Part1: Potential Benefits Of Flavonoids On The Progression Of Atherosclerosis By Their Effect On Vascular Smooth Muscle Excitability

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Abstract: Flavonoids are a group of secondary metabolites derived from plant-based foods, and they offer many health benefits in different stages of several diseases. This review will focus on their effects on ion channels expressed in vascular smooth muscle during atherosclerosis. Since ion channels can be regulated by redox potential, it is expected that during the onset of oxidative stress-related diseases, ion channels present changes in their conductive activity, impacting the progression of the disease. A typical oxidative stress-related condition is atherosclerosis, which involves the dysfunction of vascular smooth muscle. We aim to present the state of the art on how redox potential affects vascular smooth muscle ion channel function and summarize if the benefits observed in this disease by using flavonoids involve restoring the ion channel activity.

Keywords: flavonoids; progression; atherosclerosis; ion channels; vascular; smooth muscle; calcium currents; oxidative stress; membrane potential; excitability

1. Introduction

Atherosclerosis is the principal cause of cardiovascular diseases[1,2]; these diseases are considered the first cause of mortality globally, especially in developed and high-income countries, taking the lives of almost 18 million people every year [3]. Atherosclerosis is a disease that progresses over the years and can affect anybody, without race or gender restriction; however, genetic and environmental factors place-specific populations at risk [4]. The complications of this disease also represent high expenses for patients and healthcare systems. Therefore, it is urgent to make efforts to prevent the progression of this disease [3,5,6]. The primary preventive measures include the improvement of nutritional habits. Diets enriched in polyunsaturated fatty acids and antioxidants with proper fiber intake and low in processed foods and saturated fatty acids have been associated with a lower incidence of cardiovascular diseases. One example is the consumption of antioxidants, such as ascorbic acid, carotenoids, vitamin E, and polyphenols. Many studies indicate the benefits of consuming fruits and vegetables containing antioxidants known as flavonoids [7,8].

Flavonoids are secondary metabolites from plants and the biggest group of polyphenolic compounds. There are more than 5000 different flavonoids with diverse activities. Polyphenols have been studied a lot, ever since Albert Szent-Gyorgyi discovered them in 1930 when he isolated citrine from lemon and called it vitamin P. This name was chosen because the molecule regulated the permeability of capillaries [9]. We consume flavonoids in our regular diet when we eat fruits and vegetables every day |10]. They are subdivided into subgroups: chalcones, aurones, flavones, flavanols, anthocyanidins, flavonols, flavanones, and isoflavones [11]. They have common characteristics that give them a high nutritional value, although they present differences in absorption, metabolism, and bioavailability[12]; they all have substantial benefits for human health if consumed regularly. The help in vascular health results from their biological activities as antioxidants since the opposition to oxidative stress lowers the risk of atherosclerosis development [13]. Some of these actions are derived from their interaction with reactive oxygen species(ROS) and reactive nitrogen species (RNS); however, effects on ion channels in the plasmatic membrane of endothelial and vascular smooth muscle cells have also been reported [13,14].

Ion channels are integral proteins in plasmatic and inner membranes. They are responsible for the ion movement across membranes called ion currents. These currents produce changes in the electrical properties of cells such as endothelial and vascular smooth muscle cells(VSMCs) in vessels. Many of these imbalances affect intracellular calcium concentrations, disturbing the vessels' contraction-relaxation regulation [14]. VSMCs constitute wall vessels and control the diameter of medium and large blood arteries. This group of cells contracts or relaxes to keep blood pressure and oxygenation of tissues. When vascular vessels start accumulating oxidized cholesterol, atherosclerosis develops, causing cardiac complications and different peripheral vascular diseases with high morbidity and mortality rates [11,15].

This paper discusses the influence of vascular smooth muscle ion currents in the progression of atherosclerosis and how this altered condition can be reversed using flavonoids.

2. Atherosclerosis

Atherosclerosis is an immune-metabolic disease because it involves cells of the immune system and organic molecules of metabolism. Atherosclerotic lesions show high amounts of monocytes, macrophages, lipoproteins, and low-density cholesterol. The progress of the disease is considered chronic. It involves a degenerative process that occurs in many phases. Damage is generated in the walls of blood vessels due to the accumulation of lipids, calcium, platelets, and other blood compounds [16]. The process of plaque development takes place in the coronary, aorta, carotid, iliac, and femoral arteries for many years[17]. The process initiates with an early fatty streak development during childhood; then, an early fibroatheroma is formed during adolescence and the twenties. Advanced atheroma or a thin cap of fibroatheroma occurs in elders above 55 years [18].

