Oxidative Regulation of Vascular Cav1.2 Channels Triggers Vascular Dysfunction in Hypertension-Related Disorders

Blood pressure is determined by cardiac output and peripheral vascular resistance. The L-type voltage-gated Ca2+ (Cav1.2) channel in small arteries and arterioles plays an essential role in regulating Ca2+ influx, vascular resistance, and blood pressure. Hypertension and preeclampsia are characterized by high blood pressure. In addition, diabetes has a high prevalence of hypertension. The etiology of these disorders remains elusive, involving the complex interplay of environmental and genetic factors. Common to these disorders are oxidative stress and vascular dysfunction. Reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria are primary sources of vascular oxidative stress, whereas dysfunction of the Cav1.2 channel confers increased vascular resistance in hypertension. This review will discuss the importance of ROS derived from NOXs and mitochondria in regulating vascular Cav1.2 and potential roles of ROS-mediated Cav1.2 dysfunction in aberrant vascular function in hypertension, diabetes, and preeclampsia.


Introduction
Free intracellular Ca 2+ functions as an important second messenger to regulate various physiological processes including muscle contractility, gene expression, hormone secretion, and neuronal transmission [1,2]. The Ca 2+ signal transduction is initiated by increasing intracellular Ca 2+ concentration ([Ca 2+ ] i ) due to Ca 2+ entry through ion channels on the cell membrane and Ca 2+ release via Ca 2+ -release channels on the endo/sarcoplasmic reticulum. Ca 2+ influx in vascular smooth muscle cells is primarily mediated by L-type voltage-gated Ca 2+ (Ca v 1.2) channels encoded by the CACNA1C gene [3]. Vascular tone in small arteries and arterioles, a major determinant of blood pressure and blood flow, is governed by the contractile state of vascular smooth muscle cells which is determined by dynamic changes in intracellular [Ca 2+ ] i [4]. An increase in [Ca 2+ ] i instigates vascular smooth muscle cell contraction, whereas a decrease in [Ca 2+ ] i promotes relaxation.
Elevated blood pressure is a prime characteristic of hypertension and preeclampsia [5][6][7]. Preeclampsia is a multisystem disorder characterized by the onset of hypertension after 20 weeks of gestation. In addition, there is a strong epidemiologic association of diabetes and hypertension, and hypertension is common among diabetic patients [8][9][10]. Prevalence of hypertension in adults is~30% worldwide in 2010 and in United States between 2011-2012 [11][12][13]. Remarkably, prevalence of hypertension in adults with diabetes is 66-76% in United States [10]. Preeclampsia affects~5% of pregnancies globally [14]. Hereafter, these three disorders are collectively referred as hypertension-related disorders in this review. Hypertension is categorized into essential (primary) and secondary hypertension. Essential hypertension accounts for 95% human cases and has no known cause, whereas the remaining are secondary hypertension which occurs due to known diseases or conditions including renovascular disease, aldosteronism, Cushing's syndrome, thyroid thyroid dysfunction, and among others [15,16]. Diabetes is commonly classified into two types. Type 1 diabetes results from insulin deficiency due to autoimmune destruction of β-cells in the pancreas, whereas type 2 diabetes originates from insulin resistance owing to reduced insulin sensitivity and accounts for >90% of all cases [17,18]. Hypertension is the leading cause of cardiovascular disease worldwide, including stroke, coronary artery disease, heart failure, arrhythmia, and peripheral vascular disease [19].
High blood pressure arises from vascular dysfunction [20]. Oxidative stress is a hallmark of hypertension, preeclampsia, and diabetes [21][22][23]. Excessive reactive oxygen species (ROS) play an important role in vascular dysfunction and contribute to the pathogenesis of hypertension-related disorders [24][25][26]. Given the pivotal role of Cav1.2 in regulating vascular tone, we discuss in this review the oxidative modification leading to alternations of vascular Cav1.2 function/expression and enhanced myogenic tone in hypertension-related disorders.

Overview of Cav1.2
L-type Ca 2+ (Cav) channels are heteromultimeric complexes comprising pore-forming α1c and auxiliary β, α2δ, and γ subunits [27]. The α1c subunit possesses four repeat domains (I-IV) linked by intracellular loops and intracellular NH2-/COOH-termini with each domain containing six transmembrane segments (S1-S6) ( Figure 1). The ion-conducting pore is formed by S5 and S6 and the loop between them, whereas the voltage sensor is located in the S4 segments [28]. The intracellular COOH terminus, along with intracellular loops, plays important roles in Ca 2+ -dependent inactivation, channel trafficking, phosphorylation and oxidation [28][29][30]. Cav is activated by membrane depolarization and its activation allows Ca 2+ influx through the channel pore. The expression of the α1c subunit itself can form functional channels to conduct Ca 2+ ions, whereas the incorporation of auxiliary subunits promotes membrane α1c expression and alters biophysical properties of the channel [28,31]. Cav channels are sensitive to blockades by dihydropyridines (i.e., nifedipine), phenylalkylamines (i.e., verapamil) and benzothiazepines (i.e., diltiazem) [32]. There are four types of Cav channels (Cav1.1-1.4). Cav1.2 is predominantly expressed in the heart and in vascular smooth muscle [33,34]. Ca 2+ influx through the channel is a primary trigger of vasoconstriction and also participates in transcriptional regulation. The auxiliary subunits β2, β3, and α2δ1 are expressed in vascular smooth muscle [35][36][37][38]. β subunits, lacking membrane-spanning segments, are located intracellularly and interact with the α interaction domain (AID) on the I-II linker of the α1c subunit [39]. The α2δ1 subunit is a single gene product bound together by disulfide bonds. Whereas the α2 subunit is extracellular, the δ subunit contains a single transmembrane segment [40]. The diversity of Cav1.2 is also conferred by alternative splicing. Smooth muscle contains Cav1.2 exons 1, 8, 9 *, 31/32, and 33 [41,42]. Due to the scope of this review, only a brief description of Cav1.2 is presented here. The reader is referred to the literature for detailed information on Cav1.2 [43][44][45].

