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ABSTRACT: Estrogen receptors (ERs) mediate genomic and nongenomic vasodilator effects, but estrogen therapy may not provide systemic vascular protection. To test whether this is due to regional differences in ER distribution or vasodilator activity, cephalic (carotid), thoracic (thoracic aorta, pulmonary) and abdominal arteries (abdominal aorta, mesenteric, renal) from female Sprague-Dawley rats were prepared to measure contraction to phenylephrine (Phe), and relaxation to acetylcholine (ACh) and the ER activators 17β-estradiol (E2) (all ERs), PPT (ERα), DPN (ERβ) and G1 (GPR30). Phe caused contraction that was enhanced in endothelium-denuded aorta, supporting endothelial release of vasodilators. In cephalic and thoracic arteries, ACh relaxation was abolished by the NOS inhibitor L-NAME, suggesting a role of NO. In mesenteric vessels, ACh-induced relaxation was partly inhibited by L-NAME+COX inhibitor indomethacin and blocked by the K channel blocker tetraethylammonium (TEA), suggesting a hyperpolarization pathway. E2 and PPT caused similar relaxation in all vessels. DPN and G1 caused smaller relaxation that was more prominent in abdominal vessels. RT-PCR revealed variable ERα mRNA expression, and increased ERβ in carotid artery and GPR30 in abdominal arteries. Western blots revealed greater amounts of ERα, ERβ and GPR30 in abdominal arteries. In thoracic aorta, E2, PPT and DPN-induced relaxation was blocked by L-NAME, and was associated with increased nitrite/nitrate production, suggesting a role of NO. In abdominal vessels, E2, PPT, DPN and G1-induced relaxation persisted in L-NAME+indomethacin+TEA-treated or endothelium-denuded arteries, suggesting direct effect on vascular smooth muscle (VSM). E2, PPT, DPN, and G1 caused greater relaxation of KCl-induced contraction in abdominal vessels, suggesting inhibitory effects on Ca entry. Thus, E2 and ERα stimulation produce similar relaxation of the cephalic, thoracic and abdominal arteries. In the cephalic and thoracic arteries, particularly the thoracic aorta, E2-induced and ERα- and ERβ-mediated vasodilation involve NO production. ERβ- and GPR30-mediated relaxation is greater in the abdominal arteries, and appears to involve hyperpolarization and inhibition of VSM Ca entry. Specific ER agonists could produce vasodilation in specific vascular beds without affecting other vessels in systemic circulation.
Journal of cardiovascular pharmacology 02/2013; · 2.83 Impact Factor
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ABSTRACT: Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.
AJP Endocrinology and Metabolism 04/2012; 303(1):E55-70. · 4.75 Impact Factor
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ABSTRACT: Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.
AJP Lung Cellular and Molecular Physiology 01/2012; 302(9):L875-90. · 3.66 Impact Factor
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ABSTRACT: The vascular endothelium plays a major role in the control of arterial tone; however, its role in venous tissues is less clear. The purpose of this study was to determine the role of endothelium in the control of venous function and the relaxation pathways involved.
Circular segments of inferior vena cava (IVC) from male Sprague-Dawley rats were suspended between two wires and isometric contraction to phenylephrine (Phe; 10(-5)M) and 96 mM KCl was measured. Acetylcholine (Ach; 10(-10) to 10(-5)M) was added and the percentage of venous relaxation was measured. To determine the role of nitric oxide (NO) and prostacyclin (PGI(2)), vein relaxation was measured in the presence of the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME; 3 × 10(-4) M) and the cyclooxygenase inhibitor indomethacin (10(-5) M). To measure the role of hyperpolarization, vein relaxation was measured in the presence of K(+) channel activator cromakalim (10(-11) to 10(-6) M), and the nonselective K(+) channel blocker tetraethylammonium (TEA; 10(-3) M). To test for the contribution of a specific K(+) channel, the effects of K(+) channel blockers: glibenclamide (adenosine triphosphate [ATP]-sensitive K(ATP), 10(-5) M), 4-aminopyridine (4-AP; voltage-dependent K(v), 10(-3) M), apamin (small conductance Ca(2+)-dependent SK(Ca), 10(-7) M), and iberiotoxin (large conductance Ca(2+)-dependent BK(Ca), 10(-8) M) on Ach-induced relaxation were tested.
Ach caused concentration-dependent relaxation of Phe contraction (maximum 49.9 ± 4.9%). Removal of endothelium abolished Ach-induced relaxation. IVC treatment with L-NAME partially reduced Ach relaxation (32.8 ± 4.9%). In IVC treated with L-NAME plus indomethacin, significant Ach-induced relaxation (33.6 ± 3.2%) could still be observed, suggesting a role of endothelium-derived hyperpolarizing factor (EDHF). In IVC treated with L-NAME, indomethacin and TEA, Ach relaxation was abolished, supporting a role of EDHF. In veins stimulated with high KCl, Ach caused relaxation (maximum 59.5 ± 3.5%) that was abolished in the presence of L-NAME and indomethacin suggesting that any Ach-induced EDHF is blocked in the presence of high KCl depolarizing solution, which does not favor outward movement of K(+) ion and membrane hyperpolarization. Cromakalim, an activator of K(ATP), caused significant IVC relaxation when applied alone or on top of maximal Ach-induced relaxation, suggesting that the Ach response may not involve K(ATP). Ach-induced relaxation was not inhibited by glibenclamide, 4-AP, or apamin, suggesting little role of K(ATP), K(v) or SK(Ca), respectively. In contrast, iberiotoxin significantly inhibited Ach-induced relaxation, suggesting a role of BK(Ca).
