functioning as a target nuclear receptor against oxidative stress, and it is a major component of the redox homeostasis of cells [101]. Chronic oxidative stress inhibits *Nrf2* activity and function [102]. It is worth mentioning that Farooqui and colleagues demonstrated the development of AH and renal function impairment due to an *Nrf2* inhibition in mice. The result showed that, in mice treated with a pro-oxidant—L-buthionine sulfoximine (10 mmol/L in drinking water) and an *Nrf2* inhibitor—ML385 (10 mg/kg body weight/day, intraperitoneally), oxidative stress, renal functional impairment, inflammation, and elevated BP were revealed [103]. Additionally, microRNA (miR)-140-5p exaggerates AH and oxidative stress in mouse models. Liu et al. established that downregulation of miR-140-5p reduced oxidative stress and ROS levels by activating the protein expression of *Nrf2* [104]. Furthermore, Biernacki et al. investigated changes in oxidative metabolism and apoptosis in the hearts of the hypertensive rats. The result showed that inhibition of lipolysis by fatty-acid amide hydrolase inhibitors can increase the enzymatic activity and nonenzymatic antioxidant activity in rats, whereas *Nrf2* expression is suppressed [105]. Therefore, treatments involving the upregulation of *Nrf2* expression might be promising. Nonetheless, further research is needed on the therapeutic potential of *Nrf2*.

It has become clear that inflammation, ROS, and BP elevation are significant in the pathophysiology of AH.

#### **4. Conclusions**

In this review, we focused on the important molecular aspects of three cardiovascular diseases: atherosclerosis, CAD, and AH. In atherosclerosis, we paid attention to the role of the oxidative processes, their pathomechanism, and the influence on the development of the disease. Important epigenetic factors, particularly those that have been highlighted in research in recent years, were also highlighted. In CAD, we focused on the endothelial dysfunction and the most important factors leading to it, as well as its new aspects. The most recent discoveries in the field of genetic background and the discovery of the role of many genes that directly or indirectly contribute to the increased risk of CAD were also considered to be of interest.

In AH, the most noteworthy aspects were the matrix metalloproteinases and the functioning of the immune system and its dysfunctions. Here, we also drew attention to the influence of oxidative stress.

These findings might shed new light on the cellular mechanisms of CVDs and prospective targets for the prevention and treatment in the near future. However, the major scientific achievements of recent years and the many new discoveries and mechanisms still require careful attention and additional studies.

**Author Contributions:** Conceptualization, E.M., B.F. and J.R.; methodology, W.F., A.W., W.L. and E.M.; software, E.M.; validation, E.M.; formal analysis, W.F., A.W., W.L., E.M., B.F. and J.R.; investigation, W.F., A.W. and W.L.; resources, E.M., B.F. and J.R.; data curation, E.M.; writing—original draft preparation, W.F., A.W. and W.L.; writing—review and editing, E.M.; visualization, W.F., A.W., W.L. and E.M.; supervision, E.M., B.F. and J.R.; project administration, E.M.; funding acquisition, E.M., B.F. and J.R. 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:** The data used in this article were sourced from materials mentioned in the references.

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

#### **References**


### *Review* **Cellular Mechanisms of Coronary Artery Spasm**

**Beata Franczyk, Jill Dybiec, Weronika Fr ˛ak, Julia Krzemi ´nska, Joanna Ku´cmierz, Ewelina Młynarska \*, Magdalena Szlagor, Magdalena Wronka and Jacek Rysz**

> Department of Nephrology, Hypertension and Family Medicine, Medical University of Lodz, ul. Zeromskiego 113, 90-549 Ł ˙ ód ´z, Poland

**\*** Correspondence: emmlynarska@gmail.com; Tel.: +48-(042)-6393750

**Abstract:** Coronary artery spasm (CAS) is a reversible phenomenon caused by spontaneous excessive vascular smooth muscle contractility and vascular wall hypertonicity, which results in partial or complete closure of the lumen of normal or atherosclerotic coronary arteries. The clinical picture of CAS includes chest discomfort which is similar in quality to that of stable effort angina. Mechanisms underlying the development of CAS are still unclear. CAS certainly is a multifactorial disease. In this review, we paid attention to the role of the main pathophysiologic mechanisms in CAS: endothelial dysfunction, chronic inflammation, oxidative stress, smooth muscle hypercontractility, atherosclerosis and thrombosis, and mutations leading to deficient aldehyde dehydrogenase 2 (ALDH2) activity. These findings might shed novel insight on the underlying mechanisms and identify potential diagnostic and therapeutic targets for cardiovascular diseases in the future.

**Keywords:** coronary artery spasm; cellular mechanism; endothelial dysfunction; oxidative stress; smooth muscle hypercontractility; inflammation; atherosclerosis; thrombosis; variant angina

**Citation:** Franczyk, B.; Dybiec, J.; Fr ˛ak, W.; Krzemi ´nska, J.; Ku´cmierz, J.; Młynarska, E.; Szlagor, M.; Wronka, M.; Rysz, J. Cellular Mechanisms of Coronary Artery Spasm. *Biomedicines* **2022**, *10*, 2349. https://doi.org/ 10.3390/biomedicines10102349

Academic Editors: Tânia Martins-Marques, Gonçalo F. Coutinho and Attila Kiss

Received: 29 August 2022 Accepted: 17 September 2022 Published: 21 September 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/).