. Regulation by Auxiliary Subunits
The molecular composition of Ca v 1.2 in vascular smooth muscle cells includes the pore-forming α1c subunit and auxiliary β2/3 and α2/δ1 subunits. The β3 subunit appears to be the principal β isoform in vascular smooth muscle cells [35]. Genetic deletion of the β3 subunit resulted in reduced α1c expression in mouse aorta and is associated with a reduction in Ca 2+ channel current and a slower inactivation rate [35]. This genetic manipulation also attenuates angiotensin II-induced upregulation of Ca v 1.2 channels in mouse mesenteric arteries and the development of hypertension [38]. Similarly, knocking-down α2/δ1 with short hairpin RNA in cerebral artery reduced plasma membrane α1c expression [36]. Moreover, Ca v 1.2 currents were inhibited by pregabalin, an α2/δ1/2 ligand, and by an α2/δ-1 antibody in cerebral vascular smooth muscle cells, leading to vasodilation [36].

Regulation by Protein Kinases
Protein phosphorylation, a covalent addition of the phosphate group to the side chain of serine, threonine, and tyrosine residues by protein kinases, is a common posttranslational modification to fine tune activities of receptors, ion channels and enzymes. Unsurprisingly, Ca v 1.2 is a target of protein kinases, and its activity is subject to the regulation by protein phosphorylation. Both α1c and β2 subunits are phosphorylated by protein kinases A (PKA), C (PKC), and G (PKG) [29,[46][47][48][49]

Regulation by Small GTPases
The Ras superfamily of small GTPases (also known as small G-proteins) are cellular switches that regulate a variety of biological processes in living cells. They have been implicated in regulating Ca 2+ homeostasis and Ca v 1.2 is recognized as an important effector of the RGK subfamily (Rem, Rem2, Rad, and Gem/Kir) [87,88]. Unlike vascular Ca v 1.2, direct phosphorylation of the cardiac Ca v 1.2 α1c subunit does not contribute to PKAstimulated channel activity as mutating all PKA consensus sites in the α1c subunit fails to block the increase in Ca v 1.2 activity in response to β-adrenergic stimulation [89,90].
Cardiac Ca v 1.2 is under tonic inhibition of Rad due to its association to both α1c and/or β subunits [91,92]. β-adrenergic stimulation induces RAD phosphorylation, promotes the release of RAD from Ca v 1.2, and increase channel activity [90]. Similarly, Ca v 1.2 activity is suppressed by other members of the RGK subfamily [93][94][95]. It remains to be determined whether vascular Ca v 1.2 is modulated by RGK members. Small GTPases also regulate surface expression of Ca v 1.2. Rab25, a member of the Rab subfamily, regulates intracellular vesicle trafficking and promotes surface expression of Ca v 1.2 in vascular smooth muscle cells [96].

Ca v 1.2 and Myogenic Tone
Vascular smooth muscle cells in resistant arteries and arterioles possess intrinsic ability to contract in response to an increase in intraluminal pressure and to relax upon a decrease in intraluminal pressure [97]. This phenomenon is defined as myogenic response and the steady-state of vascular smooth muscle cell contractility in these vessels is termed as myogenic tone. Myogenic tone sets the basal vascular tone and distribution of blood flow to and within tissues/organs. In principle, peripheral vascular resistance can be described by the Poiseuille equation: R = 8Lη/πr 4 , where R is the resistance, L is length of the vessel, η is viscosity of blood, and r is the radius of the vessel. According to the Poiseuille's law, peripheral vascular resistance is inversely proportional to the radius to the fourth power. For example, decreases in vessel caliber by 10% and 20% will theoretically lead to increases in vascular resistance by~50% and~145%, respectively. Thus, the radius of a given small artery/arteriole has a great impact on peripheral vascular resistance and blood flow.
Vascular smooth muscle cells in resistance arteries/arterioles become depolarized in response to increased intraluminal pressure [98][99][100]. Various mechanisms have been proposed to regulate myogenic tone ( Figure 2). It is widely believed that pressure-induced membrane depolarization in vascular smooth muscle cells is instigated by mechanosensitive or stretch-activated cation channels including transient receptor potential (TRP) channels and epithelial Na + channels (ENaCs) [101][102][103][104][105][106][107]. The membrane depolarization leads to increased [Ca 2+ ] i and vasoconstriction [99,108]. The increases in both [Ca 2+ ] i and/or myogenic tone are blocked by the removal of extracellular Ca 2+ or by Ca v 1.2 blockers [99,[109][110][111][112][113][114][115]. These findings suggest that altered intraluminal pressure initiates myogenic tone via membrane depolarization and subsequent opening of Ca v 1.2. This notion is corroborated by findings from the genetic deletion of Ca v 1.2 α1c subunit in smooth muscle [116]. Such a manipulation abolishes myogenic reactivity in murine tibialis arteries [116]. However, Ca 2+ influx mediated by T-type voltage-gated Ca 2+ channels (Ca v 3.x) may also contribute to myogenic tone development [106,117]. Integrins, linking the extracellular matrix and cellular cytoskeleton, could also function as mechanotransducer to regulate myogenic tone via c-Src-mediated phosphorylation of Ca v 1. 2 [83,85,118]. Moreover, some G protein-coupled receptors are mechanosensitive and produce inositol triphosphate (IP 3 ) upon stretch to induce Ca 2+ release via activating IP 3 R on the sarcoplasmic reticulum (SR) membrane [119,120]. Over myogenic tone, extrinsic factors such as vasodilators and vasoconstrictors can induce vasodilation and further vasoconstriction, respectively. It should be noted that the large-conductance Ca 2+ -activated K + channel is also activated by the increase in intralumenal pressure, which functions as a negative feedback mechanism to limit the magnitude of intralumenal pressure-induced vasoconstriction under physiological conditions [121]. For more detailed information on the regulation of myogenic tone by ion channels, readers are referred to recent reviews on this topic [106,122,123]. As aforementioned, myogenic response is the intrinsic property of vascular smooth muscle cells to respond to changes in intraluminal vascular pressure. In general, myogenic tone development is independent of endothelium [124-126]. However, myogenic tone can be modified by the endothelium. For example, activation of the endothelial intermediate-conductance Ca 2+ -activated K + channel is found to reduce myogenic tone [127,128]. Moreover, myogenic tone in mouse mesenteric and uterine arteries from pregnant mice is enhanced by the loss of endothelium-derived NO [129].

