Raouf A Khalil

Brigham and Women's Hospital, Boston, Massachusetts, United States

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Publications (150)561.31 Total impact

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    ABSTRACT: Estrogen (E2) promotes vasodilation during pregnancy; however, the E2 receptor (ER) subtype, post-ER signaling mechanism and vascular bed involved are unclear. We tested whether pregnancy-associated vascular adaptation involves changes in expression/distribution/activity of ER subtypes in a blood vessel-specific manner. BP and plasma E2 were measured in virgin and day-19 pregnant (Preg) rats, and the aorta, carotid, mesenteric and renal artery were isolated for measurement of ERα, ERβ and GPER expression and relaxation responses to E2 (all ERs), PPT (ERα), DPN (ERβ) and G1 (GPER). Western blots revealed increased ERα and ERβ in aorta and mesenteric artery and GPER in aorta of Preg vs. virgin. Immunohistochemistry revealed increases in ERs mainly in intima and media. In phenylephrine precontracted vessels, E2 and PPT caused relaxation that was greater in aorta and mesenteric artery of Preg vs. virgin, and aortic relaxation to G1 was in Preg>virgin. The NOS inhibitor L-NAME±COX inhibitor indomethacin±EDHF blocker tetraethylammonium or endothelium removal reduced E2, PPT and G1-induced relaxation in aorta, suggesting an endothelium-dependent mechanism. E2, PPT, DPN and G1 caused relaxation of Ca(2+) entry-dependent KCl contraction, and the effect of PPT was greater in mesenteric artery of Preg vs. virgin. Thus, during pregnancy, an increase in ER in endothelial and vascular smooth muscle layers of aorta and mesenteric artery is associated with increased ERα-mediated relaxation via endothelium-derived vasodilators and inhibition of Ca(2+) entry, supporting a role of ERα in pregnancy-associated vasodilation. GPER contributes to aortic relaxation while enhanced ERα expression could mediate other genomic vascular effects during pregnancy.
    No preview · Article · Sep 2015 · AJP Heart and Circulatory Physiology
  • Elisabeth MacColl · Raouf A Khalil
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    ABSTRACT: Lower extremity veins have efficient wall structure and function and competent valves that permit upward movement of deoxygenated blood towards the heart against hydrostatic venous pressure. Matrix metalloproteinases (MMPs) play an important role in maintaining vein wall structure and function. MMPs are zinc-binding endopeptidases secreted by fibroblasts, vascular smooth muscle (VSM) and leukocytes as proMMPs, which are activated by other MMPs, proteinases and other activators. MMPs cause degradation of extracellular matrix (ECM) proteins such as collagen and elastin, and could have additional effects on the endothelium, as well as VSM cell migration, proliferation, Ca2+ signaling and contraction. Increased lower extremity hydrostatic venous pressure is thought to induce hypoxia inducible factors and other MMP inducers/activators such as EMMPRIN, prostanoids, chymase, and hormones, leading to increased MMPs expression/activity, ECM degradation, VSM relaxation, and venous dilation. Leukocyte infiltration and inflammation of the vein wall cause further increases in MMPs, vein wall dilation, valve degradation and different clinical stages of chronic venous disease (CVD) including varicose veins (VVs). VVs are characterized by ECM imbalance, incompetent valves, venous reflux, wall dilation, and tortuosity. VVs often show increased MMP levels, but may show no change or decreased levels, depending on the VVs region (atrophic vs. hypertrophic) and MMP form (pro- vs. active). Management of VVs includes compression stockings, venotonics, and surgical obliteration or removal. Because these approaches do not treat the cause of VVs, alternative methods are being developed. In addition to endogenous tissue inhibitors of MMPs (TIMPs), synthetic MMP inhibitors have been developed, and their effects in treatment of VVs need to be examined. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Aug 2015 · Journal of Pharmacology and Experimental Therapeutics
  • Sajjadh MJ Ali · Raouf A Khalil
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    ABSTRACT: Introduction: Preeclampsia (PE) is a major complication of pregnancy that could lead to maternal and fetal morbidity and mortality. The pathophysiological mechanisms of PE are not completely understood, but recent research has begun to unravel some of the potential mechanisms. Areas covered: Genetic polymorphisms and altered maternal immune response may cause impaired remodeling of the spiral arteries; a potential early defect in PE. Inadequate invasion of cytotrophoblasts into the decidua leads to reduced uteroplacental perfusion pressure (RUPP) and placental ischemia/hypoxia. Placental ischemia causes the release of biologically active factors such as anti-angiogenic factors, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and angiotensin II receptor autoantibodies. These vasoactive factors could cause systemic vascular endotheliosis and consequent increase in vascular resistance and blood pressure, glomerular endotheliosis causing proteinuria, cerebrovascular endotheliosis causing cerebral edema, seizures and visual disturbances, and hepatic endotheliosis, which may contribute to the manifestations of HELLP syndrome. PE-associated vascular endotheliosis causes a decrease in vasodilator mediators such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor, an increase in vasoconstrictors such as endothelin-1, angiotensin II and thromboxane A2, and enhanced mechanisms of vascular smooth muscle contraction such as intracellular Ca(2+), protein kinase C and Rho-kinase. Changes in matrix metalloproteinase activity and extracellular matrix cause vascular remodeling and further vasoconstriction. Expert opinion: Some of the genetic, immune and vasoactive factors involved in vascular endotheliosis could be used as biomarkers for early detection, and as potential targets for prevention and treatment of PE.