#### **1. Introduction**

Coronary artery spasm (CAS) is a reversible phenomenon caused by spontaneous excessive vascular smooth muscle contractility and vascular wall hypertonicity, which results in partial or complete closure of the lumen of normal or atherosclerotic coronary arteries [1,2]. The concept of CAS was first postulated by Prinzmetal et al. who described angina that occurs at rest or during regular daily activities which could not be explained by the increased oxygen demand of the myocardium [3]. Prevalence of CAS is diverse between countries: in the Japanese, 24.3%, followed by the Taiwanese, 19.3%, and Caucasian, 7.5%, populations [2]. Among patients aged 40 to 70 years, CAS is more common in men than in women [1]; however, it is mostly a disease of middle- and older-aged men and postmenopausal women [4].

The clinical picture of CAS includes chest discomfort which is similar in quality to that of stable effort angina [4]. A typical CAS attack is transient, often lasts only a few seconds, and is unpredictable; however, it arises particularly from midnight to early morning [4,5]. It occurs at rest and is a vague sensation of compression in the precordium or upper abdomen with radiation mostly to the neck, jaw, and left shoulder [6]. Angina may be accompanied by cold sweats, syncope, and a lowering of blood pressure [6]. Kishida et al. revealed that 82% (872 of 1062 episodes) of CAS episodes were asymptomatic and that syncope occurred in 12.5% (30 of 240 patients) of patients with CAS [7].

The main risk factors for CAS are smoking, age, high-sensitivity C-reactive protein (hs-CRP), hypertension, LDL cholesterol, and diabetes mellitus [1,2].

Mechanisms underlying the development of CAS are still unclear. CAS certainly is a multifactorial disease [5]. The main pathophysiologic mechanisms in CAS are dysfunction of the autonomic nervous system, endothelial dysfunction, chronic inflammation, oxidative stress, smooth muscle hypercontractility, atherosclerosis and thrombosis, and mutations leading to deficient aldehyde dehydrogenase 2 (ALDH2) activity [1].

23

In patients with CAS due to dysfunction of the endothelium, deficiency in nitric oxide (NO) is observed [8]. One of the damaging factors for the endothelium is oxidative stress. Free radicals degrade NO which results in artery spasms. Cigarette smoking is one of the main risk factors that intensify this process [9]. In addition, it is critical for people suffering from CAS to cease smoking, as the inflammation in the body, an essential part of smoking, triggers coronary spasms [10–13].

Fortunately, it has been reported that antioxidants such as vitamin C or E can restore disturbed arterial reactivity [14,15]. Furthermore, estrogen, a hormone responsible for enhancing NO synthase activity, can be considered as a protective factor. It has been shown that high estrogen levels (typical of the pre-menopausal period) were associated with a lower frequency of ischemic episodes [16].

One of the triggers of CAS is also vascular smooth muscle cell hyperreactivity. The excessive intracellular influx of calcium ions, disturbances in the functioning of calcium channels, and malfunctioning of ATP-sensitive potassium channels may result in the occurrence of coronary artery spasms [17–19]. RhoA/Rho-kinase (ROK) activity and a number of neurotransmitters are also involved in the pathogenesis of hypercontractility leading to CAS [2,20–23].

Another important risk factor is also deficiency of magnesium, an endogenous calcium channel antagonist [1,4,5,24]. Intravenous magnesium administration is beneficial in patients with CAS [5].

The main pathogenetic mechanisms of CAS are presented in Figure 1.

**Figure 1.** Pathogenetic mechanisms of CAS.

Lifestyle change and elimination of risk factors, as well as adherence to prescribed pharmacotherapy, form the basis of the management of CAS and reduce the risk of further episodes in the future [25]. The pharmacotherapy of CAS is based on the use of calcium channel blockers (CCBs) and/or nitrates. In exceptional cases, invasive therapies can be used [26].

Diagnosis of vasospastic angina may be problematic [5]. The primary role in the initial evaluation of a patient with an attack of a coronary artery spasm is to perform an electrocardiography (ECG) [5]. However, coronary angiography with a provocation test is considered the gold standard for diagnosing the disease. There are also other modern imaging tests such as intravascular ultrasound (IVUS) and optical coherence tomography (OCT), which are much more accurate but less commonly used [1].

#### **2. Endothelial Dysfunction**

The vascular endothelium is known as a regulatory organ that is essential for the proper function of the cardiovascular system. Due to its ability to produce biologically active substances, the endothelium is a significant factor that maintains homeostasis. Moreover, it is crucial for the fluidity of blood because of its anticoagulant, fibrinolytic, and antithrombotic properties. Therefore, its dysfunction plays an important role in the pathomechanism of blood vessel alterations [27].

One of the multiple roles of a normal functional endothelium is the production of NO. This compound is responsible for vasodilatation by suppressing vasoconstrictors such as angiotensin II and endothelium I [1]. Furthermore, NO deficiency may intensify their synthesis [28,29].