Thus, endothelium-dependent venous relaxation plays a major role in the control of venous function. In addition to NO, an EDHF pathway involving BK(Ca) may play a role in endothelium-dependent venous relaxation.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 12/2011; 55(6):1716-25. · 3.52 Impact Factor
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ABSTRACT: Phytoestrogens are estrogenic compounds of plant origin classified into different groups including isoflavones, lignans, coumestans and stilbenes. Isoflavones such as genistein and daidzein are the most studied and most potent phytoestrogens, and are found mainly in soy based foods. The effects of phytoestrogens are partly mediated via estrogen receptors (ERs): ERα, ERβ and possibly GPER. The interaction of phytoestrogens with ERs is thought to induce both genomic and non-genomic effects in many tissues including the vasculature. Some phytoestrogens such as genistein have additional non-ER-mediated effects involving signaling pathways such as tyrosine kinase. Experimental studies have shown beneficial effects of phytoestrogens on endothelial cells, vascular smooth muscle, and extracellular matrix. Phytoestrogens may also affect other pathophysiologic vascular processes such as lipid profile, angiogenesis, inflammation, tissue damage by reactive oxygen species, and these effects could delay the progression of atherosclerosis. As recent clinical trials showed no vascular benefits or even increased risk of cardiovascular disease (CVD) and CV events with conventional menopausal hormone therapy (MHT), phytoestrogens are being considered as alternatives to pharmacologic MHT. Epidemiological studies in the Far East population suggest that dietary intake of phytoestrogens may contribute to the decreased incidence of postmenopausal CVD and thromboembolic events. Also, the WHO-CARDIAC study supported that consumption of high soybean diet is associated with lower mortalities from coronary artery disease. However, as with estrogen, there has been some discrepancy between the experimental studies demonstrating the vascular benefits of phytoestrogens and the data from clinical trials. This is likely because the phytoestrogens clinical trials have been limited in many aspects including the number of participants enrolled, the clinical end points investigated, and the lack of long-term follow-up. Further investigation of the cellular mechanisms underlying the vascular effects of phytoestrogens and careful evaluation of the epidemiological evidence and clinical trials of their potential vascular benefits would put forward the use of phytoestrogens as an alternative MHT for the relief of menopausal symptoms and amelioration of postmenopausal CVD.
Mini Reviews in Medicinal Chemistry 10/2011; 12(2):149-74. · 2.53 Impact Factor
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Luminita H Pojoga,
Jonathan S Williams,
Tham M Yao,
Abhinav Kumar,
Joseph D Raffetto,
Graciliano R A do Nascimento,
Ossama M Reslan,
Gail K Adler,
Gordon H Williams,
Yujiang Shi, Raouf A Khalil
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ABSTRACT: Histone methylation, a determinant of chromatin structure and gene transcription, was thought to be irreversible, but recent evidence suggests that lysine-specific demethylase-1 (LSD1, Kdm1a) induces demethylation of histone H3 lysine 4 (H3K4) or H3K9 and thereby alters gene transcription. We previously demonstrated a human LSD1 phenotype associated with salt-sensitive hypertension. To test the hypothesis that LSD1 plays a role in the regulation of blood pressure (BP) via vascular mechanisms and gene transcription, we measured BP and examined vascular function and endothelial nitric oxide (NO) synthase (eNOS) expression in thoracic aorta of male wild-type (WT) and heterozygous LSD1 knockout mice (LSD1(+/-)) fed either a liberal salt (HS; 4% NaCl) or restricted salt diet (LS; 0.08% NaCl). BP was higher in LSD1(+/-) than WT mice on the HS diet but not different between LSD1(+/-) and WT mice on the LS diet. Further examination of the mechanisms of this salt-sensitive hypertension in LSD1(+/-) mice on the HS diet demonstrated that plasma renin activity and plasma levels and urinary excretion of aldosterone were less in LSD1(+/-) than WT, suggesting suppressed renin-angiotensin-aldosterone system. In contrast, phenylephrine (Phe)-induced aortic contraction was greater in LSD1(+/-) than WT mice on the HS diet. Treatment of aortic rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a blocker of guanylate cyclase) enhanced Phe contraction in LSD1(+/-) compared with WT mice on the HS diet. Acetylcholine (Ach)-induced relaxation was less in LSD1(+/-) than WT mice on the HS diet. Endothelium removal or pretreatment with N(ω)-nitro-L-arginine methyl ester (blocker of NOS) or ODQ abolished Ach-induced relaxation in aorta of WT but had minimal effect in LSD1(+/-). Vascular relaxation to sodium nitroprusside, an exogenous NO donor and guanylate cyclase activator, was decreased in LSD1(+/-) vs. WT mice on the HS diet. RT-PCR and Western blots revealed decreased eNOS mRNA expression and eNOS and guanylate cyclase protein in the heart and aorta of LSD1(+/-) compared with WT mice on HS diet. Thus, during the HS diet, LSD1 deficiency is associated with hypertension, enhanced vascular contraction, and reduced relaxation via NO-cGMP pathway. The data support a role for LSD1-mediated histone demethylation in the regulation of NOS/guanylate cyclase gene expression, vascular function, and BP during the HS diet.