    No preview · Article · Aug 2015 · Expert Opinion on Therapeutic Targets
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    ABSTRACT: Aldosterone (ALDO) interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules, and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors, and also eNOS thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1-replete or -deficient states would alter vascular function in a mouse model of low nitric oxide (NO)-high angiotensin II (AngII)-induced cardiovascular injury. Wild type (WT) and cav-1 knockout mice (cav-1-/-) consuming a high salt diet (4% NaCl) received L-NAME (0.1-0.2 mg/ml in drinking water at days 1-11) plus AngII (0.7-2.8 mg/kg/day via osmotic minipump at days 8-11) ±MR antagonist eplerenone (EPL) 100 mg/Kg/day in food. In both genotypes, BP increased with L-NAME+AngII. EPL minimally changed BP, although its dose was sufficient to block MR and to reverse cardiac expression of the injury markers CD68 and PAI-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine- and KCl-contraction was enhanced with EPL in L-NAME+AngII treated WT, but not cav-1-/-. AngII-induced contraction was not different and AT1 receptor expression was reduced in L-NAME+AngII treated WT and cav-1-/-. In WT, acetylcholine (ACh)-induced relaxation was enhanced with L-NAME+AngII treatment and reversed with EPL. ACh relaxation in cav-1-/- was greater than in WT, not modified by L-NAME+AngII or EPL, and blocked by ex vivo L-NAME, ODQ or endothelium removal, suggesting a role of NO-cGMP. Cardiac eNOS was increased in cav-1-/- vs. WT, further increased with L-NAME+AngII and not affected by EPL. Vascular relaxation to the NO donor sodium nitroprusside was increased with L-NAME+AngII in WT, but not cav-1-/-. Plasma ALDO levels increased and cardiac MR expression decreased in L-NAME+AngII treated WT and cav-1-/-, and did not change with EPL. Thus, during L-NAME+AngII-induced hypertension, MR blockade increases contraction and alters vascular relaxation via NO-cGMP, and these changes are absent in cav-1 deficiency states. The data suggest a cooperative role of MR and cav-1 in regulating vascular contraction and NO-cGMP-mediated relaxation during low NO-high AngII-dependent cardiovascular injury. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Jul 2015 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: Striatin is a protein regulator of vesicular trafficking in neurons that also binds caveolin-1 and Ca(2+)-calmodulin and could activate endothelial nitric oxide synthase. We have shown that striatin colocalizes with the mineralocorticoid receptor and that mineralocorticoid receptor activation increases striatin levels in vascular cells. To test whether striatin is a regulator of vascular function, wild-type and heterozygous striatin-deficient mice (Strn(+/-)) were randomized in crossover intervention to restricted (0.03%) and liberal sodium (1.6%) diets for 7 days on each diet, and blood pressure and aortic vascular function were measured. Compared with wild-type, sodium restriction significantly reduced blood pressure in Strn(+/-). On liberal salt intake, phenylephrine and high KCl caused a greater vascular contraction in Strn(+/-) than wild-type, and endothelium removal, nitric oxide synthase inhibitor L-NAME, and guanylate cyclase inhibitor ODQ enhanced phenylephrine contraction to a smaller extent in Strn(+/-) than wild-type. On liberal salt, acetylcholine relaxation was less in Strn(+/-) than in wild-type, and endothelium removal, L-NAME, and ODQ blocked acetylcholine relaxation, suggesting changes in endothelial NO-cGMP. On liberal salt, endothelial nitric oxide synthase mRNA expression and the ratio of endothelial nitric oxide synthase activator pAkt/total Akt were decreased in Strn(+/-) versus wild-type. Vascular relaxation to NO donor sodium nitroprusside was not different among groups. Thus, striatin deficiency is associated with salt sensitivity of blood pressure, enhanced vasoconstriction, and decreased vascular relaxation, suggesting a critical role for striatin, through modulation of endothelial NO-cGMP, in regulation of vascular function and BP during changes in sodium intake. © 2015 American Heart Association, Inc.
