Vein graft intimal hyperplasia remains the leading cause of graft failure, despite many pharmacological approaches that have failed to translate to human therapy. We investigated whether local suppression of inflammation and fibrosis with MMI-0100, a novel peptide inhibitor of Mitogen Activated Protein Kinase Activated Protein Kinase II (MK2), would be an alternative strategy to reduce cell proliferation and intimal hyperplasia. The cell permeant peptide MMI-0100 was synthesized using standard Fmoc chemistry. Pharmacological doses of MMI-0100 induced minimal human endothelial and smooth muscle cell proliferation (30% and 12% respectively). MMI-0100 suppressed IL-6 expression to control levels, without effect on IL-8 expression. MMI-0100 caused sodium nitroprusside induced smooth muscle cell relaxation and inhibited intimal thickening in human saphenous vein rings in a dose-dependent fashion. In a murine aortic bypass model, MMI-0100 reduced intimal thickness in vein grafts by 72%, and there were fewer F4/80-reactive cells in vein grafts treated with MMI-0100. MMI-0100 prevents vein graft intimal thickening ex vivo and in vivo. These results suggest that inhibition of MK2 with the cell-permeant peptide MMI-0100 may be a novel strategy to suppress fibrotic processes such as vein graft disease.
Estrogens are known to contribute to endothelial function and sympathetic activity, both of which are strongly associated with the pathogenesis of ischemic heart disease. In addition, estrogens improve impaired lipid profile, a risk factor of endothelial dysfunction. In this study, we investigated the effects of OS-0544, a structurally new selective estrogen receptor modulator (SERM), on endothelial function, sympathetic activity, and plasma cholesterol level in ovariectomized (OVX) rats. Female Sprague-Dawley rats were ovariectomized and orally treated with OS-0544 (or OS-0689, the (R)-enantiomer of OS-0544), or 17beta-estradiol (E2) for 4 weeks, starting the next days after ovariectomy or for 1 week, starting 6 weeks after ovariectomy. Ovariectomy significantly increased vasopressin-induced mean blood pressure (AVP-MBP) (57+/-3.3 mm Hg vs. 46+/-3.5 mm Hg, P<0.05) and decreased acetylcholine (Ach)-induced maximum vasorelaxation response (69+/-5.6% vs. 81+/-4.0%, P<0.05). OS-0544 significantly inhibited AVP-MBP elevation (46+/-3.5 mm Hg vs. 57+/-3.3 mm Hg, P<0.05) and decreased Ach-induced maximum vasorelaxation response (90+/-3.3% vs. 69+/-5.6%, P<0.05) in OVX rats. In addition, OS-0689 as well as E2 significantly reduced (up to 67%) the increase in sympathetic activity in OVX rats. Moreover, like E2, OS-0544 significantly decreased plasma cholesterol level in OVX rats. These results demonstrate that OS-0544 has vascular protective effect on vascular function after ovariectomy. It is therefore believed that OS-0544 has vascular protective effect in postmenopausal woman.
Calcimimetics are a class of compounds that positively modulate the calcium-sensing receptor (CaR) by allosterically increasing the affinity of the receptor for extracellular Ca(2+). In this study we have investigated the effects of the clinically used calcimimetic, AMG 073, on contractility of the rat aorta by wire myography. AMG 073 elicited a concentration-dependent vasodilatation of the precontracted aorta. Inhibition of endothelium function by L-NAME and indomethacin reduced AMG 073-induced relaxation of the vessel precontracted with phenylephrine, but not with 125 mM K(+). The vasodilatory effect could be mediated by the CaR or/and a direct action on the ion channels. Intriguingly, CaR agonists, neomycin and gadolinium, did not have any effect on the contractility of the aorta. Immunohistochemical staining of the aorta with two CaR specific antibodies demonstrated the presence of the CaR protein, predominantly in endothelial and adventitial layers.
