Part2: Potential Benefits Of Flavonoids On The Progression Of Atherosclerosis By Their Effect On Vascular Smooth Muscle Excitability
Mar 22, 2022
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3. Flavonoids in Atherosclerosis
3.1. General Concepts
3.1.1. Classification and Structure
Flavonoids have a basic structure that consists of two aromatic or phenyl rings, A and B, and one heterocyclic ring C; the last ring is formed with an oxygen atom (Figure2). Their basic structure contains 15 carbons that can be abbreviated as C6-C3-C6 [12,102], and they may have more than one substituent forming different compounds because the flavonoid's basic structure may suffer modifications. These modifications include the increase or decrease in the number of hydroxyl groups, flavonoid core, or hydroxyl groups methylation, ortho hydroxyl groups methylation, dimerization, the formation of bisulfates, and hydroxyl groups glycosylation to produce flavonoids O-glycosides or the glycosylation of flavonoid's cores to produce flavonoids C-glycosides. Most of them belong to the following groups: chalcones, aurones, flavanols, catechins, flavones, flavonols, flavanones, isoflavones, and anthocyanidins. Some characteristics to distinguish them based on their structure,i.e., isoflavones, have the B ring in position 3 of the Cring [103](Table 3).
3.1.2.Flavonoids Diet Source and Absorption
Anthocyanidins are commonly found in plant pigments, while flavanols are in fruits and tea, flavonols in vegetables and fruits, flavanones in citrus, flavones in vegetables, isoflavones in legumes, chalcones in vegetables and fruits, and aurones in flowering plants. However, their physiological effects depend on their bioavailability, beginning with the absorption process. In general, we consume higher quantities of anthocyanins, flavonols, flavan-3-ols, and flavanones. The natural form of flavonoids in plants is glycosides. We consume them as β-glycosides, except for catechins. EnzVmes hydrolyze these compounds in the brush border of small intestine epithelial cells. The released aglycones are lipophilic, and they can cross membranes by passive diffusion into cells without the help of transporters; however, permeability levels depend on size and hydrophobicity. Before they pass into the bloodstream, they are metabolized by enzymes and converted to sulfate, glucuronide, and/or methylated metabolites. The absorption for most of them occurs in the small intestine (Table 3). If not absorbed, they move into distal intestinal portions where interaction with the microbiota and production of other metabolites takes place [104,105]. Aurones have been used for dye and drug development; their predicted absorption is in the intestine demonstrated by in silico pharmacokinetic ADMET parameters [106].
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3.1.3. Antioxidant Mechanisms of Flavonoids
The characteristic flavonoid structure gives them antioxidative properties. In some cases, they combat two targets simultaneously; for example, it has been observed that an inhibition of cholesterol-LDL oxidation [110,111] and platelet aggregation can occur with only one compound [112]. In other cases, they inhibit oxidases, i.e., lipoxygenase and cyclooxygenase[113,114], or make a transition metal chelation of iron or copper[115], regulating metal blood levels [116].
The intake of flavonoids in a healthy diet is higher than other antioxidants such as vitamins C or E and carotenes[117]. Some flavonoids have a great capacity to act on free radicals neutralizing them by electron donation and hydrogen transfer; this is the case of quercetin and myricetin because they have ortho hydroxyl groups in ring B at position C3'and C4', or C4'and C5'(Figure 3). This characteristic, together with the flavonol structure, gives them a better antioxidant capacity [118].
Another antioxidant mechanism is possible for any C3-OH or C5-OH flavone by electron donation where a tautomeric form can behave as an antioxidant in vivo by inhibiting pro-oxidant enzymes (Figure 4) [119].
Ferric ion chelators prevent the binding of iron to components of the membrane and prevent the precipitation of Fe(OH)3; this process avoids hydroxyls radicals or peroxides formation (Figure 5) [120].
Some requirements have been described for flavonoids to have the ability to inhibit some oxidases, such as the OH group at least at C7 or one additional OH at C5, including a double bond between C2 and C3 in the benzopyrone ring. The catechol group in the B ring could be present to have inhibitory activity on xanthine oxidase(Figure6). This enzyme catalyzes the oxidation of xanthine and hypoxanthine to uric acid [121-123]; this can be used as the base to synthesize inhibitors for this enzyme.
Flavonoids can inhibit lipoxygenases if they fulfill structural specifications such as a double bond between C2 and C3,a carbonyl group in C4,and a catecholgroup in the B ring (OH in C4'is fundamental, in combination with OH in C3'or C5).An excess of OH groups lowers the lipophilic affinity of flavonoids(Figure 7)[124].