    No preview · Article · Jul 2015 · Hypertension
  • Dania A Shah · Raouf A Khalil
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    ABSTRACT: Preeclampsia is a pregnancy-associated disorder characterized by hypertension, and could lead to maternal and fetal morbidity and mortality; however, the pathophysiological mechanisms involved are unclear. Predisposing demographic, genetic and environmental risk factors could cause localized abnormalities in uteroplacental cytoactive factors such as integrins, matrix metalloproteinases, cytokines and major histocompatibility complex molecules leading to decreased vascular remodeling, uteroplacental vasoconstriction, trophoblast cells apoptosis, and abnormal development of the placenta. Defective placentation and decreased trophoblast invasion of the myometrium cause reduction in uteroplacental perfusion pressure (RUPP) and placental ischemia/hypoxia, an important event in preeclampsia. RUPP could stimulate the release of circulating bioactive factors such as the anti-angiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin that cause imbalance with the pro-angiogenic factors vascular endothelial growth factor and placental growth factor, or cause the release of inflammatory cytokines, reactive oxygen species, hypoxia-induced factor-1 and AT1 angiotensin receptor agonistic autoantibodies. The circulating bioactive factors target endothelial cells causing generalized endotheliosis, endothelial dysfunction, decreased vasodilators such as nitric oxide and prostacyclin and increased vasoconstrictors such as endothelin-1 and thromboxane A2, leading to increased vasoconstriction. The bioactive factors also stimulate the mechanisms of VSM contraction including Ca(2+), protein kinase C, and Rho-kinase and induce extracellular matrix remodeling leading to further vasoconstriction and hypertension. While therapeutic options are currently limited, understanding the underlying mechanisms could help design new interventions for management of preeclampsia. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Apr 2015 · Biochemical pharmacology
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    ABSTRACT: Estrogen interacts with vascular estrogen receptors (ERs) to produce vasodilator effects that could impact vascular resistance. However, the specific ER subtype and the downstream post-ER relaxation pathways involved particularly in resistance microvessels are unclear. We tested if ER subtypes mediate distinct changes in microvascular reactivity and [Ca2+]i via specific post-ER relaxation pathways in endothelium or vascular smooth muscle (VSM). Pressurized mesenteric microvessels from female Sprague-Dawley rats were loaded with fura-2 and the simultaneous changes in diameter and [Ca2+]i in response to the ER activators 17β-estradiol (E2) (all ERs), PPT (ERα), DPN (ERβ) and G1 (GPR30) were measured. In intact vessels preconstricted with phenylephrine (Phe), E2 and PPT caused similar relaxation and decrease in maintained [Ca2+]i, suggesting that E2-induced vasodilation largely involves ERα. DPN caused less relaxation and decreased [Ca2+]i, suggesting a smaller role of ERβ, while G1 caused minimal changes in vessel diameter and [Ca2+]i, suggesting little role of GPR30. To test the endothelium-dependent pathways, acetylcholine (ACh) caused vasodilation and decreased [Ca2+]i that were abolished by endothelium removal, partly inhibited with the NOS blocker L-NAME, and blocked with the K+ channel blockers tetraethylammonium (TEA) (non specific), or apamin (SKCa) plus TRAM-34 (IKCa), suggesting a role of EDHF-dependent activation of KCa channels. E2-, PPT-, DPN- and G1-induced vasodilation and decreased [Ca2+]i were not blocked by L-NAME, TEA, apamin+TRAM-34, or endothelium removal, suggesting an endothelium-independent mechanism. In endothelium-denuded vessels preconstricted with Phe or high KCl (51 mM), ER agonists caused relaxation and decrease in [Ca2+]i that were with E2=PPT>DPN>G1, were not inhibited by guanylate cyclase inhibitor ODQ, and showed similar relationship between decreased [Ca2+]i and vasorelaxation, supporting direct effects on Ca2+ entry in VSM. Immunohistochemistry revealed ERα, ERβ and GPR30 mainly in the vessel media and VSM. Thus in mesenteric microvessels, ER subtypes mediate distinct vasodilation and decreased [Ca2+]i (ERα>ERβ>GPR30) through endothelium-independent inhibition of Ca2+ entry mechanisms of VSM contraction. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Feb 2015 · Journal of Pharmacology and Experimental Therapeutics
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    Marc Q Mazzuca · Karina M Mata · Raouf A Khalil
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    ABSTRACT: Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism, and a role of cytokines and endothelin-1 (ET-1) has been suggested. We have recently shown downregulation of endothelial type B ET-1 receptor (ET R) in Preg rats with reduced uterine perfusion pressure (RUPP). To test if cytokines are a possible mechanism linking RUPP to downregulation of ET R, day 14-Preg rats were either nontreated or infused with TNFα (200 ng/kg/day), and RUPP rats were either nontreated or infused with the TNFα decoy receptor etanercept (0.4 mg/kg/day) for 5 days by osmotic minipump. On day 19, BP was recorded and mesenteric microvessels were isolated for simultaneous measurement of diameter and [Ca ] (fura-2 340/380 ratio). BP was in TNFα-Preg (127±8) > Preg (97±5mmHg) and in etanercept-RUPP (113±2) < RUPP (124±3mmHg). ET-1 vasoconstriction was in TNFα-Preg (86.1±4.7) > Preg (58.1±5.2%), and in etanercept-RUPP (65.9±5.0) < RUPP (86.2±3.7%). ET-1 caused parallel increases in microvascular [Ca ] that were in TNFα-Preg (0.90±0.01) > Preg (0.86±0.01), and in etanercept-RUPP (0.85±0.01) < RUPP (0.92±0.01). Endothelium removal or microvessel treatment with ET R antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca ] in Preg and etanercept-RUPP, but not in TNFα-Preg or RUPP. ET R-mediated relaxation with IRL-1620 was in TNFα-Preg (4.11±6.1) < Preg (28.8±4.2%), and in etanercept-RUPP (20.2±4.6) > RUPP (10.17±2.9%). The NOS inhibitor L-NAME partially reduced ACh-induced and ET R-mediated relaxation in Preg and etanercept-RUPP, but not TNFα-Preg or RUPP, suggesting decreased NO-dependent and ET R-mediated vasorelaxation in HTN-Preg. Addition of the K channel blocker teraethylammonium (non specific), or apamin (SK) plus TRAM-34 (IK) abolished the remaining ET R-mediated relaxation in all groups, suggesting equal role of EDHF. Thus similar to RUPP, increasing TNFα in Preg rats increases ET-1 microvascular constriction and decreases ET R-mediated NO-dependent vasodilation, and counteracting TNFα reduces BP and ET-1 vasoconstriction, and enhances ET R-mediated vasodilation in RUPP rats. The results support that endothelial ET R is a major microvascular target in placental ischemia and TNFα-mediated HTN-Preg Disclosure: M.
