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Raouf A. Khalil - One of the best experts on this subject based on the ideXlab platform.
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Mineralocorticoid receptor expression in human Venous Smooth Muscle cells: a potential role for aldosterone signaling in vein graft arterialization
American journal of physiology. Heart and circulatory physiology, 2011Co-Authors: Richard Bafford, Xin Xin Sui, Min Jung Park, Takuya Miyahara, Brenna G. Newfell, Iris Z. Jaffe, Jose R. Romero, Gail K. Adler, Gordon H. Williams, Raouf A. KhalilAbstract: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.
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mmp 2 induced vein relaxation via inhibition of ca2 e dependent mechanisms of Venous Smooth Muscle contraction role of rgd peptides
Journal of Surgical Research, 2010Co-Authors: Raouf A. Khalil, Joseph D. Raffetto, Yaskara V R Barros, Amanda K WellsAbstract:Background Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of varicose veins. We have shown that MMP-2 causes relaxation of Venous segments and suggested a role of Venous Smooth Muscle (VSM) hyperpolarization; however, the downstream mechanisms are unclear. We tested whether MMP-2 induced Venous relaxation involves inhibition of the Ca 2+ mobilization mechanisms of VSM contraction due to generation of Arg-Gly-Asp (RGD)-containing peptides. Methods Circular segments of inferior vena cava (IVC) were isolated from male Sprague-Dawley rats, suspended between two wires in a tissue bath, and isometric contraction was measured. Contraction data in mg/mg tissue were presented as means ± SEM. Results In IVC incubated in normal Krebs (2.5 mM Ca 2+ ), the α-adrenergic agonist phenylephrine (Phe, 10 −5 M) caused initial peak (133.2 ± 17.5) followed by a maintained contraction (73.4 ± 11.6), that was inhibited by MMP-2 (1 μg/mL) to 32.4 ± 12.8 in 30 min. The inhibitory effects of MMP-2 were reversible by washing the tissue with Krebs or in the presence of the MMP inhibitors TIMP-1 (1 μg/mL), Ro 28-2653, and BB-94 (10 −6 M), and were not associated with changes in IVC structure, demonstrating specificity. Angiotensin II (AngII, 10 −6 M) caused a monophasic contraction (114.2 ± 12.2), that was also inhibited by MMP-2 (66.0 ± 7.4), suggesting a post-receptor effect on the downstream mechanisms of VSM contraction. To test the role of Ca 2+ release from the sarcoplasmic reticulum, IVC was incubated in Ca 2+ -free 2 mM ethylene glycol-bis(2-aminoethyl ether-N,N,N',N'-tetra-acetic acid (EGTA) Krebs with or without MMP-2. In Ca 2+ -free Krebs, caffeine did not cause contraction, suggesting a limited role of the Ca 2+ -induced Ca 2+ -release mechanism, and Phe and AngII caused a small contraction (7.2 ± 1.7 and 14.9 ± 2.8) that was slightly increased by MMP-2 (10.4 ± 3.0 and 33.8 ± 10.0), suggesting little effect on IP 3 -induced Ca 2+ release. To test the role of Ca 2+ entry through membrane channels, after eliciting a transient Phe contraction in nominally 0 Ca 2+ Krebs, increasing concentrations of CaCl 2 (0.1, 0.3, 0.6, 1, 2.5 mM) were added and the extracellular Ca 2+ concentration [Ca 2+ ] e -contraction relationship was constructed. The [Ca 2+ ] e -contraction relation was reduced in MMP-2 treated IVC, suggesting inhibition of Ca 2+ entry. In IVC treated with MMP-2, the Ca 2+ channel blocker diltiazem (10 −5 M) did not cause any further inhibition of Phe contraction, suggesting that Ca 2+ entry is already inhibited by MMP-2. To test whether MMP-2 actions involve generation of RGD and modulation of integrin receptors, experiments where repeated in IVC segments saturated with RGD (10 −5 M), or pretreated with the α v β 3 integrin blocker cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (cyclo-RGD). RGD-peptide caused only small relaxation of Phe contracted IVC (6.4 ± 3.4%), and addition of MMP-2 to RGD-treated IVC caused further relaxation (69.7 ± 3.0%). Pretreatment of IVC with cyclo-RGD did not significantly affect MMP-2 induced relaxation (55.0 ± 5.0%). Conclusions In rat IVC, MMP-2 attenuates [Ca 2+ ] e -dependent VSM contraction without affecting Ca 2+ release from intracellular Ca 2+ stores. MMP-2 induced VSM relaxation may not involve RGD generation or activation of α v β 3 integrin receptor. MMP-2 induced inhibition of the Ca 2+ entry mechanism of VSM contraction may play a role in the Venous dilation associated with varicose vein formation.