It is known that aglycones can protect lipids, since the flavonoids without glycosides groups are less water-soluble, more reactive, and they can be closer to lipids than glycosyl-flavonoids. They can participate in a lipoxygenase reaction donating hydrogen with one electron in the last step of the reaction to get a stable lipid that was previously oxidized (Figure 8) [125,126].
3.2.Effect of Flavonoids in Atherosclerosis
The consumption of flavonoids in a regular diet has been associated with reducing risk factors in atherosclerosis, which is probably because of their antioxidant and vasoactive properties[127]. The beneficial effects are related to vascular health, including inhibition of LDL oxidation[128], anti-platelet activity[129], reduction of the atherosclerotic lesion [130], lowering blood pressure [131], better endothelial function [132], and improving vascular smooth muscle functions [133]. Effects on VSMC could be related to ion channels activity modulation since the effect exerts vasodilation in most cases. The effect of apigenin or Diocletian on potassium channels reduces their activity and produces vasorelaxation. Other flavonoids produce full vasorelaxation, for example, flavones and flavanones such as acacetin, chrysin, apigenin, hesperetin, pinocembrin, luteolin, 4'-hydroxyflavanone, 5-hydroxy flavone, 5-methoxyflavone, 6-hydroxyflavanone, and 7-hydroxy flavone; partial relaxation is observed with quercetin, quercitrin, hesperidin, and rhoifolin; and some of them do not produce relaxation such as quercetagetin and baicalein [134].
The anti-atherosclerosis effect has been studied mainly in two major groups of flavonoids: flavonols and flavan-3-ols because they are the most abundant compounds in the human diet. They are also structurally similar; both contain a hydroxyl group at C3; however, flavonols contain a carbonyl group at C4 and a double bond between C2 and C3 from the heterocyclic ring, while flavan-3-ols do not. Their effect has been studied in many biological activities with the following findings: LDL oxidation was reduced ex vivo, using quercetin and glabridin [93,94], serum LDL-oxidation in apoE-/-mice was reduced with myricitrin treatment [91], aortic ROS was reduced with kaempferol [92], and plasma fat concentration was reduced with quercetin [135].
Flavonoids diminish oxidative stress by scavenging free radicals and reactive oxygen species [136], downregulating cyclooxygenases and lipoxygenases[137-139], upregulating cellular antioxidants [140], and improving anti-inflammatory actions[141].In the progress of atherosclerosis, flavonoids can avoid thrombus formation and improve lipid and glucose metabolism [142-144].
When we consume flavonoids, we metabolize them into glycosides or aglycones. Agly-cones are more liposoluble and capable of interacting with cell membranes than glycoside flavonoids[145,146]. This characteristic helps them to be in contact with ion channels.
3.3. Effect of Flavonoids in VSMC's Ion Channels
Ion channels on the plasma membrane of VSMC are affected by flavonoids. The modulation depends on which flavonoid exerts their effect on them. Smooth muscle cell membrane potential is modulated directly by the movement of calcium ions from the extracellular compartment into the cytoplasmic space and indirectly by calcium release from sarcoplasmic reticulum and mitochondria, as we mentioned before [86].
Proper amounts of dietary flavonoids influence the development of cardiovascular diseases by protecting the bioactivity of endothelial nitric oxide. Flavonoids also interfere with the signaling cascades of inflammation. They can prevent the overproduction of NO and its harmful consequences. In healthy tissues, flavonoids can increase endothelial nitric oxide synthase (Enos)activity, which is necessary to produce vasodilation. In oxidative stress and inflammatory conditions, flavonoids inhibit the NFkB pathway to prevent inflammation. Flavonoids reduce peroxynitrite and superoxide levels and prevent the overexpression of ROS-generating enzymes [147].
Fusi et al. (2017)studied by docking analysis the interaction between flavonoids and the Cav1.2 channel αlc subunit. They analyzed two groups of flavonoids; the first group inhibited calcium currents: scutellarein, morin, 5-hydroxy flavone, trihydroxyflavone, (±)-naringenin, daidzein, genistein, chrysin, resokaempferol,galangin, and baicalein, and the second group stimulated calcium currents: myricetin, quercetin, isorhamnetin, luteolin, apigenin, kaempferol, and tamarixetin. This study showed differences between flavonoid interactions; epigallocatechin gallate affects Cav1.2 currents in an endothelium-independent manner, while epicatechin gallate does not affect them. Hesperetin and cardamon in block Cav1.2 channels and increase Kv currents, producing vasorelaxation. At the same time, kaempferol 3-O-(6'-trans-p-coumaroyl)-β-D-glucopyranoside(salidroside)causes partial inhibition of Cav1.2 channels in vascular smooth muscle [148].