In young healthy people, acetylcholine (ACh) induces an increase in coronary artery diameter by releasing NO [8]. Nevertheless, there are other endothelium-dependent vasodilators such as serotonin, histamine, or ergonovine which are also a virtue of nitric oxide-releasing mechanisms [30]. On the other hand, a dysfunctional endothelium is characterized by the deficiency of NO. Consequently, in subjects with coronary atherosclerosis, intracoronary infusion of ACh results in spasms [8]. The difference between those two groups proved useful in the diagnosis of CAS. Injections of ACh are used as a provocative test [4]. However, it has been reported that coronary hyperconstriction induced by ACh involves all coronary segments. Spasms caused by ergonovine or serotonin concern only the given coronary site or segment which is similar to spontaneous spasms. Due to this fact, ACh may not be an appropriate option [10].

Nonetheless, nitrates via conversion into NO in vivo are independent of endothelial mechanisms. Synthesis of NO from L-arginine can be inhibited by L-monomethyl-arginine (L-NMMA) [30]. Kugiyama et al. [31] conducted a study in which they infused L-NMMA into coronary arteries in 21 patients with coronary spastic angina (CSA) and in 28 control patients. A coronary spasm was induced by ACh. Administration of L-NMMA in the control group resulted in a decrease in the basal diameter of a coronary artery but ended up with no effect in the other group. Moreover, the dilator response to nitroglycerin was significantly higher in patients with CSA. This is a result of the super-sensitivity of spasm arteries to nitroglycerin. It might be due to the deficiency of endogenous NO activity.

According to Kawano's research [16], variation in estrogen levels is strictly connected with the frequency of myocardial ischemia. As it is known, estrogen is responsible for enhancing NO synthase activity [32]. Due to the similarity between the endothelial function of a brachial and coronary artery [33], flow-mediated dilation of the brachial artery was assessed in the study. This magnitude is mostly based on endothelium-derived nitric oxide [34], and as Kawano showed, it is related to the variation in estradiol levels during the menstrual cycle. The ischemic episodes occurred more frequently with low estrogen levels and less frequently at high estrogen levels. This is why CAS occurs more often in post-menopausal women. However, no similar association was demonstrated in progesterone levels.

#### **3. Oxidative Stress**

Oxidative stress is the state of imbalance between the action of reactive oxygen species (ROS) and the biological ability to dispose of reactive intermediates or to repair the damage. Due to enzyme activity, the reducing environment in cells is maintained. Disruption of this mechanism can contribute to the production of free radicals and peroxides. Free radicals are defined as substances that have one or more unpaired electrons. This feature makes them highly reactive and allows them to donate their electrons to other molecules. Consequently, it leads to chain reactions and then oxidative damage [35].

Studies showed that oxidative stress plays an important role in the pathogenesis of endothelial dysfunction [36,37]. ROS are responsible for the degradation of NO, thus stimulating vasoconstriction and causing endothelial damage [30].

Thioredoxin is a ubiquitous enzyme, and one of its functions is cytoprotection against oxidative stress [38]. Miyamoto et al. [39] reported that plasma levels of thioredoxin were increased in subjects with coronary spastic angina. Furthermore, it was shown that higher thioredoxin levels were strictly connected with a more frequent occurrence of anginal attacks. It can be concluded that the high activity of the disease is associated with intensified oxidative stress.

Smoking has been recognized as one of the major risk factors for coronary spasms [9]. Cigarette smoke is the source of a large number of free radicals causing the degradation of NO [40]. It has been reported that the number of smokers was significantly higher in the coronary spastic angina group than in the chest pain syndrome group [39]. Oxidative activity of estrogen protects pre-menopausal women from CAS, but this does not apply to those who smoke [16]. Motoyama et al. [14] concluded that vitamin C can improve impaired endothelium-dependent vasodilation in chronic smokers. Serum levels of vitamin C were lower in smokers than in nonsmokers. Additionally, plasma levels of thiobarbituricacid-reactive substances (TBARS) were remarkably higher in those addicted to cigarettes. TBARS are known as an indicator of oxidative stress. However, the infusion of vitamin C resulted in a decrease in TBARS levels in smokers but did not change the levels in nonsmokers. These results are presented in Table 1.

**Table 1.** Association between particular indicators and risk of CSA.


TBARS, thiobarbituric-acid-reactive substances; CSA, coronary spastic angina.

Not only vitamin C is helpful for patients with coronary spastic angina. Studies have also shown that vitamin E is able to restore disturbed arterial reactivity. Miwa et al. [41] concluded that plasma vitamin E levels were markedly lower in subjects suffering from active variant angina than in those without coronary spasms. This finding suggests a connection between oxidative stress and CAS. In Motoyama's research [15], the effect of the oral administration of 300 mg/day of vitamin E on endothelium-dependent vasodilation was examined. It was shown that supplementation of vitamin E resulted in improvement in flow-dependent vasodilation. Furthermore, this management also caused a decrease in plasma TBARS levels.

The mechanism of either vitamin C and E is based on increasing the availability of intracellular reduced glutathione (GSH) and thiols [42]. Glutathione is mainly responsible for protection from oxidative stress and the prevention of nitric oxide inactivation. It has been reported that intracoronary infusion of GSH can restore the proper function of the endothelium [43] and suppress constrictor response to ACh in epicardial coronary arteries [44].