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Functional adaptation of Venous Smooth Muscle response to vasoconstriction in proximal, distal, and varix segments of varicose veins
Journal of vascular surgery, 2010Co-Authors: Joseph D. Raffetto, Xiaoying Qiao, Katie G. Beauregard, Alain F. Tanbe, Abhinav Kumar, Virak Mam, Raouf A. KhalilAbstract:Background 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. Methods 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. Results 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. α-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. Conclusions 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 α-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.
Alfred K. Cheung - One of the best experts on this subject based on the ideXlab platform.
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PDGF-induced proliferation in human arterial and Venous Smooth Muscle cells: molecular basis for differential effects of PDGF isoforms.
Journal of cellular biochemistry, 2011Co-Authors: Donald K. Blumenthal, Christi M. Terry, Mary Carlson, Alfred K. CheungAbstract:The proliferative phenotype of artery and veins is different from each other and appears to have clinically significant consequences. Coronary artery bypass grafting with the autologous saphenous vein or internal mammary artery is widely used to treat atherosclerotic coronary artery disease. Graft failure due to stenosis occurs much more frequently in saphenous vein grafts than internal mammary artery grafts [Loop et al., 1986; Motwani and Topol, 1998]. Graft stenosis is most frequently caused by neointimal hyperplasia, another type of vascular proliferative disease. Similarly, most neointimal hyperplasia occurs at the graft-Venous anastomosis of hemodialysis arterioVenous grafts, resulting in 60% graft loss in the first year [US Renal Data System, 2007]. In contrast, atherosclerosis occurs in arteries but rarely in veins despite the exposure of both arteries and veins to the same systemic risk factors such as smoking and dyslipidemia. While hemodynamic and other local factors probably play a role in the different susceptibilities to atherosclerosis and neointimal hyperplasia between artery and veins, it is likely that the observed dissimilarities can be at least partially explained by intrinsic phenotypic differences between arterial and Venous Smooth Muscle cells (SMCs). These phenotypic differences would include the activation of different mitogenic signaling pathways. Platelet-derived growth factor (PDGF) is the most potent SMC mitogen released at the site of injury by platelets, endothelial cells, SMCs, and many other cell types [Heldin and Westermark, 1999; Raines, 2004]. The role for PDGF in the pathogenesis of arterial injury disorders, including atherosclerosis and post-angioplasty restenosis, has been well established. However, the effects of various PDGF isoforms and the differences between arterial and Venous SMCs in their responses to these isoforms are largely unknown and may explain the clinically observed differences between the arterial and Venous diseases. PDGF is a dimer composed of disulfide-linked polypeptides termed A-chain and B-chain. At least three isoforms (PDGF-AA, PDGF-AB, and PDGF-BB) are present in human platelets in comparable quantities [Hart et al., 1990]. The PDGF-C and PDGF-D chains were discovered more recently and their biology is not well established [Li et al., 2000; Bergsten et al., 2001; LaRochelle et al., 2001]. The role and expression of PDGF-C and PDGF-D in arteries and in atherosclerotic lesions remain unclear. Recently, Karvinen et al. [2009] reported that PDGF-C was strongly expressed in endothelial cells in both normal arteries and atherosclerotic lesions whereas PDGF-D was only weakly expressed in these cells. The functions of PDGF-CC and PDGF-DD have been explored and reviewed [Reigstad et al., 2005; Wagsater et al., 2009] and will not be further discussed in this paper. PDGF stimulates cell