2.1. General Concepts

The pathogenesis of atherosclerosis can be resumed in four hypotheses:(a)oxidative modification of low-density lipoproteins(LDL)[19,20], (b)response to damage [21], (c) response to LDL retention [22, and (d)autoimmune nature of the disease [23,24](Figure 1).Two experiments support the oxidative LDL modification hypothesis: firstly it was demonstrated that oxidized-LDL (ox-LDL) causes damage to cultured endothelial cells [25,26; secondly, ox-LDL was recognized by different scavenger receptors (Lectin-like oxidized LDL receptor-1(LOX-1)including scavenger receptors that bind LDL(CD36), scavenger receptors for phosphatidylserine and oxidized LDL in human atherosclerotic lesions (SR-PSOX), and the multifunctional receptor in atherosclerosis (SR-A macrophage receptors), which mediates the influx of lipids into macrophages. Activation of all these receptors promoted the formation of foam cells in cultured endothelial cells [27].

The second hypothesis considers that the damage caused to the vascular endothelium is responsible for the endothelial activation and the initiation of the atherosclerotic process. The process included an increased permeability of lipoproteins and the expression of adhesion molecules, such as E-selectin, P-selectin, vascular endothelial cell adhesion molecule-1(VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). These molecules bind to their corresponding receptors on circulating monocytes and T lymphocytes and induce the recruitment of these cells to the site of injury, where ROS species from fibroblast and other cells cause atherogenesis [28].

The third hypothesis considers the retention of LDL first. The accumulation of lipoproteins within the arterial wall and arterial proteoglycans can trigger the pro-inflammatory cascade and promote atherosclerosis [29,30]. The fourth hypothesis is related to the dis-ease's autoimmune nature; this involves the immune response before plaque development. During the onset of the illness, antigens, antibody complexes, T lymphocytes, B lymphocytes, and proteins of the complement system participate, and infiltration of mononuclear cells into the lesion, such as CD8+ lymphocytes, CD4*(Th1) helper T lymphocytes, mast cells, monocytes, and macrophages, occurs. DAMPs(damage-associated molecule patterns)such as heat stress proteins(HSP) and ox-LDL appear; in fact, anti-HSP60 antibodies can be used as a disease marker for progression. Innate immune cells recognize ox-LDL and HSPs and activate inflammation; all these events support the importance of immunity during disease development [31-33].

Any of these four processes cause atherosclerosis, but it can be prevented by diminishing risk factors. The major atherogenic risk factors are central obesity, oxidative stress, dyslipidemia, hyperglycemia, and pro-inflammatory states 34,35]. Studies have reported that high serum concentrations of LDL-cholesterol, glucose and C-reactive protein(CRP)are directly associated with the risk of developing cardiovascular disease [35,36].

It is necessary to understand the details of the disease's pathophysiology to develop appropriate preventive and/or therapeutic strategies that can avoid vascular calcification [37J. During this process, the predominant cell type in the arterial wall is smooth muscle cells; they are responsible for vessels structure and function integrity [21]. Calcification is generated in the intima of blood vessels at specific points that form crystal patches with necrotic core spaces [37,38]. During the initial atherosclerotic stage, smooth muscle cells are 90% of the cellular content in the lesion area. However, this changes in advanced lesions; in those cases, the extracellular matrix predominates over smooth muscle cells, forming the fibrous covering of plaques. Smooth muscle cells with a non-proliferative contractile phenotype are transformed into cells that actively proliferate, migrate attracted by chemotactic agents, and produce extracellular matrix proteins(collagen, elastin, and proteoglycans). This transformation activates the expression of genes that encode mem-brane receptors for growth factors [22]. The migration of smooth muscle cells promotes calcification in the injured area, which is associated with higher mortality and morbidity rates [39].

The spontaneous rupture of an atherosclerotic plaque causes the activation of pro-thrombotic elements of the endothelium. When platelets aggregate, they release their granules rich in mitogens and induce the migration and proliferation of smooth muscle cells, including inflammation and oxidative stress, which are present during all stages of the disease [40,41].