The antiplatelet and antithrombotic activities of a newly synthesized CP201, 2-(3,5-di-tert-butyl-4-hydroxyl)-3-chloro-1,4-naphthoquinone on human platelet aggregation in vitro and murine pulmonary thrombosis in vivo were examined. In addition, the antiplatelet activity of CP201 involved in calcium-signaling cascade was also investigated. CP201 showed concentration-dependent inhibitory effects on platelet aggregation induced by collagen and thrombin, with IC50 values of 4.1+/-0.3 and 4.6+/-0.4 microM, respectively. Orally administered CP201 protected mice against the collagen plus epinephrine-induced thromboembolic death in a dose-dependent manner. On the other hand, CP201 did not alter such coagulation parameters as activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in human plasma in vitro. These results suggest that the antithrombotic activity of CP201 may be due to antiplatelet rather than anticoagulation activity. CP201 potently inhibited platelet aggregation challenged by calcium ionophore A23187 and thapsigargin, which is a selective inhibitor of the Ca(2+)-ATPase pump, in a concentration-dependent manner, indicating that CP201 may have an inhibitory effect on calcium-signaling cascade. This was supported by measuring [Ca2+]i in platelets loaded with fura-3AM, where CP201 inhibited the rise in cytosolic Ca2+ mediated by thrombin. Taken together, these results suggest that CP201 may be a promising antithrombotic agent, and the antithrombotic effect of CP201 may be due to antiplatelet activity, which was mediated, at least partly, by the inhibition of cytosolic calcium mobilization.
Vascular smooth muscle cell (VSMCs) proliferation is an essential factor in cardiovascular diseases, such as primary atherosclerosis and in-stent restenosis. In this study, we examined the effects of the novel synthetic naphthoquinone, 2-pyrrilidino-3-(p-hydroxyphenylamino)-1,4-naphthoquinone (TW-96), on cultured VSMCs and endothelial cells (ECs). Pharmacological concentrations of the derivative TW96 were found to induce VSMCs death, probably by increasing ROS levels while decreasing mitochondrial potential (DeltaPsi(m)) without affecting ECs. Treatment of tissue cultures with ROS is known to induce MAPK activity. Our observations showed prolonged phosphorylation and perinuclear accumulation of ERK1/2 and p38 simultaneously with an inhibition of MKP1. Increased expression of Bax found in TW96-stimulated VSMCs was inhibited by the NADPH oxidase inhibitor diphenyliodonium (DPI). An examination of the suppressive effects of TW96 on PDGF-BB-stimulated VSMCs cycle progression showed that TW96 leads to migration arrest at concentrations lower than LC(50). We hope that this prototype derivative will establish the basis for creating more specific naphthoquinone derivatives aimed at preventing the VSMCs proliferation associated with stenosis and restenosis.
Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of atherosclerosis. The aim of this study was to assess the effect of NQ304 [2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone], a newly synthesized 1,4-naphthoquinone derivative, on the proliferation of PDGF-BB-stimulated rat aortic VSMCs. Antiproliferative effects of NQ304 on rat aortic VSMCs were examined by direct cell counting and by using [(3)H] thymidine incorporation assays. It was found that NQ304 potently the growth of VSMCs. Preincubation with NQ304 (1-10 microM) significantly inhibited proliferation and DNA synthesis of 50 ng/ml PDGF-BB-stimulated rat aortic VSMCs in a concentration-dependent manner. In addition, we investigated the mechanism of proliferation suppression by NQ304 in PDGF-BB-stimulated rat aortic VSMCs, and found that PDGF-BB-stimulated immediate-early gene expression (c-fos), activator protein (AP)-1 activation, extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and Akt kinase were significantly inhibited by NQ304. An examination of the suppressive effects of NQ304 on PDGF-BB-stimulated VSMC cycle progression showed that NQ304 (10 microM) induced the G1 phase arrest of PDGF-BB-stimulated cell cycle progression by elevating p21(cip1) mRNA expression. These findings suggest that the inhibitory effects of NQ304 on DNA synthesis, proliferation, and cell cycle progression on PDGF-BB-stimulated VSMCs are mediated via the downregulations of AP-1 activation and c-fos expression achieved in turn via the suppressions of the phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2 signaling pathways.