#### **4. Inflammation**

The evidence based on many studies [4,10,11,45], as well as clinical settings, suggests an association between CAS and inflammation, especially connected with Rho-kinase regulation [10,11]. CAS indicates an association with chronic inflammation by elevated biomarkers such as hs-CRP [2,12,45], interleukin-6, peripheral leukocytes, monocytes [2], and soluble CD40 ligands [12]. Moreover, adhesive molecules, such as P-selection, are elevated in patients with CAS [46]. A chronic low-grade inflammatory state may lead to

CAS through RhoA/Rho-kinase pathway activation and a reduction in endothelial NO activity [11]. C-reactive protein, a sensitive marker of inflammation, suppresses endothelial NO activity and activates RhoA signaling [47] and Rho-kinase activity in white blood cells, which is a prognostic factor for the severity of CAS, correlating with the Il-6 level in the plasma [2]. Moreover, another inflammatory marker, an increased mastocyte level, has been reported in patients with CAS [48]. Cigarette smoking, a major risk factor for coronary artery spasms, is connected with low-grade inflammation and an increased hs-CRP level [12,13]. Thus, it confirms that even minor elevations of the hs-CRP level in serum are essentially and independently associated with coronary spasms. Furthermore, a recent study suggested that coronary spasms are associated with inflammation of coronary adventitia and perivascular adipose tissue [49].

#### **5. Smooth Muscle Hypercontractility**

The activity of vascular smooth muscles, contraction and relaxation, is regulated by the phosphorylation and dephosphorylation of the myosin light chain (MLC). Physiologically, phosphorylation is induced by an increase in the intracellular concentration of calcium ions, which, being in a complex with calmodulin, activate myosin light chain kinase leading to phosphorylation of MLC. In coronary artery spasms, excessive contraction of the smooth muscles of the coronary vessels occurs in response to an increase in the intracellular Ca2+ influx [4].

Elevated expression of L-type Ca2+ channels and an increase in Ca2+ entry into vascular smooth muscle cells (VSMCs) through the channels may also initiate the spasm [50]. Moreover, a Ca2+ influx through the alpha1H Ca2+ system is crucial to coronary arteries' relaxation. The deficiency of α1HT-type calcium channels inhibits the relaxing effect of ACh [17], which may contribute to the pathogenesis of coronary artery spasms.

Phospholipase C overactivity, which is dependent on Ca2+, may also cause CAS through enhanced contraction of VSMCs [51].

ROK and RhoA, being VSMC contractility regulators, are involved in the pathogenesis of coronary artery spasms. Properly, the Rho-kinase metabolic pathway modulates the level of MLC phosphorylation by the inhibition of myosin phosphatase.

The hyper-reactivity of Rho kinase in smooth muscle cells promotes its contraction by sensitizing the myosin light chain to calcium ions, as well as indirectly increasing the phosphorylation of this chain, promoting vasoconstriction. A study on animal models showed that hydroxyfasudil, the Rho kinase inhibitor, prevented dose-dependent excessive coronary contractions, supporting the role of Rho kinase in the pathogenesis of CAS [52,53].

It is essential to mention that inflammation may be also a trigger for vascular smooth muscle cell hyperactivity. The activity of Rho kinase in the coronary artery can be increased by the proinflammatory mediator, interleukin 1β (Il-1β) [10], which confirms the participation of the inflammatory process in the generation of excessive smooth muscle contractions in CAS.

Coronary artery spasms may also be the result of a defect in the endothelial enzyme responsible for the production of NO, which is one of the key mediators inducing vasodilation. Rho kinase can inhibit the production of NO [54], and the absence of this relaxing factor might result in CAS. Treatment that shows the appropriate effect in this situation is statins, which increase the activity of endothelial NO, decrease ROS, and suppress the RhoA/ROCK pathway [2].

ATP-sensitive potassium channels (KATP) are responsible for the regulation of vascular tone. KATP channels are made up of two types of subunits: the lumen-forming subunit (usually Kir 6.2) and the sulfonylurea receptor (SUR). Studies [18,19] suggest that the SUR2 KATP channel is a crucial regulator of episodic vasomotor activity, and the loss of function of SUR2 KATP channels is of key importance for the proper function of the coronary vessels. The loss of KATP activity in the smooth muscles of the coronary vessels correlates with the occurrence of excessive contraction of the coronary artery. Based on these conclusions, it

can be confirmed that there is a relationship between malfunctioning KATP channels and the occurrence of Prinzmetal variant angina.

Transmitters such as serotonin [20], dopamine [21], or histamine [22] may also be important factors influencing the VSMCs involved in the pathogenesis of CAS. Direct injection of ACh, a parasympathetic neurotransmitter of the nervous system, into the coronary artery also causes excessive contraction [23]. Focal administration of Il-1β may damage the inner membrane of the coronary vessels, sensitizing these places to the coronary administration of serotonin and histamine, which may lead to vasoconstriction [55].

There are multiple different pathways in which vascular smooth muscle hypercontractility may cause coronary artery spasms, but their connotation remains to be elucidated.