proliferation by binding to PDGF receptor α (PDGF-Rα) or PDGF receptor β (PDGF-Rβ), causing receptor dimerization and autophosphorylation. Previous studies have demonstrated that PDGF-AA activates only αα receptor dimers, PDGF-AB activates both αα and αβ receptor dimers, whereas PDGF-BB activates αα, αβ, and ββ receptor dimers [Heldin et al., 1998]. Many studies have demonstrated the pathogenic roles of PDGF-AB, PDGF-BB, and PDGF-Rβ in the formation of atherosclerotic and neointimal hyperplasia lesions. However, their roles in the pathogenesis of Venous diseases and the roles of PDGF-AA and PDGF-Rα in vascular disorders are largely unknown. We have previously reported that the proliferation of human Venous SMCs was more responsive to PDGF-AB stimulation compared to arterial SMCs [Li et al., 2006a]. In the present study, we investigated the proliferative effects of PDGF-BB and PDGF-AA on arterial and Venous SMCs and identified differences in the underlying signaling pathways that led to different responses to these PDGF isoforms between arterial and Venous SMCs. These results provide unique insights into the molecular basis of the varying susceptibility to proliferative disorders between arteries and veins.
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Different signaling responses to anti‐proliferative agents in human aortic and Venous Smooth Muscle cells
Journal of cellular biochemistry, 2006Co-Authors: Ray M. Lee, Takahisa Masaki, Donald K. Blumenthal, Hung Sheng Yang, Jihua Liu, Jun Chen, Alfred K. CheungAbstract:Proliferation of Smooth Muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous Venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs. J. Cell. Biochem. 99: 835–844, 2006. © 2006 Wiley-Liss, Inc.
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different signaling responses to anti proliferative agents in human aortic and Venous Smooth Muscle cells
Journal of Cellular Biochemistry, 2006Co-Authors: Takahisa Masaki, Donald K. Blumenthal, Ray M. Lee, Hung Sheng Yang, Jihua Liu, Jun Chen, Alfred K. CheungAbstract:Proliferation of Smooth Muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous Venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs. J. Cell. Biochem. 99: 835–844, 2006. © 2006 Wiley-Liss, Inc.
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Mechanism of dipyridamole's action in inhibition of Venous and arterial Smooth Muscle cell proliferation.
Basic & clinical pharmacology & toxicology, 2006Co-Authors: Sergey B. Zhuplatov, Takahisa Masaki, Donald K. Blumenthal, Alfred K. CheungAbstract:Abstract: Dipyridamole is a potential pharmacological agent to prevent vascular stenosis because of its antiproliferative properties. The mechanisms by which dipyridamole inhibits the growth of vascular Smooth Muscle cells, especially Venous Smooth Muscle cells, are unclear. In the present study, dipyridamole transiently but significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in human Venous and arterial Smooth Muscle cells in a time- and dose-dependent manner. Peak concentrations of both cyclic nucleotides were achieved at 15–30 min. and correlated with inhibition of proliferation in both cell types. The antiproliferative effects of dipyridamole observed at 48 hr were similar whether drug exposure was only 15 min. or sustained for 48 hr. Specific competitive inhibitors of protein kinases A and G attenuated the antiproliferative effects of subsaturating concentrations of dipyridamole, with the effects of protein kinase inhibition being particularly pronounced in Venous Smooth Muscle cells. Flow cytometry analysis showed that dipyridamole caused an enrichment of cells in G0/G1 and a corresponding reduction of cells in S phase. These data indicate that a transient increase in cGMP and cAMP is sufficient to induce downstream kinase activation and subsequent cell cycle arrest, and that protein kinase G may be more important than protein kinase A in mediating the growth inhibitory effect of dipyridamole in Venous protein kinase.
