Raouf A Khalil

Harvard Medical School, Boston, Massachusetts, United States

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Publications (126)465.51 Total impact

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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.
    Journal of Pharmacology and Experimental Therapeutics 12/2014; · 3.89 Impact Factor
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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.
    HBPR; 09/2014
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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.
    HBPR, San Francisco; 09/2014
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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, [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.
    Hypertension 06/2014; · 6.87 Impact Factor
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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.
    Hypertension 06/2014; · 6.87 Impact Factor
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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.
    Biochemical pharmacology 04/2014; · 4.25 Impact Factor
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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.
    Journal of Pharmacology and Experimental Therapeutics 11/2013; · 3.89 Impact Factor
  • 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.
    Biochemical pharmacology 10/2013; · 4.25 Impact Factor
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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.
    Journal of Cellular Physiology 09/2013; · 4.22 Impact Factor
  • 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.
    Biochemical pharmacology 07/2013; · 4.25 Impact Factor
  • Raouf A Khalil
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    ABSTRACT: The goal of "Recent Patents on Cardiovascular Drug Discovery" has long been to publish top quality original research papers and review articles highlighting the molecular and cellular mechanisms underlying cardiovascular disease, and new patents and discoveries for management of this major disorder. In this issue, the journal continues its goal and presents to the scientific community and health professionals a selection of excellent reviews describing the risk factors, manifestations, complications and novel interventional approaches for prevention and treatment of cardiovascular disease. The issue highlights the role of obesity and hypercholesterolemia as major risk factors for atherosclerosis, hypertension and heart disease, and the availability of new anticoagulants to reduce the risk of stroke and other complications of vascular disease. An important aspect of this issue is that it emphasizes not only on new therapies, but also on the use of botanicals as an alternative preventive or therapeutic tool as well as mechanical approaches for correction of incompetence of aortic and heart valves. Another important aspect of this issue is that it includes work from research investigators and clinician-scientists from all over the world including Australia, Europe and America, thus presenting diverse points of view and different treatment strategies. Thanks to the authors, reviewers and Editors, the journal was able to bring these important topics forward and present them to our readers in a concise yet very clear and informative fashion. The important contributions to this issue include: 1. Phytoestrogens and other botanicals: on the problems of evidence-based evaluation (Jargin). Peoples' Friendship University of Russia 2. The role of omega-3 polyunsaturated fatty acids supplementation In childhood: A review. (Ciccone et al.). University of Bari, Italy 3. Phentermine and topiramate extended-release for the obesity: new kids on the block. (Katsi et al.). Cardiology Department, Asklepeion General Hospital, Athens, Greece 4. Advances in irrigated tip catheter technology for treatment of cardiac arrhythmias. (Peichl and Kautzner). Prague, Czech Republic 5. Current status and ongoing development of reversing agents for novel oral anticoagulants (NOACs). (Capodanno et al.). Catania, Italy. 6. Patents and heart valve surgery I: Mechanical valves. (Cheema et al.) New York, New York. Milan, Italy, Chicago, Illinois 7. Anti-atherosclerotic therapy based on botanicals. (Bobryshev). Sydney, Australia. I would like to take this opportunity to express my deepest gratitude to Dr. Paul Higgins, for sharing with me the highly demanding position of Co-Editor-in-Chief, the Bentham Science organization for its sincere commitment to the highest quality science and novel discoveries, to our outstanding Publication Manager Ms. Sumaiya Azhar, and our hard-working Bentham Editorial Staff that have done an exceptional job in insuring the highest standards and integrity of the peer review process. I also would like to acknowledge the untiring help from our distinguished members of the Editorial Board who have been very generous with their time and effort in providing critical reviews of the submitted articles and assisting the authors with helpful comments and constructive criticism. I particularly wish to thank our readers for their continued interest in our journal and their helpful feed-back. I encourage all of you to contact me directly if you have any questions, comments, suggestions, criticism or ideas that could further strengthen our publications and help us make "Recent Patents on Cardiovascular Drug Discovery" achieve its goals and meet the high expectations of our readers.