#### **6. Atherosclerosis and Thrombosis**

Atherosclerosis is characterized by the chronic accumulation of cholesterol-rich plaque in the arteries and is linked to a broad spectrum of cardiovascular diseases [56]. The disturbance of vascular endothelial structure and its function has also a major role in the pathogenesis of atherosclerosis [57,58].

In recent years, there has been substantial research showing correlations between atherosclerosis and vasomotor dysfunction. Although normal vessels are not excluded, CAS occurs more frequently among arteries with atherosclerotic segments [59]. Pelligrini et al. found that patients with CAS have more advanced atherosclerosis and a higher prevalence of vulnerable plaques, compared to those without CAS [60]. Furthermore, many studies have revealed that the presence of atherosclerosis together with CAS is associated with worse patient outcomes [59]. It is also worth mentioning that the CAS could trigger the rapture of stable plaque. In this way, coronary thrombosis and myocardial infarction (MI) might occur [61]. These findings by Yamagishi et al. suggest that atherosclerosis exists at the location of the focal vasospasm even in the absence of coronary disease on the angiography. Therefore, the development of focal vasospasms is linked to the presence of atherosclerotic lesions [62].

Spasms and atherosclerosis most likely have similar etiological pathways, such as endothelial dysfunction and arterial remodeling [63]. However, in accordance with recent research by Morita et al., atherosclerosis and CAS pathophysiologies might differ. Cigarette smoking, low diastolic blood pressure, and systemic low-grade inflammation are risk factors for CAS. On the contrary, the predictors for coronary atherosclerotic stenosis are age, diabetes mellitus, low HDL cholesterol, hypertension or high systolic blood pressure, and uric acid [64]. Interestingly, in the arterial vasculature, the atherosclerotic lesions develop at different locations where the spasm occurs. Moreover, recent studies showed that percutaneous coronary intervention (PCI) with stenting due to atherosclerotic stenosis does not contribute to the recurrence of CAS and that spasms often occur diffusely in the distal segments of the stented lesion [4].

CAS and atherosclerosis play an essential role in the pathogenesis of coronary heart diseases [65].

We also do not forget that coronary thrombosis is recognized as one of the causes of acute coronary syndromes including acute myocardial infarction, unstable angina, and sudden ischemic death [66,67]. Intimal tear, intimal erosion, and microthrombi are the three main abnormal morphologic findings of optical coherence tomography in patients with vasospasm-induced acute coronary syndrome as compared to individuals with chronic stable variant angina [68]. Kitano et al. established that spastic segments with focal spasms were more prone to have intracoronary thrombi [69]. Furthermore, intracoronary thrombi were found at all spastic segments, despite the presence of atherosclerotic lesions. Teragawa et al. indicated that CAS plays a significant role in thrombogenicity [70]. However, there were no significant differences between the spastic section with the intracoronary thrombi and the spastic segment in terms of the rate and severity of plaque formation as determined by CAS [70].

Patients with CAS have an increased risk of rapid plaque progression and ischemic events because coronary artery spasms trigger local thrombus formation and extensive inflammatory response [71]. Interestingly, patients with coronary spasms have increased plasma levels of hs-CRP and P-selection. Platelets are activated after attacks of CAS. Further, thrombogenesis is intensified [5]. Therefore, we can conclude that CAS might induce thrombosis in the coronary circulation.

#### **7. Deficient Aldehyde Dehydrogenase 2 Activity**

Susceptibility to disease is often genetically determined and is specific to a population [72]. This is also the case with CSA, which is a common disease among East Asians [73]. A mutation in the ALDH2 gene is believed to be the cause of this condition; moreover, it is likely that nearly 1 billion people worldwide carry the mutation, most of whom are Easterners [74]. It has been suggested that the risk of coronary heart disease and myocardial infarction may be increased due to the Glu504Lys variant in the ALDH2 gene, which is responsible for reducing the ability of ALDH2 to metabolize acetaldehyde [72]. East Asians are at higher risk for this variant, which is quite common in this population—it occurs in up to 40% of East Asians, while it is not observed in other parts of the world [73]. The mutation in question is a point mutation involving the substitution of glutamic acid for lysine and occurs on chromosome 12q24.2, leading to a mutant, dominant allele (A) [75]. Homozygotes carrying the defective variant are most at risk for enzyme deficiency, while heterozygotes have moderate enzyme deficiency [73]. It is also suggested that carriers of the A allele have a 48% risk of coronary artery disease (CAD) compared to those without the mutation [75].

Alcohol consumption has been proven to be one of the risk factors for the development of coronary heart disease [72]. After consuming ethanol, we can divide its metabolism into two stages. The first involves the conversion of alcohol to acetaldehyde with the involvement of alcohol dehydrogenases ADH; then, in the second stage, this product is oxidized to acetic acid via aldehyde dehydrogenases [72,74,75]. It is believed that acetaldehyde has 30 times more toxicity than ethanol; moreover, it forms free radicals that can react with DNA [74]. It is aldehyde dehydrogenase 2 that is thought to be the main enzyme that oxidizes the harmful aldehyde [72,76]. However, the action of ALDH2 is not limited to removing toxic acetaldehyde from the body but also aldehydes formed by lipid peroxidation [73]. Unfortunately, ALDH2 activity can be reduced by the presence of the 504lys variant, which leads to an increase in harmful acetaldehyde in the blood after ethanol ingestion due to impaired metabolism. As a result, homozygotes have an 18-fold higher concentration of noxious aldehyde, while heterozygotes have a 5-fold higher concentration of noxious aldehyde compared to wild-type homozygotes. The risk of CAD is likely related to the amount of alcohol consumed, with large amounts of alcohol having a higher risk [75]. It has also been suggested that high levels of acetaldehyde may affect circulation and blood pressure, thereby increasing the risk of CAD [72]. What is more, patients with ALDH2 deficiency are at risk for many other diseases such as esophageal and gastric cancer, cirrhosis, Alzheimer's disease, and osteoporosis [74].