Aberrant Vascular Tone in Hypertension-Related Disorders
Hypertension is associated with increased peripheral vascular resistance. As myogenic tone is the fundamental element of vascular tone, it is reasonable to speculate that myogenic tone is altered in hypertension-related disorders. In patients with hypertension, myogenic tone is increased in coronary arterioles [110]. Increased myogenic tone is also noted in resistance arteries from the adipose tissue of paravertebral muscles of hypertensive patients [130]. Commonly used rat models of experimental hypertension include the spontaneously hypertensive rat (SHR), stroke-prone spontaneously hypertensive rat (SHRSP), Milan hypertensive strain (MHS), vasopressin-deficient (Di/H) rats, two-kidney, one-clip (2K1C rats), and among others. Compared to the Wistar-Kyoto rat (WKY), myogenic tone of afferent arterioles and arcuate arteries from SHR kidneys is increased [131,132]. Mesenteric arteries of SHR and MSH also exhibits higher myogenic tone than those of WKY and Milan normotensive strain (MNS), respectively [133,134]. Elevated myogenic tone is observed in cerebral arteries/basilar arteries of SHR/SHRSP/Di/H compared to WKY and Di normotensive (Di/N) rats, respectively [135][136][137][138]. Similarly, there is an increase in the myogenic tone of cremaster arterioles of SHR [139,140]. Furthermore, chronic infusion of angiotensin II in C57BL/6 mice also causes increased myogenic tone in middle cerebral arteries [141]. In contrast, mice with smooth muscle cell-specific deficiency of the mineralocorticoid receptor reduces myogenic tone and lowers blood pressure [142,143].

Aberrant Vascular Tone in Hypertension-Related Disorders
Hypertension is associated with increased peripheral vascular resistance. As myogenic tone is the fundamental element of vascular tone, it is reasonable to speculate that myogenic tone is altered in hypertension-related disorders. In patients with hypertension, myogenic tone is increased in coronary arterioles [110]. Increased myogenic tone is also noted in resistance arteries from the adipose tissue of paravertebral muscles of hypertensive patients [130]. Commonly used rat models of experimental hypertension include the spontaneously hypertensive rat (SHR), stroke-prone spontaneously hypertensive rat (SHRSP), Milan hypertensive strain (MHS), vasopressin-deficient (Di/H) rats, two-kidney, one-clip (2K1C rats), and among others. Compared to the Wistar-Kyoto rat (WKY), myogenic tone of afferent arterioles and arcuate arteries from SHR kidneys is increased [131,132]. Mesenteric arteries of SHR and MSH also exhibits higher myogenic tone than those of WKY and Milan normotensive strain (MNS), respectively [133,134]. Elevated myogenic tone is observed in cerebral arteries/basilar arteries of SHR/SHRSP/Di/H compared to WKY and Di normotensive (Di/N) rats, respectively [135][136][137][138]. Similarly, there is an increase in the myogenic tone of cremaster arterioles of SHR [139,140]. Furthermore, chronic infusion of angiotensin II in C57BL/6 mice also causes increased myogenic tone in middle cerebral arteries [141]. In contrast, mice with smooth muscle cell-specific deficiency of the mineralocorticoid receptor reduces myogenic tone and lowers blood pressure [142,143].
A study by Kublickiene and colleagues find that preeclampsia does not alter myogenic tone of myometrial arteries [162]. However, the measurement is made in only one pressure point. Nevertheless, the authors reveal that flow-mediated dilatation is lost in pressurized myometrial arteries from women with preeclampsia [162]. A frequently used rodent model of preeclampsia is induced by chronic reductions in uteroplacental perfusion (RUPP) in rats/mice by clipping/ligating the aorta below the renal arteries or the arterial and venous branches of the uterine vascular arcade [163,164]. Myogenic tone is increased in both uterine arteries and mesenteric arteries in this animal model [165][166][167]. Intriguingly, exposure to small extracellular vesicles purified from the plasma of pregnant women with preeclampsia elevates myogenic tone in the mesenteric arteries of C57Bl/6J mice [168], suggesting that circulating bioactive factor(s) could contribute to the increased peripheral vascular tone in preeclampsia. Hypoxia plays a key role in the pathogenesis of preeclampsia [169][170][171]. High-altitude hypoxia increases maternal systolic and diastolic blood pressure at term in human pregnancy [172] and is associated with increased incidence of preeclampsia [173][174][175]. Pregnant sheep exposed to normobaric hypoxia at low altitude and hypobaric hypoxia at high altitude developed preeclampsia-like symptoms including elevated maternal systemic blood pressure and increased uterine vascular resistance as well as various biochemical changes in the circulation and uteroplacental tissues [176][177][178]. Myogenic tone of resistance uterine arteries is increased in pregnant sheep acclimatized to high-altitude hypoxia [179]. This effect is replicated by ex vivo hypoxia treatment of resistance uterine arteries of low-altitude pregnant sheep [180].