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Different responses by cultured aortic and Venous Smooth Muscle cells to gamma radiation.
Kidney international, 2005Co-Authors: Seung Jung Kim, Takahisa Masaki, Roy Rowley, John K. Leypoldt, Syed F. Mohammad, Alfred K. CheungAbstract:Different responses by cultured aortic and Venous Smooth Muscle cells to gamma radiation. Background Stenosis of hemodialysis arterioVenous grafts is usually focal and caused by the proliferation of vascular Smooth Muscle cells (SMCs). External radiation of the graft is a potential strategy to prevent stenosis; however, the relative responsiveness of arterial and Venous SMCs to radiation is unknown. Methods Human aortic and saphenous vein SMCs were cultured in a medium containing growth factors and serum and treated with 0 to 50Gy in a γ irradiator. At 2 to 20 days post-irradiation, cell counting, methylthiazoletetrazolium dye reduction, [ 3 H]-thymidine uptake, and bromodeoxyuridine (BrdU) incorporation assays were performed. Results All assays showed that 1 to 50Gy inhibited the proliferation of both aortic and Venous SMCs in a dose-dependent manner. Importantly, Venous cells were less susceptible to radiation in all assays, compared to aortic cells. At day 10, 1 to 50Gy of radiation inhibited the increase in the number of aortic cells by 24% to 66% and Venous cells by 8% to 25% ( P Conclusion Inasmuch as myointimal hyperplasia occurs at both arterial and Venous anastomoses, future strategies using radiation to prevent hemodialysis vascular access stenosis should take these differences into consideration.
Kristof Graf - One of the best experts on this subject based on the ideXlab platform.
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Effects of abciximab and tirofiban on vitronectin receptors in human endothelial and Smooth Muscle cells.
European Journal of Pharmacology, 2000Co-Authors: Ulrich Kintscher, Kai Kappert, Gunther Schmidt, Gesine Doerr, Matthias Grill, Brigitte Wollert-wulf, Michael Graefe, Eckart Fleck, Kristof GrafAbstract:Abstract Glycoprotein IIb/IIIa blockade by abciximab and tirofiban, non-peptidergic inhibitors, leads to sustained clinical benefits in the treatment of acute coronary syndromes. The purpose of this study was to clarify the functional effects of abciximab and tirofiban on vascular vitronectin receptors, αvβ3- and αvβ5-integrins. Integrin expression and 7E3 binding in human umbilical Venous endothelial cells, human umbilical Venous Smooth Muscle cells, and human iliac arterial Smooth Muscle cells were observed in the following intensity: αvβ3 — human umbilical Venous endothelial cells>human umbilical Venous Smooth Muscle cells>human iliac arterial Smooth Muscle cells/αvβ5 — human iliac arterial Smooth Muscle cells>human umbilical Venous Smooth Muscle cells>human umbilical Venous endothelial cells. 7E3 binding correlated with αvβ3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical Venous endothelial cells, these functions were mediated by αvβ3 and in human iliac arterial Smooth Muscle cells by αvβ5. In human umbilical Venous Smooth Muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited αvβ3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin–cell interactions in vascular cells are mediated via two distinct integrin-receptors, αvβ3 and αvβ5. Abciximab, which solely inhibits αvβ3-mediated cell functions, may be particularly effective in human endothelium and in β3-integrin expressing vascular Smooth Muscle cells.
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Effects of abciximab and tirofiban on vitronectin receptors in human endothelial and Smooth Muscle cells.
European Journal of Pharmacology, 2000Co-Authors: Ulrich Kintscher, Kai Kappert, Gunther Schmidt, Gesine Doerr, Matthias Grill, Brigitte Wollert-wulf, Michael Graefe, Eckart Fleck, Kristof GrafAbstract:human umbilical Venous endothelial cells. 7E3 binding correlated with alphavbeta3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical Venous endothelial cells, these functions were mediated by alphavbeta3 and in human iliac arterial Smooth Muscle cells by alphavbeta5. In human umbilical Venous Smooth Muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited alphavbeta3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin-cell interactions in vascular cells are mediated via two distinct integrin-receptors, alphavbeta3 and alphavbeta5. Abciximab, which solely inhibits alphavbeta3-mediated cell functions, may be particularly effective in human endothelium and in beta3-integrin expressing vascular Smooth Muscle cells.