2.2. Stages of Atherosclerosis

There is a way to classify the progression of atherosclerosis based on histological studies from human and animal autopsies in the following stages: pre-atherosclerosis, early atherosclerosis, late atherosclerosis, and clinical sequelae [42,43]. In all phases, vascular smooth muscle cells are crucial for plaque development. Pre-atherosclerosis initiates at birth because the diffuse intimal thickenings and intimal xanthomas are used as an adaptation to the blood flow[44-46]; this is considered a pre-plaque [42]. During early atherosclerosis, pathological intima thickening is formed. This early plaque contains extracellular lipid pools deep in the intima with a large quantity of VSMCs and extra-cellular matrix(ECM)[42,43]. The progression involves the retention and oxidation of LDL, induction of inflammation, and VSMCs proliferation, with phenotypic changes and death [22,47]. VSMCs produce ECM in the intima, where it plays an essential role in the initiation of atherosclerosis. During this process, it has been demonstrated that negatively charged side chains of proteoglycans interact with the positively charged side of apolipoproteins [48] to retain lipoproteins from plasma [30]. The trapped lipoproteins suffer oxidation, macrophages are recruited, and inflammation initiates [22]. Sometimes, a micro-calcification near media tissue occurs, which has been associated with VSMC apoptosis [49]. Pathological intima thickening in late stages always presents with abundant macrophages, representing a crucial step for the progression to fibroatheroma [50-54] and VSMCs' proliferation, migration, and phenotype change [55]. During the late stages, the accumulation of macrophages in the luminal space is necessary. The lesion is characterized by a fibrous cap and necrotic core, which is formed by dead VSMCs and macrophages that phagocyte lipids and become foam cells [56,57]; then, the fibroatheroma develops, and calcification can be observed first in the necrotic core and then in the surrounding ECM [58-60]. This mature plaque forms sheets whose fragments can protrude into the lumen and precipitate thrombosis [42,60]. Finally, clinical sequelae depend on which artery has been affected [6].

2.3. Role of Oxidation

ROS and RNS species are produced in low concentrations in VSMCs, adventitia, and endothelial cells in normal conditions. They function as mediators in cell signaling to regulate vascular activity[13,62-64], participate in vascular smooth muscle growth, and regulate contraction and relaxation [65,66]. However, during pathological states, there is a disequilibrium between antioxidants and oxidants, and when oxidants are favored, oxidative stress is produced. The sources of ROS include lipooxygenases, cytochrome P450, cyclooxygenase, xanthine oxidase, mitochondrial respiration, NADPH oxidase, and uncoupled nitric oxide synthases[13]. Additionally, intracellular ROS production can derive from the electron-transport chain [67]. One of the first tissues affected in atherosclerosis is the endothelium, where nitric oxide(NO), endothelin I, angiotensin II, adhesion molecules, and cytokines are produced [13,68]. Oxidative stress affects cell functions, generates endothelial dysfunction, and reduces NO synthesis; the reduced bioavailability of NOexerts atherogenic effects [69]. A significant factor that has not been deeply explored is how oxidative stress modulates ion channel oxidation in VSMCs during the development of atherosclerosis. Ion channels represent transcendental elements for the proper function of VSMCs; if their function is compromised, it is important to elucidate how this affects disease development [70].

ROS and RNS can affect ion channels directly or indirectly: directly by producing post-translational modifications on the proteins, such as nitrosylation, sulfhydration, or the nitration of specific amino acid residues; or indirectly by altering different signal-ing pathways. Sulfur atoms in cysteine and methionine confer the sensitivity to redox potentials as well as aromatic rings from histidine, phenylalanine, tryptophan, and hydroxyl groups in tyrosine residues [14]. The high reactivity of the thiol groups of cysteine contributes to the formation of sulfenic acid, sulfinic acid, or sulfonic acid, depending on oxidant concentration and reaction conditions; methionine forms methionine sulfoxide and methionine sulfone; histidine oxidizes to 2-hexahistidine, and tryptophan oxidizes to 5-hydroxytryptophan and oxindolealanine [71]. Cysteine can suffer other modifications such as nitrosylation and glutathionylation [70]. For example, in vascular smooth muscle cells, ROS and NO can oxidize voltage-gated calcium channels Cav1.2b cysteine residues within the alpha subunit and produce conformational changes [14].

2.4. Damage in Vascular Smooth Muscle Cells

Vascular smooth muscle cells present differences in the expression of signaling proteins, receptors, and ion channels compared to cardiac and skeletal muscle. Its contractility is fundamentally different because VSMCs do not exert an action potential. They are partially contracted at rest, increasing their contractility in response to neuronal, humoral, or endothelial stimulus acting on membrane receptors. This contractility is relatively slow and sometimes can be sustained and tonic [72]. The maintenance of vascular tone is controlled by VSMC membrane potential. Depolarization activates the L-type high voltage-gated calcium channels(Cav1.2) at the plasma membrane, initiating an increase in Ca2+ entry [73]