    Full-text · Conference Paper · Sep 2014
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    ABSTRACT: Sch, Brigham and Woman's Hosp, BOSTON, MA Pregnancy (Preg) is associated with hormonal and vascular changes, and estrogen (E2) may promote systemic vasodilation during Preg; however, the specific E2 receptor (ER), post-ER signaling mechanisms and vascular bed involved are unclear. To test if Preg is associated with distinct expression/activity of ERs in different blood vessels, BP and plasma E2 were measured in virgin and day-19 Preg rats, and the aorta, carotid, mesenteric and renal artery were isolated for measurement of ERα, ERβ and GPR30 expression, and the responses to E2 and specific ER agonists PPT (ERα), DPN (ERβ) and G1 (GPR30). BP was in Preg (89±6) < virgin (98±4mmHg), and plasma E2 was in Preg (120.5±5.8) > virgin (94.3±7.5pg/ml). Western blots revealed increased ERα and ERβ in aorta and mesenteric artery and GPR30 in aorta of Preg vs virgin. Immunohistochemistry revealed that the increases in ERs were mainly in intima and media. E2 and PPT caused greater relaxation of aorta, but similar relaxation in carotid and renal artery of Preg vs virgin. DPN and G1 caused greater relaxation in mesenteric and renal artery (15 to 30%) than aorta and carotid artery (<10%), but only aortic relaxation to G1 was in Preg (26.2±4.4) > virgin (5.3±6.7%). The NOS inhibitor L-NAME ± EDHF blocker tetraethylammonium or endothelium removal reduced PPT relaxation in aorta, suggesting an endothelium-dependent mechanism, but did not affect E2, PPT, DPN or G1-induced relaxation in other vessels, suggesting endothelium-independent mechanisms. PPT caused relaxation of Ca entry-dependent KCl contraction of mesenteric artery that was in Preg (69.7±5.5) > virgin rats (52.9±8.11%). Thus, during pregnancy, an increased ERα expression in endothelial and smooth muscle layers of aorta and mesenteric artery is associated with increased ERα-mediated relaxation via endothelium-derived vasodilators and direct inhibition of Ca entry pathways, supporting a role of aortic and mesenteric arterial ERα in pregnancy-associated systemic vasodilation. GPR30 may contribute to aortic dilation while the enhanced ERβ may mediate other genomic vascular effects during pregnancy Disclosure: K.
    Full-text · Conference Paper · Sep 2014
  • Marc Q Mazzuca · Karina M Mata · Raouf A Khalil
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    ABSTRACT: Preeclampsia is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism, and a role of cytokines and endothelin-1 (ET-1) has been suggested. We have recently shown downregulation of endothelial type B ET-1 receptor (ETBR) in Preg rats with reduced uterine perfusion pressure (RUPP). To test if cytokines are a possible mechanism linking RUPP to downregulation of ETBR, day 14-Preg rats were either nontreated or infused with TNFα (200 ng/kg/day), and RUPP rats were either nontreated or infused with the TNFα decoy receptor etanercept (0.4 mg/kg/day) for 5 days by osmotic minipump. On day 19, BP was recorded and mesenteric microvessels were isolated for simultaneous measurement of diameter and [Ca2+]i (fura-2 340/380 ratio). BP was in TNFα-Preg (127±8) > Preg (97±5mmHg) and in etanercept-RUPP (113±2) < RUPP (124±3mmHg). ET-1 vasoconstriction was in TNFα-Preg (86.1±4.7) > Preg (58.1±5.2%), and in etanercept-RUPP (65.9±5.0) < RUPP (86.2±3.7%). ET-1 caused parallel increases in microvascular [Ca2+]i that were in TNFα-Preg (0.90±0.01) > Preg (0.86±0.01), and in etanercept-RUPP (0.85±0.01) < RUPP (0.92±0.01). Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca2+]i in Preg and etanercept-RUPP, but not in TNFα-Preg or RUPP. ETBR-mediated relaxation with IRL-1620 was in TNFα-Preg (4.11±6.1) < Preg (28.8±4.2%), and in etanercept-RUPP (20.2±4.6) > RUPP (10.17±2.9%). The NOS inhibitor L-NAME partially reduced ACh-induced and ETBR-mediated relaxation in Preg and etanercept-RUPP, but not TNFα-Preg or RUPP, suggesting decreased NO-dependent and ETBR-mediated vasorelaxation in HTN-Preg. Addition of the K+ channel blocker teraethylammonium (non specific), or apamin (SKCa) plus TRAM-34 (IKCa) abolished the remaining ETBR-mediated relaxation in all groups, suggesting equal role of EDHF. Thus similar to RUPP, increasing TNFα in Preg rats increases ET-1 microvascular constriction and decreases ETBR-mediated NO-dependent vasodilation, and counteracting TNFα reduces BP and ET-1 vasoconstriction, and enhances ETBR-mediated vasodilation in RUPP rats. The results support that endothelial ETBR is a major microvascular target in placental ischemia and TNFα-mediated HTN-Preg.