In addition to its role as a vasoconstrictor, angiotensin II also acts as a potent growth factor by activating several tyrosine kinases, including Jak2. Interestingly, Jak2 has been linked to similar cardiovascular pathologies as have been previously linked to the renin-angiotensin system. Identifying the downstream targets of Jak2 via the AT(1) receptor may therefore elucidate its role in the progression of various pathologies. Previously, microarray analysis from our laboratory identified the Type 1 inositol 1,4,5 trisphosphate (IP(3)) receptor as a potential target of Jak2 following chronic stimulation by angiotensin II. Therefore, we hypothesized that Jak2 regulates IP(3) receptor expression in response to angiotensin II. To test this hypothesis, rat aortic smooth muscle (RASM) cells over-expressing a dominant negative (DN) Jak2 protein were used. The Jak2-dependent signaling in these cells is reduced approximately 90% when compared to RASM control cells. Analysis of protein expression showed that the IP(3) receptor was degraded approximately 2-fold (P<0.05) in cells lacking functional Jak2 within 1 h of treatment by angiotensin II. Notably, degradation of the IP(3) receptor was reversible since protein levels were restored to normal following 2 h of recovery from angiotensin II. To eliminate the possibility of clonal artifact in the DN cells, wild type RASM cells were treated with the Jak2 pharmacological inhibitor, AG490. We found that angiotensin II treatment degraded IP(3) receptor in AG490-treated cells, but not in the vehicle controls. Treatment with lactacystin, a proteasome inhibitor, completely blocked angiotensin II-mediated degradation of IP(3) receptor, thereby suggesting that the degradation occurs through a proteasome-dependent mechanism. Moreover, the degradation of IP(3) receptor in DN cells correlated with a significant loss of intracellular calcium mobilization when treated with angiotensin II (DN 27.4+/-1.1% vs. WT 42.2+/-4.7%; n=5, P=0.002). We next examined through what mechanism Jak2 regulates the IP(3) receptor. When wild type RASM cells were treated with PP2, an Src-family inhibitor, IP(3) receptor expression was markedly reduced. Since previous data show that Fyn, a downstream target of Jak2, is able to phosphorylate the IP(3) receptor at Tyr 353, we believe our data suggest that Jak2 prevents the angiotensin II-mediated IP(3) receptor degradation through the activation of Fyn. In conclusion, these data suggest that Jak2 has a protective role in maintaining IP(3) receptor expression, potentially through activation of Fyn and subsequent phosphorylation of the IP(3) receptor.
Using various pharmacological methods, we previously demonstrated that the smooth muscle and endothelium of porcine coronary artery contain vasorelaxant adenosine A2 receptors, which are predominantly the A2A subtype. The present study was intended to investigate the effect of adenosine receptor stimulation on agonist-induced inositol 1,4,5-trisphosphate (IP3) generation in porcine coronary artery using the nonselective adenosine analogs, 2-chloroadenosine (CAD) and 5'-(N-ethylcarboxamido)adenosine (NECA), and the A2A selective analog 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS). In both endothelium-intact and denuded coronary artery rings, CAD, NECA and CGS elicited a dose-dependent inhibition of prostaglandin F2 alpha (PG)-induced IP3 production. However, the inhibitory effect of NECA was relatively less in endothelium-denuded preparations. The nonselective xanthine adenosine receptor antagonist, 8-sulfophenyltheophylline (8-SPT), significantly attenuated the IP3-inhibitory effect of CAD and, to a lesser extent, that of NECA. Further, the A2A selective nonxanthine antagonist, 5-amino-7-(2-phenylethyl)-2-(furyl)-pyrazolo[4,3]-1,2,4-triazolo[1,5-c] pyrimidine (SCH), markedly decreased the effects of all CAD, NECA and CGS on PG-induced IP3 generation. These results provide evidence that activation of adenosine A2 receptors by CAD, NECA and CGS in porcine coronary artery causes inhibition of agonist-induced IP3 production, and these receptors involve at least the A2A subtype.