Joseph D. Raffetto - One of the best experts on this subject based on the ideXlab platform.
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mmp 2 induced vein relaxation via inhibition of ca2 e dependent mechanisms of Venous Smooth Muscle contraction role of rgd peptides
Journal of Surgical Research, 2010Co-Authors: Raouf A. Khalil, Joseph D. Raffetto, Yaskara V R Barros, Amanda K WellsAbstract:Background Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of varicose veins. We have shown that MMP-2 causes relaxation of Venous segments and suggested a role of Venous Smooth Muscle (VSM) hyperpolarization; however, the downstream mechanisms are unclear. We tested whether MMP-2 induced Venous relaxation involves inhibition of the Ca 2+ mobilization mechanisms of VSM contraction due to generation of Arg-Gly-Asp (RGD)-containing peptides. Methods Circular segments of inferior vena cava (IVC) were isolated from male Sprague-Dawley rats, suspended between two wires in a tissue bath, and isometric contraction was measured. Contraction data in mg/mg tissue were presented as means ± SEM. Results In IVC incubated in normal Krebs (2.5 mM Ca 2+ ), the α-adrenergic agonist phenylephrine (Phe, 10 −5 M) caused initial peak (133.2 ± 17.5) followed by a maintained contraction (73.4 ± 11.6), that was inhibited by MMP-2 (1 μg/mL) to 32.4 ± 12.8 in 30 min. The inhibitory effects of MMP-2 were reversible by washing the tissue with Krebs or in the presence of the MMP inhibitors TIMP-1 (1 μg/mL), Ro 28-2653, and BB-94 (10 −6 M), and were not associated with changes in IVC structure, demonstrating specificity. Angiotensin II (AngII, 10 −6 M) caused a monophasic contraction (114.2 ± 12.2), that was also inhibited by MMP-2 (66.0 ± 7.4), suggesting a post-receptor effect on the downstream mechanisms of VSM contraction. To test the role of Ca 2+ release from the sarcoplasmic reticulum, IVC was incubated in Ca 2+ -free 2 mM ethylene glycol-bis(2-aminoethyl ether-N,N,N',N'-tetra-acetic acid (EGTA) Krebs with or without MMP-2. In Ca 2+ -free Krebs, caffeine did not cause contraction, suggesting a limited role of the Ca 2+ -induced Ca 2+ -release mechanism, and Phe and AngII caused a small contraction (7.2 ± 1.7 and 14.9 ± 2.8) that was slightly increased by MMP-2 (10.4 ± 3.0 and 33.8 ± 10.0), suggesting little effect on IP 3 -induced Ca 2+ release. To test the role of Ca 2+ entry through membrane channels, after eliciting a transient Phe contraction in nominally 0 Ca 2+ Krebs, increasing concentrations of CaCl 2 (0.1, 0.3, 0.6, 1, 2.5 mM) were added and the extracellular Ca 2+ concentration [Ca 2+ ] e -contraction relationship was constructed. The [Ca 2+ ] e -contraction relation was reduced in MMP-2 treated IVC, suggesting inhibition of Ca 2+ entry. In IVC treated with MMP-2, the Ca 2+ channel blocker diltiazem (10 −5 M) did not cause any further inhibition of Phe contraction, suggesting that Ca 2+ entry is already inhibited by MMP-2. To test whether MMP-2 actions involve generation of RGD and modulation of integrin receptors, experiments where repeated in IVC segments saturated with RGD (10 −5 M), or pretreated with the α v β 3 integrin blocker cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (cyclo-RGD). RGD-peptide caused only small relaxation of Phe contracted IVC (6.4 ± 3.4%), and addition of MMP-2 to RGD-treated IVC caused further relaxation (69.7 ± 3.0%). Pretreatment of IVC with cyclo-RGD did not significantly affect MMP-2 induced relaxation (55.0 ± 5.0%). Conclusions In rat IVC, MMP-2 attenuates [Ca 2+ ] e -dependent VSM contraction without affecting Ca 2+ release from intracellular Ca 2+ stores. MMP-2 induced VSM relaxation may not involve RGD generation or activation of α v β 3 integrin receptor. MMP-2 induced inhibition of the Ca 2+ entry mechanism of VSM contraction may play a role in the Venous dilation associated with varicose vein formation.