AJP Heart and Circulatory Physiology 08/2011; 301(5):H1862-71. · 3.71 Impact Factor
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ABSTRACT: Cardiovascular disease (CVD) is more common in men and postmenopausal women (Post-MW) than premenopausal women (Pre-MW). Despite recent advances in preventive measures, the incidence of CVD in women has shown a rise that matched the increase in the Post-MW population. The increased incidence of CVD in Post-MW has been related to the decline in estrogen levels, and hence suggested vascular benefits of endogenous estrogen. Experimental studies have identified estrogen receptor ERα, ERβ and a novel estrogen binding membrane protein GPR30 (GPER) in blood vessels of humans and experimental animals. The interaction of estrogen with vascular ERs mediates both genomic and non-genomic effects. Estrogen promotes endothelium-dependent relaxation by increasing nitric oxide, prostacyclin, and hyperpolarizing factor. Estrogen also inhibits the mechanisms of vascular smooth muscle (VSM) contraction including [Ca2+]i, protein kinase C and Rho-kinase. Additional effects of estrogen on the vascular cytoskeleton, extracellular matrix, lipid profile and the vascular inflammatory response have been reported. In addition to the experimental evidence in animal models and vascular cells, initial observational studies in women using menopausal hormonal therapy (MHT) have suggested that estrogen may protect against CVD. However, randomized clinical trials (RCTs) such as the Heart and Estrogen/ progestin Replacement Study (HERS) and the Women's Health Initiative (WHI), which examined the effects of conjugated equine estrogens (CEE) in older women with established CVD (HERS) or without overt CVD (WHI), failed to demonstrate protective vascular effects of estrogen treatment. Despite the initial set-back from the results of MHT RCTs, growing evidence now supports the 'timing hypothesis', which suggests that MHT could increase the risk of CVD if started late after menopause, but may produce beneficial cardiovascular effects in younger women during the perimenopausal period. The choice of an appropriate MHT dose, route of administration, and estrogen/progestin combination could maximize the vascular benefits of MHT and minimize other adverse effects, especially if given within a reasonably short time after menopause to women that seek MHT for the relief of menopausal symptoms.
08/2011; 7(1):47-70. · 1.07 Impact Factor
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Richard Bafford,
Xin Xin Sui,
Min Park,
Takuya Miyahara,
Brenna G Newfell,
Iris Z Jaffe,
Jose R Romero,
Gail K Adler,
Gordon H Williams, Raouf A Khalil,
Michael S Conte
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ABSTRACT: Experimental studies have suggested a role for the local renin-angiotensin-aldosterone system in the response to vascular injury. Clinical data support that aldosterone, via activation of the mineralocorticoid receptor (MR), is an important mediator of vascular damage in humans with cardiovascular disease. In mineralocorticoid-sensitive target tissue, aldosterone specificity for MR is conferred enzymatically by the cortisol-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11βHSD2). However, the role of MR/aldosterone signaling in the venous system has not been explored. We hypothesized that MR expression and signaling in venous smooth muscle cells contributes to the arterialization of venous conduits and the injury response in vein bypass grafts. MR immunostaining was observed in all samples of excised human peripheral vein graft lesions and in explanted experimental rabbit carotid interposition vein grafts, with minimal staining in control greater saphenous vein. We also found upregulated transcriptional expression of both MR and 11βHSD2 in human vein graft and rabbit vein graft, whereas control greater saphenous vein expressed minimal MR and no detectable 11βHSD2. The expression of MR and 11βHSD2 was confirmed in cultured human saphenous venous smooth muscle cells (hSVSMCs). Using an adenovirus containing a MR response element-driven reporter gene, we demonstrate that MR in hSVSMCs is capable of mediating aldosterone-induced gene activation. The functional significance for MR signaling in hSVSMCs is supported by the aldosterone-induced increase of angiotensin II type-1 receptor gene expression that was inhibited by the MR antagonist spironolactone. The upregulation of MR and 11βHSD2 suggests that aldosterone-mediated tissue injury plays a role in vein graft arterialization.
AJP Heart and Circulatory Physiology 05/2011; 301(1):H41-7. · 3.71 Impact Factor
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ABSTRACT: Saponosides (horse chestnut seed extract, escin) and flavonoids (diosmin, Daflon 500, Servier, France) exhibit venotonic properties that have been utilized in treatment of varicose veins. However, the cellular mechanisms underlying the venotonic properties of escin and diosmin are unclear. Because Ca(2+) is a major regulator of venous smooth muscle (VSM) function, we tested the hypothesis that escin and diosmin promote Ca(2+)-dependent venous contraction.