R-102444 is a prodrug that is metabolized into R-96544, a potent and selective 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. The effects of R-102444 on peripheral vascular disease were examined using two different rat models: one induced by lauric acid and the other by ergotamine plus epinephrine. R-96544 (0.3-30 nM) relaxed the 5-HT (3 microM)-precontracted rat caudal artery in a concentration-dependent manner. The intravenous administration of R-96544 (0.3-3 microg/kg) to anesthetized rats inhibited the pressor response to 5-HT (50 microg/kg i.v.) dose dependently. The oral administration of R-102444 (1 mg/kg) to rats resulted in a marked inhibition of platelet aggregation induced by 5-HT plus ADP, and statistically significant inhibition was still evident 8 h after the dosing. In contrast, sarpogrelate, at a dose of 100 mg/kg p.o., produced only a moderate antiplatelet effect. Oral administration of R-102444 (1 mg/kg/day, o.d.) significantly prevented the progression of peripheral vascular lesion induced by the injection of lauric acid into a rat femoral artery, whereas sarpogrelate (100 mg/kg/day) showed only a minimal effect. Both 5-day treatments with R-102444 (1-30 mg/kg/day p.o., o.d.), one commenced 1 h before the injection of epinephrine plus ergotamine and one just after injection, resulted in the prevention of rat tail gangrene in a dose-dependent manner, whereas sarpogrelate (100 mg/kg) produced a minimal protection in this model. Based on these results, we conclude that 5-HT2A receptor activation is involved in peripheral vascular disease in the rat and that R-102444 is a useful oral agent for the investigation of diseases involving 5-HT2A receptor activation.
MicroRNAs (miRNAs) are small non-coding RNAs, that control diverse cellular functions by either promoting degradation or inhibition of target messenger RNA translation. An aberrant expression profile of miRNAs has been linked to human diseases, including cardiovascular dysfunction. This review summarizes the latest insights in the identification of vascular-specific miRNAs and their targets, as well as their roles and mechanisms in the vasculature. Furthermore, we discuss how manipulation of these miRNAs could represent a novel therapeutic approach in the treatment of vascular dysfunction.
Basic fibroblast growth factor (bFGF), a metastatic growth factor is known to be one of the promoting factors in the tumor induced angiogenesis. The antiangiogenic activity of acetyl 11-keto beta-boswellic acid was screened against bFGF-induced angiogenesis using in-vivo Matrigel Plug Assay. Histological & colorimetric examination confirmed that numerous blood vessels were present in Matrigel+bFGF group in comparison to Matrigel alone treated mice. Acetyl 11-keto beta-boswellic acids (10 mg/kg/d) inhibited the Matrigel+bFGF-induced angiogenesis significantly (P<0.01) in contrast to anti-inflammatory agent indomethacin (10 mg/kg/d) and alkylating agent cyclophosphamide (10 mg/kg/d).
Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important modulator of vascular function. eNOS is regulated post-translationally through phosphorylation/dephosphorylation at a number of specific phosphorylation sites including Ser-116 in the bovine eNOS sequence. Whether phosphorylation of eNOS at Ser-116 in endothelial cells is stimulatory or inhibitory has not previously been definitively determined. In this study we show that mimicking phosphorylation of eNOS at Ser-116 by Asp mutation reduces basal NO release from endothelial cells. Preventing phosphorylation at this site by Ala mutation increases the amount of NO release from endothelial cells in response to agonist stimulation. In addition, mimicking phosphorylation of Ser-116 increases eNOS association with caveolin-1 and reduces the vascular reactivity of intact aortic rings. eNOS phosphorylation at Ser-116, therefore, appears to contribute to negative modulation of eNOS activity and hence to regulation of vascular tone.