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Functional adaptation of Venous Smooth Muscle response to vasoconstriction in proximal, distal, and varix segments of varicose veins
Journal of vascular surgery, 2010Co-Authors: Joseph D. Raffetto, Xiaoying Qiao, Katie G. Beauregard, Alain F. Tanbe, Abhinav Kumar, Virak Mam, Raouf A. KhalilAbstract:Background 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. Methods 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. Results 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. α-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. Conclusions 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 α-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.
Ulrich Kintscher - One of the best experts on this subject based on the ideXlab platform.
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Effects of abciximab and tirofiban on vitronectin receptors in human endothelial and Smooth Muscle cells.
European Journal of Pharmacology, 2000Co-Authors: Ulrich Kintscher, Kai Kappert, Gunther Schmidt, Gesine Doerr, Matthias Grill, Brigitte Wollert-wulf, Michael Graefe, Eckart Fleck, Kristof GrafAbstract:Abstract Glycoprotein IIb/IIIa blockade by abciximab and tirofiban, non-peptidergic inhibitors, leads to sustained clinical benefits in the treatment of acute coronary syndromes. The purpose of this study was to clarify the functional effects of abciximab and tirofiban on vascular vitronectin receptors, αvβ3- and αvβ5-integrins. Integrin expression and 7E3 binding in human umbilical Venous endothelial cells, human umbilical Venous Smooth Muscle cells, and human iliac arterial Smooth Muscle cells were observed in the following intensity: αvβ3 — human umbilical Venous endothelial cells>human umbilical Venous Smooth Muscle cells>human iliac arterial Smooth Muscle cells/αvβ5 — human iliac arterial Smooth Muscle cells>human umbilical Venous Smooth Muscle cells>human umbilical Venous endothelial cells. 7E3 binding correlated with αvβ3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical Venous endothelial cells, these functions were mediated by αvβ3 and in human iliac arterial Smooth Muscle cells by αvβ5. In human umbilical Venous Smooth Muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited αvβ3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin–cell interactions in vascular cells are mediated via two distinct integrin-receptors, αvβ3 and αvβ5. Abciximab, which solely inhibits αvβ3-mediated cell functions, may be particularly effective in human endothelium and in β3-integrin expressing vascular Smooth Muscle cells.
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Effects of abciximab and tirofiban on vitronectin receptors in human endothelial and Smooth Muscle cells.
European Journal of Pharmacology, 2000Co-Authors: Ulrich Kintscher, Kai Kappert, Gunther Schmidt, Gesine Doerr, Matthias Grill, Brigitte Wollert-wulf, Michael Graefe, Eckart Fleck, Kristof GrafAbstract:human umbilical Venous endothelial cells. 7E3 binding correlated with alphavbeta3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical Venous endothelial cells, these functions were mediated by alphavbeta3 and in human iliac arterial Smooth Muscle cells by alphavbeta5. In human umbilical Venous Smooth Muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited alphavbeta3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin-cell interactions in vascular cells are mediated via two distinct integrin-receptors, alphavbeta3 and alphavbeta5. Abciximab, which solely inhibits alphavbeta3-mediated cell functions, may be particularly effective in human endothelium and in beta3-integrin expressing vascular Smooth Muscle cells.