Rings of inferior vena cava (IVC) from male rats were suspended in a tissue bath for measurement of isometric contraction. Following control contraction to 96 mM KCl, the effects of escin and diosmin (10(-10) to 10(-4) M) on vein contraction were measured. To test the role of intracellular Ca(2+) release, the vein response to escin and diosmin was measured in Ca(2+)-free (2mM EGTA) Krebs. To test for Ca(2+)-dependent effects, IVC segments were pretreated with escin or diosmin (10(-4) M) in 0 Ca(2+) Krebs, then extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added and the [Ca(2+)](e)-contraction relationship was constructed. To test for synergistic effects of diosmin, IVC segments were pretreated with diosmin (10(-4) M), then stimulated with KCl (16-96 mM) or escin (10(-10) to 10(-4) M) and vein contraction was measured. Contraction data were presented as mg/mg tissue (means ± SEM).
In IVC segments incubated in normal Krebs (2.5 mM Ca(2+)), escin caused concentration-dependent contraction (max 104.3 ± 19.6 at 10(-4) M). Escin-induced contraction was not a rigor state, because after washing with Krebs, the veins returned to a relaxed state. In Ca(2+)-free Krebs, there was essentially no contraction to escin. In escin-treated veins incubated in 0 Ca(2+) Krebs, stepwise addition of extracellular CaCl(2) caused corresponding increases in contraction (max 80.0 ± 11.1 at 2.5 mM). In the absence of escin, the α-adrenergic agonist phenylephrine (PHE, 10(-5) M), angiotensin II (AngII, 10(-6) M), and membrane depolarization by KCl (96 mM) caused significant contraction (122.5 ± 45.1, 114.2 ± 12.2 and 221.7 ± 35.4, respectively). In IVC segments pretreated with escin (10(-4) M), the contractile response to PHE (9.7 ± 2.6), AngII (36.0 ± 9.1), and KCl (82.3 ± 10.2) was significantly reduced. Diosmin (10(-4) M) caused small contractions in normal Krebs (11.7 ± 1.9) and Ca(2+)-free Krebs (4.2 ± 2.2). In diosmin-treated veins incubated in 0 Ca(2+) Krebs, addition of extracellular CaCl(2) caused minimal contraction. Diosmin did not enhance the IVC contraction to PHE, AngII, or escin, but enhanced the contractile response to KCl (24-51 mM).
In rat IVC, escin induces extracellular Ca(2+)-dependent contraction, but disrupts α-adrenergic and AT(1)R receptor-mediated pathways and depolarization-induced contraction. The initial venotonic benefits of escin may be offset by disruption of vein response to endogenous venoconstrictors, limiting its long-term therapeutic benefits in varicose veins. Diosmin does not cause venous contraction or potentiate the venotonic effects of endogenous venoconstrictors or escin ex vivo, and its use as venotonic may need to be further evaluated.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 04/2011; 54(2):489-96. · 3.52 Impact Factor
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ABSTRACT: Decreased venous tone and vein wall dilation may contribute to varicose vein formation. We have shown that prolonged vein wall stretch is associated with upregulation of matrix metalloproteases (MMPs) and decreased contraction. Because hypoxia-inducible factors (HIFs) expression also increases with mechanical stretch, this study tested whether upregulation of HIFs is an intermediary mechanism linking prolonged vein wall stretch to the changes in MMP expression and venous contraction.
Segments of rat inferior vena cava (IVC) were suspended in tissue bath under 0.5-g basal tension for 1 hour, and a control contraction to phenylephrine (PHE, 10(-5)M) and KCl (96 mM) was elicited. The veins were then exposed to prolonged 18 hours of tension at 0.5 g, 2 g, 2 g plus HIF inhibitor U0126 (10(-5)M), 17-[2-(dimethylamino)ethyl] amino-17-desmethoxygeldanamycin (17-DMAG, 10(-5)M), or echinomycin (10(-6)M), or 2 g plus dimethyloxallyl glycine (DMOG; 10(-4)M), a prolyl-hydroxylase inhibitor that stabilizes HIF. The fold-change in PHE and KCl contraction was compared with the control contraction at 0.5-g tension for 1 hour. Vein tissue homogenates were analyzed for HIF-1α, HIF-2α, MMP-2, and MMP-9 messenger RNA (mRNA) and protein amount using real-time reverse transcription polymerase chain reaction and Western blots.
Compared with control IVC contraction at 0.5-g tension for 1 hour, the PHE and KCl contraction after prolonged 0.5-g tension was 2.0 ± 0.35 and 1.1 ± 0.06, respectively. Vein contraction to PHE and KCl after prolonged 2-g tension was significantly reduced (0.87 ± 0.13 and 0.72 ± 0.05, respectively). PHE-induced contraction was restored in IVC exposed to prolonged 2-g tension plus the HIF inhibitor U0126 (1.38 ± 0.15) or echinomycin (1.99 ± 0.40). U0126 and echinomycin also restored KCl-induced contraction in IVC exposed to prolonged 2-g tension (1.14 ± 0.05 and 1.11 ± 0.15, respectively). Treatment with DMOG further reduced PHE- and KCl-induced contraction in veins subjected to prolonged 2-g tension (0.47 ± 0.06 and 0.57 ± 0.01, respectively). HIF-1α and HIF-2α mRNA were overexpressed in IVC exposed to prolonged 2-g tension, and the overexpression was reversed by U0126. The overexpression of HIF-1α and HIF-2α in stretched IVC was associated with increased MMP-2 and MMP-9 mRNA. The protein amount of HIF-1α, HIF-2α, MMP-2, and MMP-9 was also increased in IVC exposed to prolonged 2-g wall tension.
Prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression, and reduced venous contraction in rat IVC. Together with our report that MMP-2 and MMP-9 inhibit IVC contraction, the data suggest that increased vein wall tension induces HIF overexpression and causes an increase in MMP expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 03/2011; 53(3):764-73. · 3.52 Impact Factor
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Raouf A Khalil
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ABSTRACT: Endothelin (ET) is one of the most investigated molecules in vascular biology. Since its discovery two decades ago, several ET isoforms, receptors, signaling pathways, agonists and antagonists have been identified. ET functions as a potent endothelium-derived vasoconstrictor, but could also play a role in vascular relaxation. In endothelial cells, preproET and big ET are cleaved by ET converting enzymes into ET-1, -2, -3 and -4. These ET isoforms bind with different affinities to ET(A) and ET(B) receptors in vascular smooth muscle (VSM), and in turn increase [Ca(2+)](i), protein kinase C and mitogen-activated protein kinase and other signaling pathways of VSM contraction and cell proliferation. ET also binds to endothelial ET(B) receptors and stimulates the release of nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. ET, via endothelial ET(B) receptor, could also promote ET re-uptake and clearance. While the effects of ET on vascular reactivity and growth have been thoroughly examined, its role in the regulation of blood pressure and the pathogenesis of hypertension is not clearly established. Elevated plasma and vascular tissue levels of ET have been identified in salt-sensitive hypertension and in moderate to severe hypertension, and ET receptor antagonists have been shown to reduce blood pressure to variable extents in these forms of hypertension. The development of new pharmacological and genetic tools could lead to more effective and specific modulators of the vascular ET system for treatment of hypertension and related cardiovascular disease.
Current Molecular Pharmacology 01/2011; 4(3):176-86.
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ABSTRACT: Normal pregnancy is associated with significant hemodynamic changes and vasodilation of the uterine and systemic circulation in order to meet the metabolic demands of the mother and developing fetus. Preeclampsia (PE) is one of the foremost complications of pregnancy and a major cause of maternal and fetal mortality. The pathophysiological mechanisms of PE have been elusive, but some parts of the puzzle have begun to unravel. Genetic factors such as leptin gene polymorphism, environmental and dietary factors such as Ca(2+) and vitamin D deficiency, and co-morbidities such as obesity and diabetes may increase the susceptibility of pregnant women to develop PE. An altered maternal immune response may also play a role in the development of PE. Although the pathophysiology of PE is unclear, most studies have implicated inadequate invasion of cytotrophoblasts into the uterine artery, leading to reduced uteroplacental perfusion pressure (RUPP) and placental ischemia/hypoxia. Placental ischemia induces the release of biologically active factors such as growth factor inhibitors, anti-angiogenic factors, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and antibodies to vascular angiotensin II (AngII) receptor. These bioactive factors could cause vascular endotheliosis and consequent increase in vascular resistance and blood pressure, as well as glomerular endotheliosis with consequent proteinuria. The PE-associated vascular endotheliosis could be manifested as decreased vasodilator mediators such as nitric oxide, prostacyclin and hyperpolarizing factor and increased vasoconstrictor mediators such as endothelin-1, AngII and thromboxane A₂. PE could also involve enhanced mechanisms of vascular smooth muscle contraction including intracellular Ca(2+), and Ca(2+) sensitization pathways such as protein kinase C and Rho-kinase. PE-associated changes in the extracellular matrix composition and matrix metalloproteinases activity also promote vascular remodeling and further vasoconstriction in the uterine and systemic circulation. Some of these biologically active factors and vascular mediators have been proposed as biomarkers for early prediction or diagnosis of PE, and as potential targets for prevention or treatment of the disease.
Cardiovascular & hematological agents in medicinal chemistry 10/2010; 8(4):204-26.