Dysfunction of Vascular Ca v 1.2 in Hypertension-Related Disorders
As aforementioned, activation of Ca v 1.2 in vascular smooth muscle cells is essential for the development of myogenic tone [99,100,109,110,113,114,116]. The enhanced myogenic tone in peripheral resistance arteries and arterioles in hypertension-related disorders suggests potential dysfunction of vascular Ca v 1.2. Indeed, this notion is substantiated by lines of evidence from functional studies. First, the increased myogenic tone in resistance arteries/arterioles of hypertensive and diabetic animals was normalized by the Ca v 1.2 blocker nifedipine [58,132,181]. Whereas specific deletion of the mineralocorticoid receptor reduces KCl-and Ca v 1.2 agonist Bay K 8644-induced vasoconstriction of mesenteric arteries [142,182], Bay K 8644-induced contraction and increase in [Ca 2+ ] i in 2K1C rat aorta is greater than in the control 2K rats [183]. Second, Bay K 8644 triggers greater contraction of inferior epigastric arteries of women with preeclampsia than that of normotensive subjects [184]. Similarly, Bay K 8644-or the membrane-depolarizing agent KCl-induced contraction of cerebral arteries, mesenteric arteries, renal arteries, femoral arteries is greater in SHR, diabetic STZ or GK rats, RUPP rats or pregnant rats treated with nitric oxide synthase inhibitor L-N ω -nitro arginine methyl ester (L-NAME) than their control counterparts [153,[185][186][187][188][189][190][191]. Third, Bay K 8644 or KCl produces a larger increase in [Ca 2+ ] i and contraction in renal arteries in RUPP rats and pregnant rats treated with L-NAME [186,188].
Animal models of hypertension-related disorders have provided mechanistic insights into the understanding of the Ca v 1.2 dysfunction in these disorders. Both aberrant expression of and dysregulation of Ca v 1.2 contribute to vascular Ca v 1.2 dysfunction. Various studies reveal increased protein expression of α1c [37,38,187,192,193], α2δ1 [37,192], and β3 [37,38] subunits in mesenteric, femoral, and cerebral arteries of SHR and angiotensin II-infused C57BL/6 mice. Consistently, increased expression of Ca v 1.2 is associated with enhanced channel activity in vascular smooth muscle cells [67,185,[193][194][195][196][197]. The expression and activity of Ca v 1.2 are reduced in mesenteric arteries of aged mice lacking mineralocorticoid receptors in smooth muscle cells [143]. Dexamethasone administration increases the expression of the cardiac Ca v 1.2 α1c subunit in rats [198]. As expected, Ca v 1.2 activ-ity in A7r5 cells is increased following chronic dexamethasone exposure [199]. Similarly, hyperthyroidism also boosts the expression of cardiac Ca v 1.2 [200]. Galectins (Gals) are a family of carbohydrate-binding lectins. Gal-1, through binding preferentially to Ca v 1.2 I-II loop without exon 9 *, reduces surface expression of Ca v 1.2 in vascular smooth muscle cells [201]. Gal-1 is later found to compete with the β subunit for binding to the α1c subunit leading to polyubiquitination and degradation of the α1c subunit and consequent reduction of Ca v 1.2 activity [202]. Notably, protein levels of Gal-1 and Ca v 1.2 are downregulated and upregulated, respectively, in arteries of SHR and hypertensive patients [202]. In hypertensive human pulmonary arteries, the downregulation of Gal-1 is mediated by hypoxia-inducible factor 1α [202]. The knockdown of Gal-1 with siRNA increases Ca v 1.2 activity and promotes vasoconstriction [201], whereas the deletion of the Gal-1 encoding gene LGALS1 increases blood pressure [202].
An increase in protein expression of the α1c subunits is also observed in cerebral arteries of STZ rats, which is associated with increased Ca v 1.2 activity and contraction to KCl [203]. Ca v 1.2 also displays increased activity in vascular smooth muscle cells of cerebral and mesenteric arteries from STZ, GK, and HFD rats and db/db mice [153,[204][205][206]. Human arteries from diabetic patients have higher Ca v 1.2 activity due to increased phosphorylation of α1C at Ser1928 by PKA [56]. Similar findings were observed in cerebral arteries of HFD mice [56]. These changes are simulated by in vitro acute exposure of rodent and human arteries to a high concentration of glucose [56,205]. A series of studies by Navedo's group reveal that the Gs-coupled purinergic receptor P2Y11, adenylyl cyclase 5 (AC5), PKA, AKAP150, and Ca v 1.2 form a signaling microdomain in vascular smooth muscle cells and these components are localized in nanometer proximity within the microdomain [56,58,161]. Apparently, elevated glucose stimulates Ca v 1.2 activity by promoting autocrine release of nucleotides, which sequentially activates P2Y11, AC5, and PKA leading to increased phosphorylation of the α1C subunit at Ser1928 [56,58,161].
Gal-1 is highly expressed in the placenta [207]. Circulating Gal-1 level also increases during gestation [208]. Given the critical role of Gal-1 in regulating Ca v 1.2 surface expression/activity discussed above, it is reasonable to speculate that the elevated Gal-1 in pregnancy may contribute to reduced uterine arterial myogenic tone by suppressing Ca v 1.2 surface expression/activity [209,210]. Intriguingly, both circulating and placental Gal-1 is downregulated in early onset preeclampsia and pregnant women with fetal growth restriction [211,212]. The downregulation of Gal-1 probably plays a role in the increased myogenic tone in uterine and other vascular beds in preeclampsia due to upregulating Ca v 1.2 surface expression/activity [165][166][167]179].