Other possible mechanisms that influence atherosclerosis include the effect of flavonoids on ion channels for blood pressure regulation. Marunaka(2017) reports a quercetin activity outside vascular tissue that stimulates Na+-K+-2Cl- cotransporter 1(NKCC1), regulating the cytosolic Cl–concentration in lung endothelial cells. The elevated chloride concentration downregulates the expression of epithelial Na* channels, controlling blood volume by Nat reabsorption with a consequent decrease in blood pressure [149].
Recently, Fusi et al. (2020)studied the beneficial effects of flavonoids on the cardiovascular system, emphasizing the study of potassium channels by docking analysis. They describe flavonoid-channel interactions at the molecular level and relate them with experimental evidence. They observed that the main vasodilator effects are associated with the opening of K channels. In some experiments, the effect is dose-dependent; for example, baicalin at daily doses of 50 to 200 mg/kg body weight lowers blood pressure in an experiment with hypertensive rats due to ATP-dependent K+ (KATp)activation [150].
4. Effects of Flavonoids on Atherosclerosis through Modulation of Ion Channels in VSMC Activity
Flavonoids can exert effects on different ion channels in VSMC and produce changes in the progression of atherosclerosis. Effects can modulate ion channel activity and make changes in ion currents and vascular tone. Several flavonoids inhibit calcium currents, producing vasorelaxation; this is the case of genistein, phloretin, and biochanin-A, which act through an endothelium-independent mechanism; this mechanism does not involve ATP-sensitive potassium channels but may involve other channels[151]. Scutellarin relaxes rat aortic rings in a dose-dependent form by inhibiting calcium currents; this process is independent of voltage-dependent calcium channels, demonstrating the participation of other calcium channels for calcium influx mediation during contraction. The candidates for this action include non-selective cation channels, receptor-operated calcium channels (ROCCs), and store-operated calcium channels(SOCCs), among others. As a result of this effect, scutellarin is used to treat ischemic diseases or hypertension-related to atherosclerosis [152]. Other biological activities related to relaxant flavonoid actions are anti-platelet aggregation and inhibition of smooth muscle cell proliferation[153]. Daidzein, genistein, apigenin, and trans-resveratrol inhibit SOCCs and impede platelet aggregation and thrombus formation, with an effect that is related to second messengers [154].
Epigallocatechin from green tea can act at two levels: first, increasing calcium influx to generate endothelium-independent vasoconstriction, and second, by inhibiting voltage-gated calcium channels to induce vasodilation. Long treatments of 200 mg/kg/day of epigallocatechin significantly reduce systolic blood pressure in spontaneously hypertensive rats; in normotensive rats, effects were shown at a dose of 25-100 mg/kg/day[155,156]. （一）-Epigallocatechin-3-gallate and（-）-epicatechin-3-gallate reduce the activity of Karp channels at low concentrations, but higher concentrations completely inhibit the channel [157]. Quercetin is a flavonoid that activates L-type Ca2+ channels in VSMCs; however, quercetin-induced vasorelaxant mechanisms are more relevant than the increase in Ca2 influx. On the other hand, rutin, the glycoside form of quercetin, acts only during endothelium-dependent relaxation due to its lower liposolubility [158]. Quercetin decreases the cell surface expression of vascular cell adhesion molecules and reduces lipid peroxidation [109]. The significant quercetin effects are observed in resistance arteries compared to conductive arteries [107].
Activation of calcium-activated potassium channels is a key mechanism in flavonoid-induced vasorelaxation. Kaempferol activates BKCa channels of endothelial cells, resulting in membrane hyperpolarization, and this mechanism contributes to vasodilation[159], while puerarin activates BKCa channels on smooth muscle cells, resulting in vasodilation [160]. Diocletian generates hypotension in normal rats, which is caused by the opening of the KCa channels [161. Saponara et al. (2006) demonstrated that naringenin activates BKCa channels and dilates aortic rings [162]. The same results were obtained with quercetin, puerarin, epigallocatechin, and proanthocyanidins through ion channel activation, hyperpolarization, and vasorelaxation [162-164]. The contribution of BKCa agonists in atherosclerosis is to lower blood pressure and improve other cardiovascular symptoms [160].
Genistein inhibits Kv current with the slow recovery of voltage-gated potassium channels [165]. The activation of potassium channels shows vasodilatory effects. Tilianin produces vasorelaxation that may be produced due to an opening of these potassium channels [166]. Kolaviron, amentoflavone, pinocembrin, luteolin, and cardamon in act via two effects: firstly, by reducing calcium currents and, secondly, by increasing potassium currents, both increasing vasodilation [167-171].