The cardiovascular effects induced by labdane-302, a diterpene isolated from the stems of Xylopia langsdorffianna St. Hill and Tull, were evaluated in male Wistar rats. In normotensive, conscious animals, labdane-302 produced dose-dependent hypotension and tachycardia. These effects were significantly attenuated after pre-treatment with L-NAME (20 mg/kg, i.v.). In isolated mesenteric artery rings, labdane-302 (10(-10)-10(-4)M) elicited concentration-dependent relaxation of phenylephrine-induced contractions (IC50 = 5.4 +/- 1.4 microM). Endothelium removal, and pre-treatment with L-NAME (100 microM) or indomethacin (10 microM) caused significant reductions in sensitivity. Labdane-302 also caused concentration-dependent relaxation in arterial rings pre-contracted with high extracellular KCl (80 mM). In Ca2+-free depolarized preparations, labdane-302 inhibited contractions produced by cumulative increases in extracellular Ca2+ concentration. In GH3 cells, labdane-302 (100 microM) inhibited whole-cell L-type Ca2+ currents by approximately 50%. These results demonstrate that labdane-302 causes hypotension through peripheral vasodilation, mediated in part by NO and PGI2 and by blockade of Ca2+ entry through L-type Ca2+ channels.
Angiostatin is an angiogenesis inhibitor in part generated by and released from platelets. Since platelets upon thrombus formation can give rise to areas of hypoxia, we investigated the effects of angiostatin on endothelial cell migration and apoptosis during hypoxia. Human microvascular endothelial cells (HMVEC-L) were exposed to angiostatin under normoxic or hypoxic conditions. Apoptosis was measured by flow-cytometry. HMVEC-L migration was studied using a modified Boyden Chamber assay, in which migration is MMP-dependent. MMP-2, MMP-14, and VEGF levels were measured using immunoblot, Q-PCR and ELISA. During hypoxia HMVEC-L were protected from angiostatin-induced apoptosis due to increased hypoxia-induced VEGF expression. However, MMP-dependent migration of HMVEC-L was inhibited by angiostatin under hypoxic but not normoxic conditions. Angiostatin decreased MMP-2 at the gene and protein levels only in HMVEC-L exposed to hypoxia. A similar result was obtained for MMP-14. Higher angiostatin concentrations, as would be seen during thrombosis, induced HMVEC-L apoptosis, which was not rescued by VEGF. Under hypoxic conditions angiostatin's primary anti-angiogenic mechanism is likely inhibition of endothelial cell MMP-dependent endothelial cell migration. Only at higher concentrations does angiostatin induce endothelial cell death. This study identifies a novel angiostatin anti-angiogenesis mechanism that is only triggered under pathological-like conditions.
The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.
We have previously reported that organ cultured coronary arteries from market-age pigs (6-9 months of age) exhibit an enhanced contraction to the atherosclerotic-associated peptide, endothelin-1 (ET-1). The objective of this study was to investigate the interaction of 17beta-estradiol with ET-1 in organ cultured coronary arteries from older female pigs (3-4 years old). A cumulative concentration-response relationship (1 x 10(-9) M to 3 x 10(-7) M) was generated to ET-1, and the isometric tension measured in fresh and organ cultured (4 days at 37 degrees C) arterial rings that were each pre-incubated for 50 min in different concentrations (1 x 10(-9) M to 1 x 10(-5) M) of 17beta-estradiol. Compared to freshly used arteries, culturing induced a 2-fold increase in tension development to ET-1 (3 x 10(-7) M). Although 17beta-estradiol previously relaxed pre-constricted (with a 60 mM KCl solution) arteries, it did not affect the constrictive response to ET-1. Also, using an ET-1 ELISA we found that 17beta-estradiol did not effect ET-1 production in intact arteries. Our results indicate that 17beta-estradiol does not attenuate the production and constrictive properties of ET-1 in coronary arteries demonstrating a dedifferentiated cell phenotype.