    No preview · Article · Sep 2014 · Hypertension
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    Celia A. Kanashiro · Khalid A. Altirkawi · Raouf A. Khalil

    Full-text · Dataset · Jul 2014
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    ABSTRACT: Preeclampsia is a pregnancy-related disorder characterized by hypertension with an unclear mechanism. Studies have shown endothelial dysfunction and increased endothelin-1 (ET-1) levels in hypertensive pregnancy (HTN-Preg). ET-1 activates endothelin receptor type-A in vascular smooth muscle to induce vasoconstriction, but the role of vasodilator endothelial endothelin receptor type-B (ETBR) in the changes in blood pressure (BP) and vascular function in HTN-Preg is unclear. To test whether downregulation of endothelial ETBR expression/activity plays a role in HTN-Preg, BP was measured in normal pregnancy (Norm-Preg) rats and rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and mesenteric microvessels were isolated for measuring diameter, [Ca(2+)]i, and endothelin receptor type-A and ETBR levels. BP, ET-1- and potassium chloride-induced vasoconstriction, and [Ca(2+)]i were greater in RUPP than in Norm-Preg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca(2+)]i in Norm-Preg, but not RUPP, suggesting reduced vasodilator ETBR in HTN-Preg. The ET-1+endothelin receptor type-A antagonist BQ-123 and the ETBR agonists sarafotoxin 6c and IRL-1620 caused less vasorelaxation and nitrate/nitrite production in RUPP than in Norm-Preg. The nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester reduced sarafotoxin 6c- and IRL-1620-induced relaxation in Norm-Preg but not in RUPP, supporting that ETBR-mediated nitric oxide pathway is compromised in RUPP. Reverse transcription polymerase chain reaction, Western blots, and immunohistochemistry revealed reduced endothelial ETBR expression in RUPP. Infusion of BQ-788 increased BP in Norm-Preg, and infusion of IRL-1620 reduced BP and ET-1 vasoconstriction and [Ca(2+)]i and enhanced ETBR-mediated vasorelaxation in RUPP. Thus, downregulation of microvascular vasodilator ETBR is a central mechanism in HTN-Preg, and increasing ETBR activity could be a target in managing preeclampsia.
    Full-text · Article · Jun 2014 · Hypertension
  • Marc Q Mazzuca · Li W · Reslan OM · Yu P · Mata KM · Khalil RA
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    ABSTRACT: Preeclampsia is a pregnancy-related disorder characterized by hypertension with an unclear mechanism. Studies have shown endothelial dysfunction and increased endothelin-1 (ET-1) levels in hypertensive pregnancy (HTN-Preg). ET-1 activates endothelin receptor type-A in vascular smooth muscle to induce vasoconstriction, but the role of vasodilator endothelial endothelin receptor type-B (ETBR) in the changes in blood pressure (BP) and vascular function in HTN-Preg is unclear. To test whether downregulation of endothelial ETBR expression/activity plays a role in HTN-Preg, BP was measured in normal pregnancy (Norm-Preg) rats and rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and mesenteric microvessels were isolated for measuring diameter, [Ca2+]i, and endothelin receptor type-A and ETBR levels. BP, ET-1- and potassium chloride-induced vasoconstriction, and [Ca2+]i were greater in RUPP than in Norm-Preg rats. Endothelium removal or microvessel treatment with ETBR antagonist BQ-788 enhanced ET-1 vasoconstriction and [Ca2+]i in Norm-Preg, but not RUPP, suggesting reduced vasodilator ETBR in HTN-Preg. The ET-1+endothelin receptor type-A antagonist BQ-123 and the ETBR agonists sarafotoxin 6c and IRL-1620 caused less vasorelaxation and nitrate/nitrite production in RUPP than in Norm-Preg. The nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester reduced sarafotoxin 6c- and IRL-1620-induced relaxation in Norm-Preg but not in RUPP, supporting that ETBR-mediated nitric oxide pathway is compromised in RUPP. Reverse transcription polymerase chain reaction, Western blots, and immunohistochemistry revealed reduced endothelial ETBR expression in RUPP. Infusion of BQ-788 increased BP in Norm-Preg, and infusion of IRL-1620 reduced BP and ET-1 vasoconstriction and [Ca2+]i and enhanced ETBR-mediated vasorelaxation in RUPP. Thus, downregulation of microvascular vasodilator ETBR is a central mechanism in HTN-Preg, and increasing ETBR activity could be a target in managing preeclampsia.