Calderone et al. (2004) investigated the endothelium-independent vasorelaxant effect of flavonoids mediated by potassium channels. Their results showed that two flavonoids were almost entirely ineffective: baicalein and quercetagetin. Quercetin, quercitrin, rhoifolin, and hesperidin had partial vasorelaxant effects, while the rest showed full vasorelaxant effects, such as acacetin, apigenin, chrysin, hesperetin, luteolin, pinocembrin, 4'-hydroxyflavanone, 5-hydroxy flavone, 5-methoxyflavone, 6-hydroxyflavanone, and 7-hydroxy flavone, all of them belonging to flavanones and flavones groups. The study concluded a relationship between the flavonoid structure and large-conductance, calcium-activated potassium channels. It seems that the presence of the C5-OH group is necessary for the interaction and also for the involvement of ATP-sensitive potassium channels [134].
On the other hand, acacetin prevents atrial fibrillation, inhibits ultrarapid delayed rectifier potassium currents, and blocks the acetylcholine-activated potassium current, achieving the prolongation of the action potential and the effective refractory period, preventing atrial fibrillation [172]. Studies have shown that isoliquiritigenin inhibits atherosclerosis by blocking TRPC5 channel expression in VSMCs. This store-operated channel activates the transcription of early response genes to proliferate and migrate [108].
Table 4 describes the effects of flavonoids on ion channels and their impact on atherosclerosis progression; Figure 9 depicts the localization of ion channels summarizing flavonoids' effects.
Endothelial, atrium smooth muscle and vascular smooth muscle cells are presented. Channels are inhibited (red line) or stimulated (green arrow) by flavonoids, resulting in different effects during atherosclerosis progression. IKur: ultrarapid delayed rectifier K+ currents; IK: potassium currents; ICa: calcium currents; Kv1.5: voltage-dependent potassium channel; BKCa: large-conductance calcium-activated potassium channel;Karp:ATP activated potassium channel; Cav1.2: voltage-dependent calcium-channel;SKCa:small conductance potassium channel; KCa: calcium-activated potassium channel; TRPC5:transient receptor potential canonical 5 channel.
5. Future Perspectives in the Treatment
The harmful effects of oxidants have been acknowledged for decades, and many pathogenic mechanisms have been identified in numerous diseases. The case of atheroscle-rosis is a typical example since disease progression would not take place without the oxidation of lipids, as has been extensively reviewed here. However, under oxidative stress conditions, lipids are not the only affected molecules. The role of other altered molecular structures needs to be considered for proper physiopathology comprehension and future drug design. With this review, we tried to emphasize the role of voltage-gated ion channels in VSMCs. Membrane potential regulation is transcendental for muscle function and depends on the proper function of each ionic conductance. There are still many unanswered questions about the specific role of the oxidized channels during the onset and development of atherosclerosis. Unraveling specific pathogenic mechanisms of each channel type will open new therapeutic targets that could prevent cardiovascular complications. Here, we have shown the major ion channels affected by oxidation; further efforts to describe how and when their to misfunction affects disease development are needed.
On the other hand, the beneficial effects of foods widen our options toward finding new natural compounds that can be used at different stages of atherosclerosis. Even though antioxidative, antithrombotic, anti-inflammatory, and vasorelaxant mechanisms of flavonoids are known, the scope of their benefits needs to be enlarged to new molecular targets that are not usually considered. As shown in Table 4, the effects of flavonoids on ion channels have been extensively described; however, the connection between their functional restoration and disease improvement needs to be approached in detail.
The antioxidant mechanisms of flavonoids are considered part of medicinal chemistry; it is necessary to deepen their structural and functional relationship and the role of pharmacokinetics and pharmacodynamics for their effect [173]. Nanotechnology may play a key role shortly to improve the bioavailability of the compounds. Future work with the aid of network pharmacology approaches will be needed to find significant targets in the treatment of atherosclerosis. In the case of quercetin, one of the most studied flavonoids, a recent network pharmacology study identified 47 cardiovascular disease-related targets and 12 pathways of the Kyoto Encyclopedia of Genes and Genomes, which may even display synergistic therapeutic effects. Studies such as docking analysis will unravel the precise mechanisms by which flavonoids interact with specific lipids and protein targets [174]. Our work demonstrates how nutritional and traditional medicine may be combined with sophisticated bioinformatical approaches to show specific molecular targets of natural compounds with high precision to support drug development.
6. Conclusions
In conclusion, flavonoids have direct or indirect effects over ion channels and vascular smooth muscle function; they are vasodilator compounds, antioxidants, reduce peroxidative reactions, inhibit platelet aggregation, and decrease thrombotic tendency.
Among these activities, they have the antioxidant capacity to protect LDL, reducing reactive oxygen species and oxidizing enzymes, their activity of trapping metal ions, reinforcing the endogenous antioxidant capacity. Combining those actions, working on different targets, including ion channels, affects the development of atherosclerosis in a significant way, improving vascular smooth muscle function.
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