Overview of ROS
Reactive oxygen species (ROS) are oxygen-containing molecules naturally produced in cellular metabolism. ROS comprise free radicals such as superoxide anion (O 2 •− ) and hydroxyl radical ( • OH) and nonradical molecule hydrogen peroxide (H 2 O 2 ). O 2 •− is formed from the one-electron reduction of molecular oxygen (O 2 ). It is a precursor to a cascade of other ROS. Its dismutation, either occurring spontaneously or being catalyzed by superoxide dismutases (SODs), produces H 2 O 2 . Through the Fenton reaction, H 2 O 2 can be reduced to • OH. Moreover, the reaction between O 2 •− and nitric oxide (NO) results in the formation of peroxynitrite (ONOO − ).
ROS are produced in different cellular compartments including mitochondria, endoplasmic reticulum (ER), lysosomes, peroxisomes, and plasma membrane [23,213,214]. Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) and mitochondria are the major sources of ROS [215] (Figure 3). NOXs are a family of transmembrane proteins that catalyze the transfer of electron donated by NADPH to molecular oxygen to form O 2 •− [216]. NOXs comprise seven isoforms (NOX1-5 and dual oxidases (DUOX) 1 and 2). NOX1, NOX2, NOX4, and NOX5 are primary isoforms in vasculature [217]. During oxidative phosphorylation, ATP synthesis is coupled to the movement of electrons derived Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) and mitochondria are the major sources of ROS [215] (Figure 3). NOXs are a family of transmembrane proteins that catalyze the transfer of electron donated by NADPH to molecular oxygen to form O2 •− [216]. NOXs comprise seven isoforms (NOX1-5 and dual oxidases (DUOX) 1 and 2). NOX1, NOX2, NOX4, and NOX5 are primary isoforms in vasculature [217]. During oxidative phosphorylation, ATP synthesis is coupled to the movement of electrons derived from NADH and FADH2 through Complexes I to IV in the mitochondrial electron transport chain (ETC). Electrons are received by O2 at Complex IV to produce H2O. Mitochondria consumes ~90% cellular O2 [218]. It is estimated that ~1-2% of O2 consumed by mitochondria reacted with leaked electrons, leading to the formation of O2 •− [219]. Complexes I and III are the major sites in ETC to generate O2 •− [220]. ROS can be beneficial and detrimental. They are highly reactive. O2 •− and • OH are short-lived and exert their actions in their immediate vicinity. In contrast, H2O2 is stable and can travel a long distance in cells and pass through the membrane. While O2 •− and • OH nonspecifically act on macromolecules such as proteins, DNA, and lipids, H2O2 can trigger reversible oxidation of cysteine residues in specific proteins including enzymes, protein kinases, transcription factors, and ion channels, leading to altered protein functions [221,222]. Thus, H2O2 is considered as an intracellular signaling molecule at physiological concentrations [221,222]. However, H2O2 causes irreversible damage to macromolecules and impairs their functions at supraphysiological concentrations.
Cellular ROSs are tightly regulated under physiological conditions. Physiological ROS concentrations are maintained by various antioxidant mechanisms. O2 •− is converted into H2O2 by Cu, Zn-SOD (SOD1) in the cytosol/mitochondrial intermembrane space and Mn-SOD (SOD2) in the mitochondrial matrix. H2O2 is subsequently decomposed to H2O by catalase, glutathione peroxidases (GPXs) and peroxiredoxins (PRXs). ROS can also be scavenged by nonenzymatic antioxidants such as glutathione (GSH), vitamins C/E, uric  •− and • OH nonspecifically act on macromolecules such as proteins, DNA, and lipids, H 2 O 2 can trigger reversible oxidation of cysteine residues in specific proteins including enzymes, protein kinases, transcription factors, and ion channels, leading to altered protein functions [221,222]. Thus, H 2 O 2 is considered as an intracellular signaling molecule at physiological concentrations [221,222]. However, H 2 O 2 causes irreversible damage to macromolecules and impairs their functions at supraphysiological concentrations.
Cellular ROSs are tightly regulated under physiological conditions. Physiological ROS concentrations are maintained by various antioxidant mechanisms. O 2 •− is converted into H 2 O 2 by Cu, Zn-SOD (SOD1) in the cytosol/mitochondrial intermembrane space and Mn-SOD (SOD2) in the mitochondrial matrix. H 2 O 2 is subsequently decomposed to H 2 O by catalase, glutathione peroxidases (GPXs) and peroxiredoxins (PRXs). ROS can also be scavenged by nonenzymatic antioxidants such as glutathione (GSH), vitamins C/E, uric acid, and melatonin [223]. Oxidative stress occurs when the ROS homeostasis is disrupted due to ROS overproduction, or antioxidant deficiency, or both. Thus, oxidative stress represents a state of an imbalance between ROS production and antioxidant defenses.

Oxidative Stress as a Hallmark in Hypertension-Related Disorders
Hypertension, diabetes, and preeclampsia exhibit a state of oxidative stress [21][22][23].

Mitochondria-Derived ROS in Hypertension-Related Disorders
Mitochondria become dysfunctional in hypertension-related disorders. SIRT3, a histone deacetylase, is an important regulator of mitochondrial redox state. It interacts with SOD2 in mitochondria and subsequently promotes SOD2 deacetylation, leading to enhanced enzymatic activity [355,356]. Essential hypertension in human is associated with increased mitochondrial oxidative stress in arterioles from mediastinal fat due to reduced SIRT3 and increased SOD2 acetylation [357]. Similar to human essential hypertension, SHR also exhibits overproduction of mitochondrial ROS in vessels [358]. Partial deletion of SOD2 in mitochondria (SOD2 +/− ) increases renal oxidative stress and blood pressure [359]. In angiotensin II-infused mice, the increased blood pressure is associated with increased SIRT3 S-glutathionylation and vascular SOD2 acetylation, reduced SOD2 activity and elevated vascular O 2 •− production [357,360]. Those alterations are diminished by SIRT3 overexpression [357]. Cyclophilin D is a regulatory subunit of the mitochondrial permeability transition pore and participates in regulating mitochondrial function [361]. Evidently, it is an important mediator of angiotensin II-induced hypertension as its depletion diminishes both mitochondrial O 2 •− generation in aorta and hypertension [362]. Mitochondrial O 2 •− generation in DOCA rat mesenteric arteries is also increased [255]. In cultured HUVECs, excess aldosterone suppresses SOD2 expression and increases mitochondrial ROS production [363]. Interestingly, mitochondrial ROS is also regulated by a phenomenon termed ROS-induced ROS release [364]. In cultured human aortic endothelial cells, silence of NOX2 with siRNA reduces angiotensin II-induced mitochondrial O 2 •− generation [365]. Likewise, NOX2 deletion by gp91 phox knockout also reduces angiotensin II-induced mitochondrial O 2 •− generation in aorta and hypertension [365]. Elevation of mitochondrial O 2 •− is detected in subcutaneous arterioles in type 2 diabetic patients [366]. Likewise, mitochondrial H 2 O 2 is increased in primary human saphenous vein endothelial cells from type 2 diabetic subjects [367]. STZ mice/rats, HFD mice, ZDF rats, and GK rats display increased mitochondrial ROS in vascular smooth muscle cells and endothelial cells [331,[368][369][370][371][372]. Hyperglycemia is apparently a causative factor leading to heightened mitochondrial ROS in vascular cells. Exposure to high concentrations of glucose stimulates mitochondrial ROS production in a variety of endothelial cells, promoting mitochondrial damage by producing 8-hydroxydeoxyguanosine and nitrotyrosine [373][374][375][376][377][378][379][380][381].
Preeclampsia is associated with reduced mitochondrial content and decreased oxidative phosphorylation in the placenta [382,383]. Preeclamptic placenta also exhibits mitochondrial oxidative stress as evidenced by increased malondialdehyde and 4-HNEmodified proteins in mitochondria [384,385]. Mitochondrial SOD2 activity is reduced in preeclamptic placenta [309,386]. Mitochondrial ROS is also elevated in the kidney and placenta of RUPP rats [387]. Mitochondrial ROS in the placenta is raised by infusing/injecting with either of AT1-AA, sFlt-1, TNFα and ad-CMV-hypoxia-inducible factor (HIF) into pregnant rodents [302,[388][389][390]. In cultured endothelial cells, serum from women with preeclampsia suppresses mitochondrial respiration and boosts mitochondrial ROS generation [391][392][393]. In a way like preeclamptic serum, sFlt-1 also promotes mitochondrial oxidative stress in endothelial cells [393]. Serum from RUPP rats similarly stimulates mitochondrial ROS production in HUVECs, which is ablated by AT1-AA inhibition [387,388]. Hypoxic treatment reduces mitochondrial respiration in cultured trophoblast-like JEG3 cells and increases mitochondrial ROS in human placenta [394,395]. In vitro hypoxia also suppresses mitochondrial respiration and increases mitochondrial ROS in ovine uterine artery smooth muscle cells [396].