    Recent patents on cardiovascular drug discovery. 05/2013;
  • Marc Q Mazzuca, Yiping Dang, Raouf A Khalil
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    ABSTRACT: BACKGROUND AND PURPOSE: Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ETA R), but could activate ETB R in the endothelium and release vasodilator substances. However, the role of ETB R in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear. EXPERIMENTAL APPROACH: Pressurized mesenteric microvessels from pregnant and virgin Sprague-Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca(2+) ]i . KEY RESULTS: High KCl (51 mM) and phenylephrine caused increases in vasoconstriction and [Ca(2+) ]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca(2+) ]i . Pretreatment with the ETB R antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. Acetylcholine (ACh) caused endothelium-dependent relaxation and decreased [Ca(2+) ]i , and was more potent in pregnant than virgin rats. ET-1+ETA R antagonist BQ-123, and the ETB R agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than virgin rats with no changes in [Ca(2+) ]i , suggesting upregulated ETB R-mediated relaxation pathways. ACh, S6c and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor L-NAME and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ETB R in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ETB R. CONCLUSIONS AND IMPLICATIONS: The enhanced ETB R-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.
    British Journal of Pharmacology 05/2013; · 5.07 Impact Factor
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    Raouf A Khalil
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    ABSTRACT: Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca(2+) concentration ([Ca(2+)]i), which forms a complex with calmodulin, activates myosin light chain (MLC) kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca(2+)-dependent and Ca(2+)-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca(2+)]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinase (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with antioxidants, MMP inhibitors and cytokine antagonists to reduce VSM hyperactivity in certain forms of HTN that do not respond to Ca(2+) channel blockers.
    Pharmaceuticals 03/2013; 6(3):407-439.
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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
  • Arda Kucukguven, Raouf A Khalil
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    ABSTRACT: Varicose veins (VVs) are a common venous disease of the lower extremity characterized by incompetent valves, venous reflux, and dilated and tortuous veins. If untreated, VVs could lead to venous thrombosis, thrombophlebitis and chronic venous leg ulcers. Various genetic, hormonal and environmental factors may lead to structural changes in the vein valves and make them incompetent, leading to venous reflux, increased venous pressure and vein wall dilation. Prolonged increases in venous pressure and vein wall tension are thought to increase the expression/activity of matrix metalloproteinases (MMPs). Members of the MMPs family include collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and others. MMPs are known to degrade various components of the extracellular matrix (ECM). MMPs may also affect the endothelium and vascular smooth muscle, causing changes in the vein relaxation and contraction mechanisms. ECs injury also triggers leukocyte infiltration, activation and inflammation, which lead to further vein wall damage. The vein wall dilation and valve dysfunction, and the MMP activation and superimposed inflammation and fibrosis would lead to progressive venous dilation and VVs formation. Surgical ablation is an effective treatment for VVs, but may be associated with high recurrence rate, and other less invasive approaches that target the cause of the disease are needed. MMP inhibitors including endogenous tissue inhibitors (TIMPs) and pharmacological inhibitors such as zinc chelators, doxycycline, batimastat and marimastat, have been used as diagnostic and therapeutic tools in cancer, autoimmune and cardiovascular disease. However, MMP inhibitors may have side effects especially on the musculoskeletal system. With the advent of new genetic and pharmacological tools, specific MMP inhibitors with fewer undesirable effects could be useful to retard the progression and prevent the recurrence of VVs.
    Current drug targets 01/2013; · 3.93 Impact Factor
  • Journal of Vascular Surgery: Venous and Lymphatic Disorders. 01/2013; 1(1):105–106.
  • Batoule H Majed, Raouf A Khalil