    No preview · Article · Jun 2014 · Hypertension
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    Wei Li · Karina M Mata · Marc Q Mazzuca · Raouf A Khalil
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    ABSTRACT: Preeclampsia is a complication of pregnancy manifested as maternal hypertension and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to hypertension and growth restriction are unclear. We have shown an upregulation of matrix metalloproteinases (MMPs) during normal pregnancy (Norm-Preg). To test the role of MMPs in hypertensive-pregnancy (HTN-Preg), maternal and fetal parameters, MMPs expression, activity and distribution, and collagen and elastin content were measured in uterus, placenta and aorta of Norm-Preg rats and in rat model of reduced uteroplacental perfusion pressure (RUPP). Maternal blood pressure was higher, and uterine, placental and aortic weight, and the litter size and pup weight were less in RUPP than Norm-Preg rats. Western blots and gelatin zymography revealed decreases in amount and gelatinase activity of MMP-2 and MMP-9 in uterus, placenta and aorta of RUPP compared with Norm-Preg rats. Immunohistochemistry confirmed reduced MMPs in uterus, placenta and aortic media of RUPP rats. Collagen, but not elastin, was more abundant in uterus, placenta and aorta of RUPP than Norm-Preg rats. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) decreased MMPs in uterus, placenta and aorta of Norm-Preg rats, and vascular endothelial growth factor (VEGF) reversed the decreases in MMPs in tissues of RUPP rats. Thus placental ischemia and anti-angiogenic sFlt-1 decrease uterine, placental and vascular MMP-2 and MMP-9, leading to increased uteroplacental and vascular collagen, and growth-restrictive remodeling in HTN-Preg. Angiogenic factors and MMP activators may reverse the decrease in MMPs and enhance growth-permissive remodeling in preeclampsia.
    Full-text · Article · Apr 2014 · Biochemical pharmacology
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    ABSTRACT: Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ETA R) and possibly type B (ETB R) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ETB R. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ETA R and ETB R. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12) and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ETA R and ETB R were examined using RT-PCR, Western blots, immunohistochemistry and immunofluorescence. Phenylephrine (Phe, 10(-5) M), KCl (51 mM) and ET-1 (10(-6) M) caused VSMC contraction that was in late-Preg < mid-Preg and virgin rats. In VSMCs treated with ETB R antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg < mid-Preg and virgin rats. In VSMCs treated with the ETA R antagonist BQ123, ET-1 caused a small contraction; and the ETB R agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg < mid-Preg and virgin rats. RT-PCR revealed similar ETA R, but greater ETB R mRNA expression in pregnant vs. virgin rats. Western blots revealed similar ETA R, and greater protein amount of ETB R in endothelium-intact vessels, but reduced ETB R in endothelium-denuded vessels of pregnant vs. virgin rats. Immunohistochemistry revealed prominent ETB R staining in the intima, but reduced ETA R and ETB R in the aortic media of pregnant rats. Immunofluorescence signal for ETA R and ETB R was less in VSMCs of pregnant vs. virgin rats. The pregnancy-associated decrease in ETA R- and ETB R-mediated VSMC contraction appears to involve downregulation of ETA R and ETB R expression/activity in VSM, and may play a role in the adaptive vasodilation during pregnancy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
    No preview · Article · Apr 2014 · Journal of Cellular Physiology
  • Raouf A Khalil

    No preview · Article · Jan 2014 · Recent Patents on Cardiovascular Drug Discovery
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    ABSTRACT: Hyperglycemia and endothelial dysfunction are associated with hypertension, but the specific causality and genetic underpinning are unclear. Caveolin-1 (cav-1) is a plasmalemmal anchoring protein and modulator of vascular function and glucose homeostasis. Cav-1 gene variants are associated with reduced insulin sensitivity in hypertensive individuals, and cav-1(-/-) mice show endothelial dysfunction, hyperglycemia and increased blood pressure (BP). On the other hand, insulin-sensitizing therapy with metformin may inadequately control hyperglycemia while affecting the vascular outcome in certain diabetics. To test if the pressor and vascular changes in cav-1 deficiency states are related to hyperglycemia, and to assess the vascular mechanisms of metformin under these conditions, wild-type (WT) and cav-1(-/-) mice were treated with placebo or metformin (400mg/Kg/day, for 21-days). BP and fasting blood glucose were in cav-1(-/-)>WT, and did not change with metformin. Phenylephrine (Phe)- and KCl-induced aortic contraction was in cav-1(-/-)<WT; endothelium removal, the NOS blocker L-NAME or soluble guanylate cyclase (sGC) inhibitor ODQ enhanced Phe contraction, and metformin blunted this effect. Acetylcholine (ACh)-induced relaxation was in cav-1(-/-)>WT, abolished by endothelium-removal, L-NAME or ODQ, and reduced with metformin. NO donor sodium nitroprusside was more potent in inducing relaxation in cav-1(-/-) than WT, and metformin reversed this effect. Aortic eNOS, AMPK and sGC were in cav-1(-/-)>WT, and metformin decreased total and phosphorylated eNOS and AMPK in cav-1(-/-). Thus metformin inhibits both vascular contraction and NO-cGMP-dependent relaxation, but does not affect BP or blood glucose in cav-1(-/-) mice, suggesting dissociation of hyperglycemia from altered vascular function in cav-1 deficiency states.