Type 2 diabetes mellitus is frequently accompanied by hypercoagulability and hypofibrinolysis. Both are related to increased cardiovascular risk, but possibly with endothelial injury as well. Studies with nondiabetic persons indicate that unopposed oestrogen replacement therapy (oERT) decreases cardiovascular risk, possibly mediated in part by effects on coagulation and fibrinolysis. In a double-blind, randomised placebo-controlled trial, we assessed the effect of oral 17β-oestradiol daily during 6 weeks on indicators of coagulation and of fibrinolysis in postmenopausal women with type 2 diabetes mellitus.
Insulin resistance is an underlying mechanism of type 2 diabetes and its vascular complications. Recent evidence suggests that crosstalk between angiotensin II (Ang II) and the insulin signaling in vascular smooth muscle cell (VSMC) may contribute to cellular insulin resistance. We hypothesized that Ang II inhibits the anti-mitogenic pathways while enhancing the mitogenic pathways stimulated by insulin via activation of Protein Tyrosine Phosphatase-1B (PTP-1B) in VSMC. We found that Ang II significantly inhibited insulin-induced phosphorylation of tyrosine 608 of IRS-1 and serine 473 of Akt, a downstream member of anti-mitogenic pathway of insulin. In contrast, Ang II increased the serine phosphorylation of IRS-1 which was not affected by the presence of insulin. Activation of p42/p44 MAPK (a mitogenic pathway) induced by insulin was further enhanced by Ang II. Transfection of VSMC with PTP-1B antisense oligonucleotide markedly reduced the effects of Ang II on insulin signaling. Furthermore, an increase in VSMC growth was attenuated by PTP-1B antisense only in the presence of both Ang II and insulin. Finally, we also showed that Ang II-induced activation of PTP-1B in VSMC was PKA/JAK2 dependent. We conclude that Ang II modulates both anti-mitogenic and mitogenic pathways of insulin via the activation of PTP-1B.
This article has been retracted at the request of the Editors as it contains manipulated figures.Panels in figures 5 and 6 are supposed to be representative photomicrographs of cell dishes at 0 h and 24 h after scratching. However, the panels do not represent independent data, but instead contain repetitive cell patterns suggestive of digital manipulation of these figures.As such this article represents a severe abuse of the scientific publishing system. The scientific community and the Editors take a very strong view on this matter, and apologies are offered to readers of the journal that this problem was not detected during the submission and review process.Note: The following articles related to this case have also been retracted:. Gold nanoparticles inhibit vascular endothelial growth factor-induced angiogenesis and vascular permeability via Src dependent pathway in retinal endothelial cells. Kalishwaralal K, Sheikpranbabu S, BarathManiKanth S, Haribalaganesh R, Ramkumarpandian S, Gurunathan S. Angiogenesis, 14 (2011) 29-45, doi:10.1007/s10456-010-9193-x.Isolation and characterization of goat retinal microvascular endothelial cells. Haribalaganesh R, Banumathi E, Sheikpranbabu S, Deepak V, Sirishkumar N, Gurunathan S. In Vitro Cell Dev. Biol. Anim., 46 (2010) 529-537, doi:10.1007/s11626-010-9292-4.Pigment epithelium-derived factor inhibits advanced glycation end-product-induced angiogenesis and stimulates apoptosis in retinal endothelial cells. Sardarpasha Sheikpranbabu, Ravinarayanan Haribalaganesh, Elayappan Banumathi, Namagiri Sirishkumar, Kyung-Jin Lee, Sangiliyandi Gurunathan. Life Sci., 85 (2009) 719-731, doi:10.1016/j.lfs.2009.09.015.