Mizuno Y et al. [73] investigated the relationship between smoking and the presence of a defective ALDH2 variant in the pathogenesis of CSA. The study showed that Asians with defective ALDH2\*2 alleles have a higher risk of CSA. It was also noted that the genetic factor interacts with and exacerbates the deleterious effects of smoking on vasoconstriction, and the joint effect of the two factors interacts more strongly than each factor alone by increasing reactive aldehydes. It has been pointed out that reactive aldehydes may be a target for prevention and treatment in people at risk or suffering from CSA [73]. On the other hand, Li Y et al. [76] conducted a meta-analysis in which they evaluated the association between the G487A polymorphism of the ALDH2 gene and CAD in the Chinese population. They found a positive correlation of this gene variant with susceptibility to CAD [76]. Gu J. et al. [72] also conducted a meta-analysis in which they examined the relationship between the ALDH2 Glu504Lys polymorphism and the risk of CAD or

myocardial infarction among the Asian population. Interestingly, it was shown that the Chinese and Korean population with the 504lys variant has a higher risk of developing CAD and MI which was not observed in the Japanese population [72]. Zhang L. et al. [75] evaluated the relationship between ALDH2 polymorphism and the risk of CAD. They analyzed 11 population-based studies, which included Chinese, Korean, and Japanese. Based on the study, they suggested that a defective dominant A allele is associated with lower concentrations of high-density lipoprotein C, which may also influence high CAD risk [75]. Another topic of interest was addressed by Fujioka K. et al. [74] who evaluated whether administration of the dietary supplement ESSENTIAL AD2 affects acetaldehyde levels in ALDH2-deficient subjects after alcohol consumption. They studied 12 subjects who were heterozygotes for mutations in the ALDH2 gene. Interestingly, after 28 days of supplement therapy, a reduction in serum acetaldehyde levels was observed after alcohol consumption. A reduction in liver enzymes was also observed during the study [74]. The Table 2 analyzes some of the studies discussed [72–76].

**Table 2.** Association between mutation in the ALDH2 gene and CAD risk.


ALDH2, aldehyde dehydrogenase 2; CAD, coronary artery disease.

#### **8. The Role of Magnesium**

Magnesium levels in the body may be related to the occurrence of CAS. Its deficiency is one of the factors causing coronary vasospasms [1,4,5,24]. Magnesium, as an endogenous calcium antagonist [4,5], causes blockage of calcium channels, and therefore coronary smooth muscle contraction does not occur [24,26]. Compulsive alcohol consumption can also lead to angina attacks in a magnesium-dependent mechanism. This is due to magnesium deficiency caused by excessive urinary excretion of magnesium [24].

The basis of CAS is an increase in intracellular calcium ion levels and increased sensitivity to it. The increased sensitivity to calcium ions is due to the increased activity of the RhoA/ROCK pathway. It is also influenced by a decrease in NO release. As a result, coronary smooth muscle contraction occurs [5,24].

Magnesium supplementation is crucial in patients with low magnesium levels [1,4,5]. Beneficial effects of magnesium supply in patients suffering from CAS were reported

to consist of a reduction in coronary vasospasms and spasm attacks and alleviation of hyperventilation-induced angina attacks [5,24,26].

An analysis of a case report by Popow et al. [77] describing coronary vasospasms caused by hypocalcemia (0.69 mmol/L; norm: 1.13–1.29 mmol/L) and hypomagnesemia (0.52 mmol/L; norm: 0.7–1.0 mmol/L) led to similar conclusions. The severe pain accompanying the pathology subsided after the infusion of calcium and magnesium. Although this is a rare cause of CAS, it should be taken into account during the differential diagnosis [77].

#### **9. Diagnosis of Vasospastic Angina**

The diagnosis of vasospastic angina (VSA) can be problematic [5]. Taking a clinical history and performing an ECG are the first step in the initial evaluation of a patient with an acute attack [78]. Patient symptoms are uncharacteristic and include transient retrosternal pain lasting a few seconds, occurring most often at rest, usually from midnight to the early morning hours [5]. ECG monitoring is important during an attack in the outpatient setting; however, it does not provide evidence of coronary artery spasms, and an attack may not occur at this time [5,79]. Changes in the ECG most often include the appearance of a peak T-wave; less often, we may see a lowering or elevation of the ST segment, as well as the appearance of a negative T-wave or U-wave [26,79]. These abnormalities are observed when the main coronary artery contracts partially or completely, while a mild spasm may not cause any changes in examination [26]. ECG-based diagnosis is often problematic because in many cases, several minutes after the episode occurs, parameters normalize [80]. In addition to the aforementioned changes during systole, we can observe various types of arrhythmias including supraventricular tachyarrhythmias, atrioventricular blocks, or ventricular tachycardias [26]. It is noteworthy that ventricular arrhythmias occur more frequently in patients with a shorter-lasting episode of ischemia, characterized by low severity [80].