    No preview · Article · Nov 2013 · Journal of Pharmacology and Experimental Therapeutics
  • Raouf A Khalil
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    ABSTRACT: Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and 'timing' could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD.
    No preview · Article · Oct 2013 · Biochemical pharmacology

  • No preview · Conference Paper · Sep 2013
  • Yiping Dang · Wei Li · Victoria Tran · Raouf A Khalil
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    ABSTRACT: Pregnancy is associated with uteroplacental and vascular remodeling in order to adapt for the growing fetus and the hemodynamic changes in the maternal circulation. We have previously shown upregulation of uterine matrix metalloproteinases (MMPs) during pregnancy. Whether pregnancy-associated changes in MMPs are localized to the uterus or are generalized in feto-placental and maternal circulation is unclear. Also, the mechanisms causing the changes in uteroplacental and vascular MMPs during pregnancy are unclear. MMPs expression, activity and tissue distribution were measured in uterus, placenta and aorta of virgin, mid-pregnant (mid-Preg) and late pregnant (late-Preg) rats. Western blots and gelatin zymography revealed increases in MMP-2 and -9 in uterus and aorta of late-Preg compared with virgin and mid-Preg rats. In contrast, MMP-2 and -9 were decreased in placenta of late-Preg versus mid-Preg rats. Extracellular MMP inducer (EMMPRIN) was increased in uterus and aorta of pregnant rats, but was less in placenta of late-Preg than mid-Preg rats. Prolonged treatment of uterus or aorta of virgin rats with 17β-estradiol and progesterone increased the amount of EMMPRIN, MMP-2 and -9, and the sex hormone-induced increases in MMPs were prevented by EMMPRIN neutralizing antibody. Immunohistochemistry revealed that MMP-2 and -9 and EMMPRIN increased in uterus and aorta of pregnant rats, but decreased in placenta of late-Preg versus mid-Preg rats. Thus pregnancy-associated upregulation of uterine MMPs is paralleled by increased vascular MMPs, and both are mediated by EMMPRIN and induced by estrogen and progesterone, suggesting similar role of MMPs in uterine and vascular tissue remodeling and function during pregnancy. The decreased MMPs and EMMPRIN in placenta of late-Preg rats suggests reduced role of MMPs in feto-placental circulation during late pregnancy.
    No preview · Article · Jul 2013 · Biochemical pharmacology

Publication Stats

5k Citations
561.31 Total Impact Points

Institutions

  • 2007-2015
    • Brigham and Women's Hospital
      • Division of Vascular Surgery and Endovascular Surgery
      Boston, Massachusetts, United States
  • 2003-2015
    • Harvard Medical School
      • Department of Medicine
      Boston, Massachusetts, United States
  • 2014
    • Beverly Hospital, Boston MA
      BVY, Massachusetts, United States
  • 2005-2014
    • Harvard University
      Cambridge, Massachusetts, United States
  • 1998-2003
    • University of Mississippi Medical Center
      • Department of Physiology and Biophysics
      Jackson, Mississippi, United States
    • University of Mississippi
      • Department of Physiology and Biophysics
      Mississippi, United States