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    ABSTRACT: Prostacyclin (PGI(2)) is a member of the prostanoid group of eicosanoids that regulate homeostasis, hemostasis, smooth muscle function and inflammation. Prostanoids are derived from arachidonic acid by the sequential actions of phospholipase A(2), cyclooxygenase (COX), and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX1 and COX2, that differ in structure, tissue distribution, subcellular localization, and function. COX1 is largely constitutively expressed, whereas COX2 is induced at sites of inflammation and vascular injury. PGI(2) is produced by endothelial cells and influences many cardiovascular processes. PGI(2) acts mainly on the prostacyclin (IP) receptor, but because of receptor homology, PGI(2) analogs such as iloprost may act on other prostanoid receptors with variable affinities. PGI(2)/IP interaction stimulates G protein-coupled increase in cAMP and protein kinase A, resulting in decreased [Ca(2+)](i), and could also cause inhibition of Rho kinase, leading to vascular smooth muscle relaxation. In addition, PGI(2) intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription. PGI(2) counteracts the vasoconstrictor and platelet aggregation effects of thromboxane A(2) (TXA(2)), and both prostanoids create an important balance in cardiovascular homeostasis. The PGI(2)/TXA(2) balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. A decrease in PGI(2)/TXA(2) ratio in the maternal, fetal, and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction, and persistent pulmonary hypertension of the newborn (PPHN), respectively. On the other hand, increased PGI(2) activity may contribute to patent ductus arteriosus (PDA) and intraventricular hemorrhage in premature newborns. These observations have raised interest in the use of COX inhibitors and PGI(2) analogs in the management of pregnancy-associated and neonatal vascular disorders. The use of aspirin to decrease TXA(2) synthesis has shown little benefit in preeclampsia, whereas indomethacin and ibuprofen are used effectively to close PDA in the premature newborn. PGI(2) analogs have been used effectively in primary pulmonary hypertension in adults and have shown promise in PPHN. Careful examination of PGI(2) metabolism and the complex interplay with other prostanoids will help design specific modulators of the PGI(2)-dependent pathways for the management of pregnancy-related and neonatal vascular disorders.
    Pharmacological reviews 06/2012; 64(3):540-82. · 17.00 Impact Factor
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    ABSTRACT: The study was to explore whether the brachial/carotid pulse pressure (B/C-PP) ratio selectively predicts the sex difference in age-related cardiovascular (CV) death. Hypertension and CV complications are more severe in men and post-menopausal women than in pre-menopausal women. C-PP is lower than B-PP, and the B/C-PP ratio is a physiological marker of PP amplification between B and C arteries that tends toward 1.0 with age. The study involved 72,437 men (ages 41.0 ± 11.1 years) and 52,714 women (39.5 ± 11.6 years). C-PP was calculated for each sex by a multiple regression analysis including B-PP, age, height and risk factors, and a method validated beforehand in a subgroup of 834 subjects. During the 12 years of follow-up, 3,028 men and 969 women died. In the total population, the adjusted hazard ratios (HR) (95% confidence interval [CI]) of B/C-PP ratio were: 1) for all-cause mortality: men, HR: 1.51 (95% CI: 1.47 to 1.56), women; HR: 2.46 (95% CI: 2.27 to 2.67) (p < 0.0001); and 2) for CV mortality: men, HR 1.81 (95% CI: 1.70 to 1.93); women, HR: 4.46 (95% CI: 3.66 to 5.45) (p < 0.0001). The B/C-PP impact on mortality did not significantly increase from younger men to those ≥ 55 years of age, from: HR: 1.44 (95% CI: 1.31 to 1.58) to HR 1.65 (95% CI: 1.48 to 1.84), but increased significantly with age in women: HR: 3.19 (95% CI: 2.08 to 4.89) versus HR: 5.60 (95% CI: 4.17 to 7.50) (p < 0.01). Thus, the mortality impact of B/C-PP ratio was 3-fold higher in women than in men ≥ 55 years old. PP amplification is highly predictive of differences in CV risk between men and women. In post-menopausal women, the attenuation of PP amplification, mainly related to increased aortic stiffness, contributes to the significant increase in CV risk.
    Journal of the American College of Cardiology 05/2012; 59(20):1771-7. · 14.09 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. · 4.51 Impact Factor

Publication Stats

3k Citations
465.51 Total Impact Points

Institutions

  • 2003–2014
    • Harvard Medical School
      • Department of Medicine
      Boston, Massachusetts, United States
  • 2005–2013
    • Brigham and Women's Hospital
      • • Department of Medicine
      • • Division of Vascular Surgery and Endovascular Surgery
      Boston, MA, United States
  • 2011
    • Imperial College London
      • Department of Surgery and Cancer
      London, ENG, United Kingdom
  • 2008
    • U.S. Department of Veterans Affairs
      Washington, Washington, D.C., United States
  • 1998–2004
    • University of Mississippi Medical Center
      • Department of Physiology and Biophysics
      Jackson, MS, United States