Coronary angiography with provocation testing is considered the most convincing and reliable test for diagnosing VSA [1]. However, provocation testing is justified when we suspect VSA in a patient, but our suspicions have not been definitively confirmed [78]. The provocation test involves an intracoronary injection of vasoconstricting agents, among which ergonovine and acetylcholine are the most commonly used [1,26]. The test assesses the percentage of vessel lumen reduction, which can be 50%, 70%, 75%, or 90% [1,79]. Provocative tests have the ability to induce contraction in a given coronary vessel over a well-defined period of time, providing the opportunity to document and monitor this phenomenon in a laboratory setting [5]. Indications for a provocative test can be divided into strong, good, and controversial, and Figure 2 presents some of them [81].

Unfortunately, there are also several contraindications to the provocation test, some of which are listed in Table 3 [1].


**Table 3.** Contraindications to the provocation test [1].

The diagnosis of the disease can also be made when fast-acting nitrate administered sublingually results in the resolution of ECG changes [5,79]. It is also possible when nitroglycerin causes a rapid relief of symptoms of the disease; however, in addition, at least one condition shown in Figure 3 must be met [5].

**Figure 2.** Some of the indications for the provocative test including their division [81].

**Figure 3.** Additional criteria necessary for the diagnosis of angina pectoris when symptoms are relieved by nitroglycerin. Meeting one of the listed criteria is sufficient [5]. ECG, electrocardiography; CCBs, calcium channel blockers.

The use of cardiac biomarkers released during coronary artery contraction, such as creatinine kinase or troponins I and C, can be helpful in the diagnosis, but their levels are not always elevated [79]. If there are indications, a non-invasive exercise test can be performed, but only less than 30% of patients will have ischemic changes in the ECG resulting from exercise-induced vasospasms, and the rest of the patients will have a normal result [78]. Modern coronary artery imaging methods, such as IVUS and OCT, also appear to be useful in detailed diagnosis. IVUS visualizes the vessel's thickened intima-media and even small atherosclerotic plaques at the site of the focal vasospasm; moreover, it detects lesions that may not be visible on angiography. OCT, on the other hand, allows imaging of structural changes such as erosions at the site of the coronary artery spasm [1].

Particularly noteworthy are the international diagnostic criteria presented by the International Study Group for Vasomotor Disorders in Coronary Artery Disease (COVADIS), which are helpful in the diagnosis of VSA. According to this group, diagnosis should be based on three basic pillars, among which are: (I) typical clinical signs, such as a spontaneous incident of nitrate-responsive angina, (II) ECG-documented myocardial ischemia during the spontaneous incident in the form of ST-segment elevation/decrease or new negative U-waves in at least two adjacent leads, and (III) documentation of coronary artery spasms [81].

#### **10. Management of Coronary Artery Spasm**

The basis of management of CAS is lifestyle changes and the elimination of risk factors [1,5,26]. It is recommended to stop smoking, consuming alcohol [1,4,5,26], and using substances such as cocaine [1,26]. Moreover, it is important to avoid emotional stress [1,4,5,26] and exercise in the early morning [1,4].

Another component of conservative treatment is pharmacotherapy. In the case of an acute CAS attack, nitrate is used sublingually or orally in spray form. Nitroglycerin or isosorbide dinitrate (ISDN) is converted to NO in vivo. Usage of nitrates is explained by the high sensitivity of the contracted coronary arteries to nitrates and the deficiency of endogenous NO [4,5]. Since these are short-acting drugs, it is necessary to use further preparations, which are CCBs [4]. CCBs are first-line treatment [1,26]. Both dihydropyridine and non-dihydropyridine CCBs have been shown to be effective in reducing recurrent angina [26]. However, some studies report that the use of non-dihydropyridine CCBs results in an almost complete reduction in CAS recurrence [1]. It is important that CCBs should be taken before sleep [4,5,82]. In a more selective (dihydropyridine) or less selective (non-dihydropyridine) way, CCBs act on the L-type calcium channels of myocytes in the vessels by inhibiting calcium influx. Thus, they reduce vascular resistance and cause coronary artery relaxation [83]. Long-acting nitrates are other drugs used to reduce the risk of angina [26], whereas the superiority of one drug over the other has not been demonstrated [1]. Since treatment with long-acting nitrates is accompanied by the phenomenon of tolerance, it is recommended to dose the drug in such a way that an 8-hour break is maintained [4,5,83]. Long-acting nitrates can be used both as monotherapy and as an adjunct to treatment with CCBs [26]. Underlying the action mechanism of nitrates is mitochondrial denitrification, which occurs in the vessel wall with the involvement of aldehyde dehydrogenase. As a result of these transformations, NO is produced. Hence, vasodilation occurs. The effect of nitrates varies with the dose used. Small doses cause a reduction in venous return and preload. In contrast, high-dose nitrates, similarly to CCBs, result in a decrease in afterload and thus a decrease in the heart's oxygen demand while improving oxygen supply to the myocardium [83].