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ABSTRACT: Epidemiological studies have shown that cardiovascular disease (CVD) is less common in pre-menopausal women (Pre-MW) compared to men of the same age or post-menopausal women (Post-MW), suggesting cardiovascular benefits of estrogen. Estrogen receptors (ERs) have been identified in the vasculature, and experimental studies have demonstrated vasodilator effects of estrogen/ER on the endothelium, vascular smooth muscle (VSM) and extracellular matrix. Several natural and synthetic estrogenic preparations have been developed for relief of menopausal vasomotor symptoms. However, whether menopausal hormone therapy (MHT) is beneficial in postmenopausal CVD remains controversial. Despite reports of vascular benefits of MHT from observational and experimental studies, randomized clinical trials (RCTs), such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women's Health Initiative (WHI), have suggested that, contrary to expectations, MHT may increase the risk of CVD. These discrepancies could be due to agerelated changes in sex hormone synthesis and metabolism, which would influence the effective dose of MHT and the sex hormone environment in Post-MW. Age-related changes in the vascular ER subtype, structure, expression, distribution, and post-ER signaling pathways in the endothelium and VSM, along with factors related to the design of RCTs, preexisting CVD condition, and structural changes in the blood vessels architecture have also been suggested as possible causes of MHT failure in CVD. Careful examination of these factors should help in identifying the causes of the changes in the vascular effects of estrogen with age. The sex hormone metabolic pathways, the active versus inactive estrogen metabolites, and their effects on vascular function, the mitochondria, the inflammatory process and angiogenesis should be further examined. Also, the genomic and non-genomic effects of estrogenic compounds should be viewed as integrated rather than discrete responses. The complex interactions between these factors highlight the importance of careful design of MHT RCTs, and the need of a more customized approach for each individual patient in order to enhance the vascular benefits of MHT in postmenopausal CVD.
Current Drug Metabolism 10/2010; 11(8):693-714. · 5.11 Impact Factor
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Journal of cardiovascular pharmacology 09/2010; 56(5):471-4. · 2.83 Impact Factor
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ABSTRACT: Endothelial caveolin-1 (cav-1) is an anchoring protein in plasma membrane caveolae where it binds endothelial nitric oxide synthase (eNOS) and limits its activation, particularly in animals fed a high salt (HS) diet. Cav-1 also interacts with steroid receptors such as the mineralocorticoid receptor (MR). To test the hypothesis that vascular reactivity is influenced by an interplay between MR and cav-1 during HS diet, we examined the effects of MR blockade on NOS-mediated vascular relaxation in normal and cav-1-deficient mice. Wild-type (WT) and cav-1 knockout mice (cav-1(-/-)) were fed for 14 days a HS (4% NaCl) diet with and without the MR antagonist eplerenone (Epl; 100 mg x kg(-1) x day(-1)). After systolic blood pressure (BP) was measured, the thoracic aorta was isolated for measurement of vascular reactivity, and the aorta and heart were used for measurement of eNOS and MR expression. BP was not different between WT + Epl and WT, but was higher in cav-1(-/-) + Epl than in cav-1(-/-) mice. Phenylephrine (Phe)-induced vascular contraction was less in cav-1(-/-) than WT, and significantly enhanced in cav-1(-/-) + Epl than in cav-1(-/-), but not in WT + Epl compared with WT. Endothelium removal and NOS blockade by N(omega)-nitro-l-arginine methyl ester (l-NAME) enhanced Phe contraction in cav-1(-/-), but not cav-1(-/-) + Epl. ACh-induced aortic relaxation was reduced in cav-1(-/-) + Epl versus cav-1(-/-), but not in WT + Epl compared with WT. Endothelium removal, l-NAME, and the guanylate cyclase inhibitor ODQ abolished the large ACh-induced relaxation in cav-1(-/-) and the remaining relaxation in the cav-1(-/-) + Epl but had similar inhibitory effect in WT and WT + Epl. Real-time RT-PCR indicated decreased eNOS mRNA expression in the aorta and heart, and Western blots revealed decreased total eNOS in the heart of cav-1(-/-) + Epl compared with cav-1(-/-). Vascular and cardiac MR expression was less in cav-1(-/-) than WT, but not in cav-1(-/-) + Epl compared with cav-1(-/-). Plasma aldosterone (Aldo) was not different between WT and cav-1(-/-) mice nontreated or treated with Epl. Thus in cav-1 deficiency states and HS diet MR blockade is associated with increased BP, enhanced vasoconstriction, and decreased NOS-mediated vascular relaxation and eNOS expression. The data suggest that, in the absence of cav-1, MR activation plays a beneficial role in regulating eNOS expression/activity and, consequently, the vascular function during HS diet.
AJP Heart and Circulatory Physiology 04/2010; 298(6):H1776-88. · 3.71 Impact Factor
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ABSTRACT: A greater incidence of varicose veins has been reported in premenopausal women than in men. We hypothesized that the sex differences in venous function reflect reduced constriction and enhanced venous dilation in women due to direct venous relaxation effects of estrogen on specific estrogen receptors (ER).
Circular segments of inferior vena cava (IVC) from male and female Sprague-Dawley rats were suspended between two wires, and isometric contraction (in mg/mg tissue) to phenylephrine, angiotensin II (AngII), and 96 mM KCl was measured. To investigate sex differences in venous smooth muscle, Ca(2+) release from the intracellular stores, and Ca(2+) entry from the extracellular space, the transient phenylephrine contraction in 0 Ca(2+) Krebs was measured. Extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added, and the [Ca(2+)](e)-dependent contraction was measured. To investigate sex differences in venous endothelial function, acetylcholine-induced relaxation was measured. To test the role of specific ERs, the amount of venous tissue ERs was measured using Western blots, and the venous relaxation in response to 17beta-estradiol (E2, activator of most ERs), 4,4,'4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERalpha agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERbeta agonist), and ICI 182,780 (ERalpha/ERbeta antagonist, and G protein-coupled receptor 30 [GPR30] agonist) was measured in IVC segments nontreated or treated with the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME).