Increased vascular permeability associated with retinal vascular leakage is known to occur in patients with diabetes, and contributes to endothelial barrier dysfunction. The purpose of this study was to examine the effect of pigment epithelium-derived factor (PEDF) on signaling cascade in porcine retinal endothelial cells (PREC) related to permeability and angiogenesis induced by vascular endothelial growth factor (VEGF)-and interleukin-1beta (IL-1beta). PREC were exposed to VEGF, IL-1beta and PEDF at different concentrations, and in vitro permeability was assessed by solute flux assay using 70-kDa RITC-dextran. Angiogenic assays such as proliferation, migration and tube formation were determined by MTT, wound-scratch method and on-gel assay system respectively. To explore the signaling pathways behind VEGF-and IL-1beta-induced PREC permeability, an inhibitor assay was carried out using PP2, a Src kinase inhibitor. Further, Src activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Src mutants. We report that VEGF-and IL-1beta-stimulates permeability, in a dose and time-dependent manner and PEDF inhibits the VEGF-and IL-1beta-induced PREC permeability. In addition, PEDF inhibits the VEGF-and IL-1beta-induced endothelial cell proliferation, migration and tube formation. In addition, overexpression of DN Src blocked both VEGF-and IL-1beta-stimulation of permeability, proliferation and migration, while overexpression of CA Src overpowers the inhibitory action of PEDF on permeability, proliferation and migration. These results demonstrate that PEDF may inhibit the VEGF-and IL-1beta-induced permeability and angiogenesis via Src-dependent pathway.
Vascular endothelium generates nitric oxide (NO) in large vessels and induces relaxation of vascular smooth muscle cells (VSMC). The aim of this study was to evaluate the contribution of NO produced in the endothelial cells (EC) to the relaxation induced by the Ca2+ ionophore A23187 and whether this relaxation is impaired in renal hypertensive (2K-1C) rat arteries. Concentration-effect curves for A23187 were constructed in intact endothelium isolated carotid rings from 2K-1C and normotensive (2K) in the absence or in the presence of the extracellular NO scavenger haemoglobin or inhibitors of NO-synthase (NOS, L-NOARG), guanylyl-cyclase (GC, ODQ). In carotid rings loaded with Fluo-3AM, both EC and VSMC were simultaneously imaged by a confocal microscope and [Ca2+]c was derived from fluorescence intensities (IF). The maximal relaxation (ME) induced by A23187 was lower in 2K-1C than in 2K arteries. A23187-induced relaxation was abolished by haemoglobin and L-NOARG in both groups. ODQ reduced the ME to A23187 in 2K and abolished its relaxation in 2K-1C. A23187 increased [Ca2+]c in a similar way in 2K and 2K-1C EC, and decreased [Ca2+]c in VSMC, which effect was higher in 2K than in 2K-1C arteries. L-NOARG inhibited the effect of A23187 in VSMC from 2K and abolished it in 2K-1C rats. On the other hand, L-NOARG did not modify the effect of A23187 in EC from 2K and 2K-1C rats. The basal content of cGMP was higher in 2K than in 2K-1C arterial rings that was similarly increased by A23187. In conclusion, the Ca2+ ionophore A23187 increases Ca2+, activates NOS and NO production in the EC activating GC in VSMC and [Ca2+]c decrease. All these effects are higher in 2K, which contribute to the impaired relaxation to A23187 in 2K-1C rat arteries.
This study sought to examine the alteration of local angiotensin converting enzyme (ACE) activity in the aortae, heart, kidney and lung as well as plasma during the development of hypertension in one-kidney, one-clip (1K1C) model, a non-renin-dependent model of renovascular hypertension.
Experiments were carried out 2, 4, 8 and 12 weeks after induction of hypertension in male Sprague-Dawley rats. ACE activity was analyzed by high-performance liquid chromatography (HPLC) and the structural changes in aortae were investigated by measurement of cross-sectional area (CSA).
Our results show that ACE activity in aortae and heart was gradually increased with the development of hypertension and was more pronounced at higher blood pressure. In addition, there was a positive correlation between aortic CSA and elevation of blood pressure.
Our findings emphasize the significant role of local ACE, particularly in organs regulating hypertension (aortae and heart) in 1K1C model, in which circulatory renin is known to be unelevated.