A Causative Role of ROS in in Animal Models of Hypertension-Related Disorders
Oxidative stress is believed to be linked to pathogenesis and progression of a myriad of human diseases including hypertension-related disorders [21,23,24,[397][398][399][400][401]. Numerous studies using animal models reveal a causative role of ROS in the pathogenesis of hypertension, diabetes, and preeclampsia.
Administration of mitochondria-targeted antioxidants have extensively explored in animal models of hypertension and preeclampsia. MitoQ decreases blood pressure in SP-SHR [418], whereas MitoEbselen reduces vascular ROS and prevents angiotensin II-induced hypertension in mice [360]. MitoQ and MitoTempol also reduce placental oxidative stress, lowers blood pressure, and improves fetal growth in RUPP rats and in pregnant rats experiencing gestational hypoxia [301,387,389]. Scavenging of mitochondrial isolevuglandins formed by peroxidation of arachidonic acid with Mito2HOBA and targeting cyclophilin D, a component of mitochondrial permeability transition pore, with its specific inhibitor sanglifehrin A reduce vascular mitochondrial ROS, improve vascular function, and attenuate hypertension in Ang II-infused mice [362,419]. These beneficial responses are replicated by overexpression of mitochondria-targeted SOD2 and catalase [362].

ROS and Ca v 1.2 Function/Expression
There is ample evidence that Ca v 1.2 is regulated by cellular redox state. The α1c subunit contains 48 cysteines with some of them being sensitive to redox modulation [420]. Redox modulation of sulfhydryl groups of cysteine residues could alter the structure and function of proteins. Site-directed mutagenesis has identified several cysteine residues including C543, C1789, C1790, and C1810 that subject to redox regulation [421,422]. However, electrophysiological studies reveal conflicting effects of ROS on Ca v 1.2. Studies from Amberg's group demonstrate that bath application of H 2 O 2 and ROS produced by xanthine oxidase/hypoxanthine stimulates Ca v 1.2 activity in rat cerebral arterial smooth muscle cells [423,424]. Both mitochondria-and NOX-derived ROS could exert stimulatory effects on Ca v 1.2. Antimycin enhances Ca v 1.2 activity by promoting mitochondrial H 2 O 2 generation. In addition, angiotensin II augments Ca v 1.2 activity by promoting mitochondria-and NOX-mediated H 2 O 2 production [423][424][425]. Rotenone also increases Ca v 1.2-mediated currents by stimulating mitochondrial O 2 •− production in A7r5 aortic smooth muscle cells [426]. In contrast, sulfhydryl-oxidizing agents 2,2 -dithiodipyridine (DTDP) and thimerosal inhibit heterologously expressed α1c subunit of rabbit smooth muscle Ca v 1.2 [427]. In rat tail arterial smooth muscle cells, 2,5-di-t-butyl-1,4-benzohydroquinone inhibits Ca v 1.2 via O 2 •− generation [428]. H 2 O 2 is found to inhibit Ca v 1.2 in A7r5 aortic smooth muscle cells [426]. The disunity in electrophysiological findings suggests complexity of ROS actions, possibly due to concentrations of ROS used, the expression systems, ROS compartmentation and absence/presence of channel subunits/partners.
The redox modulation of Ca v 1.2 has immense impacts on vascular function. H 2 O 2 is reported to trigger an increase in [Ca 2+ ] i and vasoconstriction in rat mesenteric arteries, rat coronary arteries, rat aorta, and canine cerebral arteries [429][430][431][432][433][434][435]. H 2 O 2 -induced increase in [Ca 2+ ] i and contractile response are inhibited by the removal of extracellular Ca 2+ and by Ca v 1.2 inhibitors nisoldipine, nifedipine, diltiazem, and verapamil, suggesting that H 2 O 2 activates Ca v 1.2 in vascular smooth muscle cells [429][430][431]434]. Likewise, an ONOO − -induced increase in [Ca 2+ ] i is inhibited by nifedipine in mesenteric arteriolar smooth muscle cells [436]. Thrombin stimulates anincrease of [Ca 2+ ] i via activating Ca v 1.2 in rat aortic smooth muscle cells, which is absent in NOX1 null cells [437]. As expected, vessels from SHR exhibits greater increase in [Ca 2+ ] i and vasoconstriction compared to WKY [431][432][433]435]. It should be noted that H 2 O 2 also functions as an EDHF in other vascular beds including mouse and human mesenteric arteries, mouse aorta, and in porcine coronary microvessels and causes vasorelaxation [438][439][440][441]. Depolarization with high concentrations of KCl causes vasoconstriction by activating Ca v 1.2 [442]. Paraquat and LY83583 enhance KCl-induced increase in [Ca 2+ ] i and vasoconstriction by increasing O 2 •− production in rat renal afferent arterioles, mesenteric arteries and intrapulmonary arteries [443,444]. Sphingosylphosphorylcholine also potentiates the contractile response to KCl in a NOX-dependent mechanism in rat mesenteric and intrapulmonary arteries [443].
NO is also a reactive, gaseous signaling molecule. S-nitrosylation, the covalent attachment of NO to a cysteine thiol in a given protein, is also an important redox signaling [445]. S-nitrosylation of Cys 1180 and/or Cys1280 in Ca v 1.2 reduces surface expression of the channel and channel activity [446]. In addition, S-nitrosylation of Ca v 1.2 also reduces channel open probability [447]. Excess ROS leads to low NO bioavailability in hypertension [448]. Intriguingly, Ca v 1.2 S-nitrosylation levels are reduced in aorta from SHR rats and in pulmonary arteries from patients with pulmonary hypertension [446]. Not surprisingly, increasing vascular s-nitrosylation levels of Ca v 1.2 by injecting S-nitrosocysteine reduces blood pressure in SHR rats [446].