Phenylephrine caused concentration-dependent contraction that was less in female (max 104.2 +/- 16.2) than male IVC (172.4 +/- 20.4). AngII (10(-6))-induced contraction was also less in female (81.0 +/- 11.1) than male IVC (122.5 +/- 15.0). Phenylephrine contraction in 0 Ca(2+) Krebs was insignificantly less in female (4.8 +/- 1.8) than male IVC (7.2 +/- 1.7), suggesting little difference in the intracellular Ca(2+) release mechanism. In contrast, the [Ca(2+)](e)-dependent contraction was significantly reduced in female than male IVC. Also, contraction to membrane depolarization by 96 mM KCl, which stimulates Ca(2+) influx, was less in female (129.7 +/- 16.7) than male IVC (319.7 +/- 30.4), supporting sex differences in Ca(2+) entry. Acetylcholine relaxation was greater in female (max 80.6% +/- 4.1%) than male IVC (max 48.0% +/- 6.1%), suggesting sex differences in the endothelium-dependent relaxation pathway. Western blots revealed greater amounts of ERalpha, ERbeta, and GPR30 in female than male IVC. ER agonists caused concentration-dependent relaxation of phenylephrine contraction in female IVC. E2-induced relaxation (max 76.5% +/- 3.4%) was more than DPN (74.8% +/- 9.1%), PPT (71.4% +/- 12.5%), and ICI 182,780 (67.4% +/- 7.8%), and was similar in L-NAME-treated and nontreated IVC.
The reduced alpha-adrenergic, AngII, depolarization-induced, and [Ca(2+)](e)-dependent venous contraction in female rats is consistent with sex differences in the Ca(2+) entry mechanisms, possibly due to enhanced endothelium-dependent vasodilation and increased ER expression/activity in female rats. E2/ER-mediated venous relaxation in female rats is not prevented by NOS blockade, suggesting activation of an NO-independent relaxation pathway. The decreased venous contraction and enhanced E2/ER-mediated venous relaxation would lead to more distensible veins in female rats.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 04/2010; 51(4):972-81. · 3.52 Impact Factor
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ABSTRACT: Varicose veins (VarVs) are a common disorder of venous dilation and tortuosity with unclear mechanism. The functional integrity and the ability of various regions of the VarVs to constrict is unclear. This study tested the hypothesis that the different degrees of venodilation in different VarV regions reflect segmental differences in the responsiveness to receptor-dependent vasoconstrictive stimuli and/or in the postreceptor signaling mechanisms of vasoconstriction.
Varix segments and adjacent proximal and distal segments were obtained from patients undergoing VarV stripping. Control great saphenous vein specimens were obtained from patients undergoing lower extremity arterial bypass and coronary artery bypass grafting. Circular vein segments were equilibrated under 2 g of tension in a tissue bath, and changes in isometric constriction in response to angiotensin II (AngII, 10(-11)-10(-7) M), phenylephrine (PHE, 10(-9)-10(-4) M), and KCl (96 mM) were recorded. The amount of angiotensin type 1 receptor (AT(1)R) was measured in vein tissue homogenate.
AngII caused concentration-dependent constriction in control vein (max 35.3 +/- 9.6 mg/mg tissue, pED(50) 8.48 +/- 0.34). AngII caused less contraction and was less potent in proximal (max 7.9 +/- 2.5, pED(50) 6.85 +/- 0.61), distal (max 5.7 +/- 1.2, pED(50) 6.74 +/- 0.68), and varix segments of VarV (max 7.2 +/- 2.0, pED(50) 7.11 +/- 0.50), suggesting reduced AT(1)R-mediated contractile mechanisms. VarVs and control veins had similar amounts of AT(1)R. alpha-adrenergic receptor stimulation with PHE caused concentration-dependent constriction in control veins (max 73.0 +/- 13.9 mg/mg tissue, pED(50) 5.48 +/- 0.12) exceeding that of AngII. PHE produced similar constriction and was equally potent in varix and distal segments but produced less constriction and was less potent in proximal segments of VarVs (max 32.1 +/- 6.4 mg/mg tissue, pED(50) 4.89 +/- 0.13) vs control veins. Membrane depolarization by 96 mM KCl, a receptor-independent Ca(2+)-dependent response, produced significant constriction in control veins and similar contractile response in proximal, distal, and varix VarV segments, indicating tissue viability and intact Ca(2+)-dependent contraction mechanisms.