The increase in intracellular Ca2 promotes a contractile response by activating calcium calmodulin-dependent myosin light chain kinase (MLCK) and releasing more Ca2+ from intracellular calcium stores [74]. On the other side, K+ efflux through Ca2+-activated K channels(KCa) modulates the membrane potential indirectly because they limit the flow of Ca2+ ions into cells, causing Cav1.2 inhibition [75]. The vascular smooth muscle cell membrane contains many types of channels. Among calcium channels, we have as the main regulators L-type channels, such as Cav1.2b, which is a different isoform from Cav1.2a in cardiac muscle [14]. They are the major regulators of vascular smooth muscle [Ca2] I and contractility. These channels work at two different levels: depolarization and hyperpolarization. The group in charge of depolarization includes the transient receptor potential family (TRP), TRPC3, TRPC6, and TRPM4 channels, and the group promoting hyperpolarization includes significant conductance calcium-activated potassium channels, TRPV4, and Cav3.2 channels. The entry of calcium into the cell is predominantly mediated by L-type channels(Cav1.2b) and, to some extent, T-type Cav3.1/3.3 channels; they control contraction, and their activity is regulated by changes in the membrane potential [76].

Other channels in the plasma membrane include chloride channels. They have several functions, including cell volume regulation, transepithelial transport, ion homeostasis, and the regulation of electrical excitability [77]. In smooth muscle cells, the electrochemical potential for chloride is higher than the resting potential. Then, the opening of chloride channels may produce enough depolarization to cause activation on Cav channels and Ca2+ influx, which is important for vascular response to mechanical stress [78].

Membrane ion channels of VSM are classified as follows:

(a) Voltage-gated Ca channels (VGCC). These channels regulate contraction and gene expression in VSCM. L-type and T-type Ca channels are representative members of this family. When L-type channels are activated, the membrane is depolarized, and calcium ions enter the cytoplasm; then, potassium channels are activated, and membrane hyperpolarization occurs with the subsequent deactivation of VGCCs [79]. The activation of PKG contributes to vasodilation mediated by NO and inhibits Cav1.2 currents [80].

T-type channels may contribute to myogenic tone at low intravascular pressure when smooth muscle cells are relatively hyperpolarized; however, their specific role needs to be further elucidated [81].

(b)Ca2-activated K channels (KCa). These channels are activated with increased Ca2+ intracellular concentration; BKCas are the most abundant in VSMCs. Up to now, only two studies have identified small conductance calcium-activated potassium channels (SK Ca) in VSMCs from systemic blood vessels [82], and intermediate conductance calcium-activated potassium channels (IKCa) are expressed only in proliferating VSMCs [83]. 

(c)Voltage-gated K+ channels (Kv). They contribute to the resting tone of small coronary arteries and are a dilator influence in the coronary circulation. Kv1.X is the main group of voltage-gated potassium channels in the VSMC of coronary microvessels. Identified family members in the vascular system include Kv1.2 and Kv1.5. The high production of peroxynitrite in hyperglycemia affects these Kv channels and impairs vascular smooth muscle dilation [84]. They regulate the pulmonary circulation and regulate vascular remodeling in pulmonary artery smooth muscle cells [71].

(d) Transient receptor potential channel (TRP). Based on homology sequences, this kind of channel is divided into six members: canonical(TRPC1-7), melanostatin (TRPM1-8), vanilloid (TRPV1-6), ankyrin(TRPA1), polycystin(TRPP1-3), and mucolipin (TRPML1-3). Each family has differences in properties and structure [85].

Table 1shows the most representative channels present in VSMC, how they are affected by oxidative stress, the consequences in membrane potential, and how they participate in atherosclerosis.

Additionally, smooth muscle excitability is modulated indirectly by cytoplasmic calcium concentration released from mitochondria and sarcoplasmic reticulum [86]. 

2.5. Natural Compounds for the Treatment of Atherosclerosis

Many plant derivatives are used as drugs; their advantages include fewer secondary effects and reduction of oxidative stress, LDL cholesterol level, and inflammation [87]. The use of natural compounds in atherosclerosis has focused on prevention or treatment to reduce the levels of blood lipids. Among the different tested substances, it has been observed that the consumption of polyphenol compounds such as flavonoids helps to reduce atherosclerosis development because of its potent antioxidant activity [88], The Mediterranean diet, which includes the consumption of olive oil and nuts, reduces cardiovascular disease incidence by 30% compared to low-fat diets related to its high phenolic content [89]. The reduction of the atherosclerotic lesion area has been observed with flavonoid treatments in different ex yivyo studies in aortas of modified Apo E mice [o0-o4]A summary of the various beneficial effects of flavonoids in cardiovascular diseases is shown in Table 2.

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