ROS generated in different cellular compartments form microdomains and act on their targets in a spatiotemporal manner [449] (Figure 4). The redox regulation of Ca v 1.2 requires adjacent location of mitochondria and NOXs to Ca v 1.2 on the plasma membrane in vascular smooth muscle cells [423,425]. Thus, it is expected that microdomains with high concentra-tions of ROS exist in subcellular regions next to Ca v 1.2. Interestingly, redox modulation of Ca v 1.2 can occur directly or indirectly. In addition to directly targeting Ca v 1.2, ROS could oxidize partners of Ca v 1.2 to indirectly alter Ca v 1.2 activity. As aforementioned, PKC and c-Src form a partnership with Ca v 1.2 to regulate the activity of the channel. PKC contains six cysteine residues in each of the two pairs of zinc fingers within the regulatory domain and the oxidation of them activates PKCs [450,451]. H 2 O 2 has been shown to activate PKCs in vascular smooth muscle cells [452,453]. SH2 domain of c-Src also contains cysteine residues and oxidation of these residues promotes the formation of disulfide bridges that subsequently override the autoinhibitory effect of Tyr527, leading to enzymatic activation [454]. Vascular c-Src is activated by NOX4-and mitochondria-derived ROS [287,455]. ROS produced by both NOXs and mitochondria are found to activate adjoining PKCs, which in turn enhances Ca v 1.2 activity in vascular smooth muscle cells [423,425,426]. Not surprisingly, H 2 O 2 -induced vasocontraction is reduced by PKC/c-Src inhibition and by Ca v 1.2 blockade [430,434,435,456,457]. Similarly, the potentiation of KCl-induced vasoconstriction of rat mesenteric and intrapulmonary arteries by NOX-derived ROS is also sensitive to the inhibition of PKC/c-Src [443]. Calmodulin is a key Ca 2+ sensor for both Ca v 1.2 inactivation and facilitation [458]. Modulation of calmodulin by H 2 O 2 also augments cardiac Ca v 1.2 activity [459].
ROS generated in different cellular compartments form microdomains and act on their targets in a spatiotemporal manner [449] (Figure 4). The redox regulation of Cav1.2 requires adjacent location of mitochondria and NOXs to Cav1.2 on the plasma membrane in vascular smooth muscle cells [423,425]. Thus, it is expected that microdomains with high concentrations of ROS exist in subcellular regions next to Cav1.2. Interestingly, redox modulation of Cav1.2 can occur directly or indirectly. In addition to directly targeting Cav1.2, ROS could oxidize partners of Cav1.2 to indirectly alter Cav1.2 activity. As aforementioned, PKC and c-Src form a partnership with Cav1.2 to regulate the activity of the channel. PKC contains six cysteine residues in each of the two pairs of zinc fingers within the regulatory domain and the oxidation of them activates PKCs [450,451]. H2O2 has been shown to activate PKCs in vascular smooth muscle cells [452,453]. SH2 domain of c-Src also contains cysteine residues and oxidation of these residues promotes the formation of disulfide bridges that subsequently override the autoinhibitory effect of Tyr527, leading to enzymatic activation [454]. Vascular c-Src is activated by NOX4-and mitochondriaderived ROS [287,455]. ROS produced by both NOXs and mitochondria are found to activate adjoining PKCs, which in turn enhances Cav1.2 activity in vascular smooth muscle cells [423,425,426]. Not surprisingly, H2O2-induced vasocontraction is reduced by PKC/c-Src inhibition and by Cav1.2 blockade [430,434,435,456,457]. Similarly, the potentiation of KCl-induced vasoconstriction of rat mesenteric and intrapulmonary arteries by NOX-derived ROS is also sensitive to the inhibition of PKC/c-Src [443]. Calmodulin is a key Ca 2+ sensor for both Cav1.2 inactivation and facilitation [458]. Modulation of calmodulin by H2O2 also augments cardiac Cav1.2 activity [459]. Remarkably, ROS also regulate gene expression [460,461]. There is a concurring decrease in both vascular ROS and Cav1.2 expression in aortas of aged mice lacking the mineralocorticoid receptor in smooth muscle cells [142]. The protein expression of the α1C subunit is increased by angiotensin II in cultured rat mesenteric arteries [462,463]. H2O2 mimics, while NOX inhibition by apocynin, diphenyleneiodonium and gp91ds-tat as well Remarkably, ROS also regulate gene expression [460,461]. There is a concurring decrease in both vascular ROS and Ca v 1.2 expression in aortas of aged mice lacking the mineralocorticoid receptor in smooth muscle cells [142]. The protein expression of the α1C subunit is increased by angiotensin II in cultured rat mesenteric arteries [462,463]. H 2 O 2 mimics, while NOX inhibition by apocynin, diphenyleneiodonium and gp91ds-tat as well as catalase annuls angiotensin II-mediated upregulation of the α1C subunit [463]. In a cardiac muscle cell line HL-1 cells, angiotensin II upregulates the α1C subunit at both mRNA and protein levels through the NOX-PKC pathway-mediated cAMP response element binding protein (CREB) phosphorylation [464]. It is expected that this mechanism could also play a role in secondary hypertension as hypertension due to Cushing's syndrome, hyperaldosteronism, and renovascular disease involves activation of the renin-angiotensin system [231,256,[465][466][467][468][469]. In rat cerebral arteries, endothelin 1 promotes mitochondrial ROS generation which subsequently stimulates the expression of the α1C subunit by activating nuclear factor kappa B (NF-κB) [470]. Consequently, the upregulation of Ca v 1.2 induced by endothelin 1 increases both myogenic tone and depolarization-induced vasoconstriction.