Compared with control veins, different regions of VarV display reduced AngII-mediated venoconstriction, which may be involved in the progressive dilation in VarVs. Postreceptor Ca(2+)-dependent contraction mechanisms remain functional in VarVs. The maintained alpha-adrenergic responses in distal and varix segments, and the reduced constriction in the upstream proximal segments, may represent a compensatory adaptation of human venous smooth muscle to facilitate venous return from the dilated varix segments of VarV.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 04/2010; 51(4):962-71. · 3.52 Impact Factor
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ABSTRACT: Normal pregnancy is associated with significant hemodynamic changes and vasodilation in the uterine and systemic circulation in order to meet the metabolic demands of the mother and developing fetus. Hypertension in pregnancy (HTN-Preg) and preeclampsia (PE) are major complications and life-threatening conditions to both the mother and fetus. PE is precipitated by various genetic, dietary and environmental factors. Although the initiating events of PE are unclear, inadequate invasion of cytotrophoblasts into the uterine artery is thought to reduce uteroplacental perfusion pressure and lead to placental ischemia/hypoxia. Placental hypoxia induces the release of biologically active factors such as growth factor inhibitors, anti-angiogenic proteins, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and antibodies to vascular angiotensin II receptor. These bioactive factors affect the production/activity of various vascular mediators in the endothelium, smooth muscle and extracellular matrix, leading to severe vasoconstriction and HTN. As an endothelial cell disorder, PE is associated with decreased vasodilator mediators such as nitric oxide, prostacyclin and hyperpolarizing factor and increased vasoconstrictor mediators such as endothelin, angiotensin II and thromboxane A(2). PE also involves enhanced mechanisms of vascular smooth muscle contraction including intracellular free Ca(2+) concentration ([Ca(2+)](i)), and [Ca(2+)](i) sensitization pathways such as protein kinase C, Rho-kinase and mitogen-activated protein kinase. Changes in extracellular matrix composition and matrix metalloproteases activity also promote vascular remodeling and further vasoconstriction in the uterine and systemic circulation. Characterization of the predisposing risk factors, the biologically active factors, and the vascular mediators associated with PE holds the promise for early detection, and should help design specific genetic and pharmacological tools for the management of the vascular dysfunction associated with HTN-Preg.
Cardiovascular & hematological disorders drug targets. 03/2010; 10(1):33-52.
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ABSTRACT: Normal pregnancy is associated with significant vascular remodeling in the uterine and systemic circulation in order to meet the metabolic demands of the mother and developing fetus. The pregnancy-associated vascular changes are largely due to alterations in the amount/activity of vascular mediators released from the endothelium, vascular smooth muscle and extracellular matrix. The endothelium releases vasodilator substances such as nitric oxide, prostacyclin and hyperpolarizing factor as well as vasoconstrictor factors such as endothelin, angiotensin II and thromboxane A(2). Vascular smooth muscle contraction is mediated by intracellular free Ca(2+) concentration ([Ca(2+)](i)), and [Ca(2+)](i) sensitization pathways such as protein kinase C, Rho-kinase and mitogen-activated protein kinase. Extracellular matrix and vascular remodeling are regulated by matrix metalloproteases. Hypertension in pregnancy and preeclampsia are major complications and life threatening conditions to both the mother and fetus, precipitated by various genetic, dietary and environmental factors. The initiating mechanism of preeclampsia and hypertension in pregnancy is unclear; however, most studies have implicated inadequate invasion of cytotrophoblasts into the uterine artery, leading to reduction in the uteroplacental perfusion pressure and placental ischemia/hypoxia. This placental hypoxic state is thought to induce the release of several circulating bioactive factors such as growth factor inhibitors, anti-angiogenic proteins, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and vascular receptor antibodies. Increases in the plasma levels and vascular content of these factors during pregnancy could cause an imbalance in the vascular mediators released from the endothelium, smooth muscle and extracellular matrix, and lead to severe vasoconstriction and hypertension. This review will discuss the interactions between the various circulating bioactive factors and the vascular mediators released during hypertension in pregnancy, and provide an insight into the current and future approaches in the management of preeclampsia.
Current Bioactive Compounds 03/2010; 6(1):60-75.
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Raouf A Khalil
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ABSTRACT: Cardiovascular disease (CVD) is more common in postmenopausal than premenopausal women, suggesting vascular protective effects of estrogen. Vascular estrogen receptors ERalpha, ERbeta and a transmembrane estrogen-binding protein GPR30 have been described. Also, experimental studies have demonstrated vasodilator effects of estrogen on the endothelium, vascular smooth muscle and extracellular matrix. However, randomized clinical trials have not supported vascular benefits of menopausal hormone therapy (MHT), possibly due to the subjects' advanced age and age-related changes in estrogen synthesis and metabolic pathways, the vascular ERs number, distribution and integrity, and the post-ER vascular signaling pathways. Current MHT includes natural estrogens such as conjugated equine estrogen, as well as synthetic and semi-synthetic estrogens. New estrogenic formulations and hormone combinations have been developed. Phytoestrogens is being promoted as an alternative MHT. Specific ER modulators (SERMs), and selective agonists for ERalpha such as PPT, ERbeta such as DPN, and GPR30 such as G1 are being evaluated. In order to enhance the vascular effectiveness of MHT, its type, dose, route of administration and timing may need to be customized depending on the subject's age and pre-existing CVD. Also, the potential interaction of estrogen with progesterone and testosterone on vascular function may need to be considered in order to maximize the vascular benefits of MHT on senescent blood vessels and postmenopausal CVD.
Cardiovascular & hematological agents in medicinal chemistry 01/2010; 8(1):29-46.