Statins are a group of drugs that cause CAS reduction and improve the prognosis of patients. The effect of statins is possible due to their properties that cause inhibition of the RhoA/ROCK pathway and an increase in NO activity [4]. They are an important adjunct to CAS pharmacotherapy [26]. Inhibitors of the RhoA/ROCK pathway, such as fasudil, a Rho-kinase inhibitor, may prove beneficial in the treatment of CAS because of the contraction-reducing properties of vascular smooth muscle cells (VSMCs). However, further studies are needed [4,5,26].

The use of aspirin in high doses, i.e., >325 mg per day, inhibits prostacyclin formation. This results in vasoconstriction; therefore, such treatment is contraindicated in CAS patients. In contrast, the use of low-dose aspirin, i.e., <100 mg per day, blocks the production of thromboxane A2, resulting in vasodilation; however, there are no conclusive reports on this topic. The use of low-dose aspirin in CAS patients is still a matter of debate [1,26].

Another drug with positive effects in CAS patients is Nicorandil. This drug causes coronary artery dilatation as a result of its nitrate- and potassium-channel-activating properties [1,4,5,83]. Nicorandil is recommended for patients with refractory CAS [26]. The use of magnesium in CAS patients and its mode of action has been discussed above, while antioxidants or estrogens have beneficial effects by improving endothelial function and reducing nitrate tolerance [4].

The use of alpha-1 adrenergic receptor antagonists is controversial, and it is thought that they may be a component of treatment when dealing with CAS refractory to conventional treatment [26]. Patients with CAS may benefit by taking magnesium or antioxidants (vitamins C and E) [1,4,5,26]. Meanwhile, in post-menopausal women, it is recommended to take estrogen, especially for patients with refractory CAS [4,5].

Drugs contraindicated in CAS include beta-blockers [1,26,82] but also, among others, catecholamines, parasympathetic stimulants, and ergot alkaloids [5,26]. They have vasoconstrictive effects and cause coronary vasospasms [1,5,82]. An exception is nebivolol, which owes its selectivity to β1 receptors and its ability to produce NO [1].

A summary of pharmacotherapy is shown in Table 4 [1,4,5,26,83].

**Table 4.** Pharmacological treatment of CAS [1,4,5,26,83].


CCBs, calcium channel blockers.

Besides the non-invasive CAS treatment methods outlined, there are also invasive methods. Patients with atherosclerotic lesions may benefit from PCI and coronary artery bypass graft (CABG) [1]. Another method is implantation of a cardioverter-defibrillator (ICD). It is designed to prevent ventricular arrhythmias that can result in sudden cardiac death. However, it is suggested that this method of treatment should only be used in selected patients [26].

A study by Lin et al. [84] shows another potential treatment option for refractory CAS—sympathectomy. The study showed that sympathectomy, compared to traditional CAS treatment, was more effective in protecting against a syndrome of episodes of a major adverse cardiac event and death. However, the authors emphasize that further studies are needed [84].

#### **11. Conclusions**

Coronary arteries can contract and relax through several mechanisms. Hence, coronary constriction is not always pathological. Nevertheless, in some diseases, coronary constriction becomes more predominant and results in symptoms of a wide variety of heart and vasculature diseases.

In this review, we focused on the important molecular aspects of CAS. We paid attention to the role of endothelial dysfunction and oxidative stress, their pathomechanism, and their influence on the development of cardiovascular diseases. We also drew attention

to the influence of smooth muscle hypercontractility, as an excessive intracellular influx of calcium ions, disturbances in the functioning of calcium channels, and malfunctioning of ATP-sensitive potassium channels may result in the occurrence of CAS. Moreover, the most recent discoveries have proven that inflammation plays a critical role in modulating all stages of CAS. The important role of atherosclerosis and thrombosis was also highlighted. Deficiency of aldehyde dehydrogenase 2 activity and magnesium contributes to CAS and was also considered to be of interest.

These findings might shed novel insight on the underlying mechanisms and identify potential diagnostic and therapeutic targets for cardiovascular diseases in the future.

**Author Contributions:** Conceptualization: B.F., E.M. and J.R.; methodology: J.D., W.F., J.K. (Julia Krzemi ´nska), J.K. (Joanna Ku´cmierz), M.S., M.W. and E.M.; software: E.M.; validation: B.F., E.M. and J.R.; formal analysis: J.D., W.F., J.K. (Julia Krzemi ´nska), J.K. (Joanna Ku´cmierz), M.S., M.W. and E.M.; investigation: J.D., W.F., J.K. (Julia Krzemi ´nska), J.K. (Joanna Ku´cmierz), M.S. and M.W.; resources: B.F., E.M. and J.R.; data curation: E.M.; writing—original draft preparation: E.M.; writing—review and editing: E.M.; visualization: J.D., W.F., J.K. (Julia Krzemi ´nska), J.K. (Joanna Ku´cmierz), M.S., M.W. and E.M.; supervision: B.F., E.M. and J.R.; project administration: B.F., E.M. and J.R.; funding acquisition: B.F. and J.R. 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:** The data used in this article are sourced from materials mentioned in the References section.

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

#### **References**