ROS and Myogenic Tone
Exogenous ROSs have been shown to alter myogenic tone. In pressurized mouse tail arterioles and rat gracilis skeletal muscle arterioles, H 2 O 2 causes vasoconstriction [471,472]. The exposure of rat cerebral arteries to the ROS-generating system xanthine oxidase plus hypoxanthine also increases myogenic tone [423]. Likewise, O 2 •− generated by paraquat enhances myogenic tone in mouse afferent arterioles [473]. • OH generated by the Fenton reaction from H 2 O 2 and the iron redox chelate Fe 3+ /nitrilotriacetate (FeNTA) elevates myogenic tone in denuded rat ophthalmic arteries [474].
Numerous studies reveal that endogenous ROS plays a key role in intralumenal pressure-induced myogenic tone. Myogenic tone in mesenteric arteries from SOD1 knockout mice is increased [475]. ROS derived from both NOXs and mitochondrial contribute to myogenic tone development. Pressurization of rat femoral arterial branches increases O 2 •− via activating NOXs [476]. Similarly, an increase in intralumenal pressure promotes ROS in vascular smooth muscle cells and myogenic constriction simultaneously in mouse tail arterioles, which are inhibited by diphenyleneiodonium/catalase and are absent in arterioles from p47 phox−/− mice or transgenic mice expressing a dominant-negative mutant of human rac1 (rac-DN) [471]. Likewise, pressure-dependent myogenic tone enhanced in mouse afferent arterioles is repressed in vessels from p47 phox−/− mice and by NOX inhibitors apocynin and diphenyleneiodonium [477,478]. The regulation of myogenic tone by ROS is tissue-, ROS type-and ROS source-dependent. H 2 O 2 , but not O 2 •− , contributes to myogenic constrictor response in rat tail arterioles as the vasoconstriction is diminished by catalase, but not by manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a cell-permeant mimic of SOD [471]. However, myogenic tone is reduced by pegylated SOD, but not by pegylated catalase in mouse afferent arterioles [477]. Selective inhibition of both NOX1 and NOX2 with 2-acetylphenothiazine (2-APT) and VAS2870, respectively, suppressed basal myogenic tone in rat cremaster skeletal muscle arterioles [479], whereas myogenic tone is inhibited by NOX1 inhibitor ML171 but not by NOX2 inhibitor gp91-dstat in rat cerebral arteries [480]. The concomitant increases in superoxide/H 2 O 2 and myogenic constriction induced by pressure is also reduced by MitoQ in rat cerebral arteries [481].
As expected, both O 2 •− and myogenic tone are increased in vessels from animal models of hypertension-related disorders including SHR cerebral arteries/afferent arterioles, OZR gracilis skeletal muscle arterioles/cerebral arteries, and db/db mouse mesenteric arteries [131,138,[482][483][484][485]. The increased myogenic constriction of mesenteric arteries of db/db mice is reduced by supplement of GSH or the Nrf2-activator sulforaphane [485]. A reduction in myogenic tone is also achieved by scavenging ROS in SHR afferent arterioles, db/db gracilis skeletal muscle arterioles, and OZR cerebral arteries/gracilis skeletal muscle arterioles [131,281,[482][483][484]. Aging is associated with increased prevalence of hypertension and increases in intralumenal pressure promote mitochondrial oxidative stress in aged mouse cerebral arteries [486]. The exacerbated myogenic tone of uterine arteries from highaltitude pregnant sheep concurs with increased expression of NOX2 and mitochondrial ROS, and pressure-induced myogenic tone in uterine arteries from high-altitude pregnant sheep is diminished by apocynin/mitoQ, whereas it is enhanced in uterine arteries from low-altitude pregnant sheep by mitochondrial respiratory chain inhibitors rotenone and antimycin [299,396].
Numerous studies demonstrate that PKC/c-Src activation contributes to myogenic tone development in small arteries/arterioles [136, [487][488][489][490][491]. The expression/activity of vascular PKC/c-Src is increased in SHR and STZ rats as well as in uterine arteries of highaltitude pregnant sheep [179,435,492,493]. PKC/c-Src activation has a greater contribution to myogenic tone in SHR cerebral arteries, STZ rat gracilis skeletal muscle arterioles, and high-altitude sheep uterine arteries [136,159,179]. Notably, the increased myogenic constriction by ROS in rat cerebral arteries is mediated by ROS-mediated PKC/c-Src activation [423,473,494]. Moreover, PKC/c-Src activation in turn stimulates Ca v 1.2 activity to promote myogenic constriction [86,112,473]. Nevertheless, both PKCs and c-Src can also contribute to myogenic tone development by increasing the sensitization of the contractile apparatus to Ca 2+ [112,136].

Conclusions/Perspective
Evidently, Ca 2+ influx through Ca v 1.2 is fundamental to vasoconstriction and myogenic tone of small arteries and arterioles. Antihypertensive drugs including Ca v 1.2 blockers have been successfully used to the management of hypertension-related disorders [495][496][497][498]. Oxidative stress is a hallmark of hypertension-related disorders. However, it is still an open question whether oxidative stress is a cause or consequence of hypertension. The data reviewed here highlights critical roles of vascular oxidative stress in the pathophysiology of hypertension-related disorders. Vascular Ca v 1.2 is targeted by excessive ROS directly and indirectly leading to exaggerated channel expression/activity. The dysfunction of Ca v 1.2 ultimately results in increased myogenic tone and elevated blood pressure. In preclinical studies, ROS-induced myogenic tone is diminished by Ca v 1.2 blocker nifedipine [473]. Moreover, apocynin is as effective as nifedipine in lowering blood pressure in an animal model of hypertension [411]. Regulation of Ca v 1.2 by ROS primarily occurs in ROS microdomains [423,425]. In this context, selectively combating compartmentalized ROS could also be a viable therapeutic approach in the treatment of hypertension. Animal studies have provided compelling evidence supporting a causative role of ROS in the pathogenesis of hypertension-related disorders. However, clinical trials on humans provide less convincing evidence [23,499]. Although animal studies have significantly contributed to our understanding mechanisms underlying hypertension-related disorders, further work is needed to translate findings from animal research to clinical benefit.