Salvianolic acid B (Sal B), a bioactive compound from Salvia miltiorrhiza, widely used to treat cardiovascular diseases, and stromal cell-derived factor-1α (SDF-1α)/CXCR4 pathway has been correlated with balloon angioplasty-induced neointimal formation. The purposes of the present study were to investigate whether Sal B can inhibit SDF-1α/CXCR4-mediated effects on the cell proliferation and migration of vascular smooth muscle cells (VSMCs) and to examine its possible molecular mechanisms. Under 0.5% FBS medium, all of the cellular studies were investigated on VSMCs (A10 cells) stimulated with 10 ng/ml SDF-1α alone or co-treated with 0.075 mg/ml Sal B. Our results showed that SDF-1α markedly stimulated the cell growth and migration of A10 cells, whose effects can be significantly reversed by co-incubation of Sal B. Similarly, Sal B also obviously down-regulated the SDF-1α-stimulated up-regulation of CXCR4 (total and cell-surface levels), Raf-1, MEK, ERK1/2, phospho-ERK1/2, FAK and phospho-FAK as well as an increase of the promoter activity of NF-κB. Besides, Sal B also effectively attenuated balloon angioplasty-induced neointimal hyperplasia. In conclusion, suppressing the expression levels of CXCR4 receptor and downstream molecules of SDF-1α/CXCR4 axis could possibly explain one of the pharmacological mechanisms of Sal B on prevention of cell proliferation, migration and subsequently neointimal hyperplasia.
Statin treatment inhibits oxidized lipoprotein-induced intracellular lipid accumulation (foam cell formation) and reduces plasma levels of inflammatory markers such as interleukin-1beta (IL-1beta). The aim of the present study was to determine if simvastatin affected lipid accumulation in macrophages incubated with aggregated low density lipoproteins (AgLDL) and whether simvastatin had a direct effect on cytokine secretion from macrophages. Simvastatin treatment did not inhibit AgLDL-induced macrophage lipid accumulation, but significantly increased the secretion of IL-1beta and IL-8 from macrophages, whilst inhibiting the secretion of tumor necrosis factor-alpha (TNF-alpha) and having no significant effect on IL-6 secretion. Increased macrophage lipid content did not block statin-induced IL-1beta and IL-8 secretion. Simvastatin-stimulated IL-1beta secretion from macrophages was inhibited by isoprenoids. We therefore hypothesized that simvastatin stimulated IL-1beta secretion by affecting isoprenylation-dependent signaling pathways. Another possible mechanism for affecting such signaling is to impair isoprenoid transfer protein activity with specific inhibitors such as GGTI-297 and FTInhI. This treatment resulted in strong stimulation of IL-1beta secretion that was further enhanced when exogenous IL-1beta was present at the beginning of treatment. These data suggest an isoprenylation-dependent negative-feedback loop for macrophage IL-1beta secretion that is inhibited by statin treatment.
The chronic effects of interleukin 1-beta (IL-beta) on vascular reactivity include augmentation of contraction and relaxation. Few studies have assessed the acute effects of IL-1beta in vessels from hypertensive and normotensive rats. We hypothesized that IL-1beta would enhance constriction in aorta from stroke prone spontaneously hypertensive rats (SHRSP).
Endothelium denuded aortic rings from 12 week-old SHRSP and Wistar Kyoto (WKY) rats were mounted in a myograph and incubated with IL-1beta (20 ng/ml) for 1 h before construction of a phenylephrine dose response curve. Indomethacin (1 microM) and PP-2 (1 microM) were utilized to inhibit cyclooxygenase (COX) and Src-kinase respectively.
In aorta from SHRSP, IL-1beta caused a significant increase in the force generated over the hour incubation; inhibition of COX or Src-kinase prevented this. The maximum phenylephrine-induced contraction was greater in aorta from SHRSP incubated with IL-1beta than control. COX or Src-kinase inhibition prevented this. IL-1beta had no effect on the vessels from WKY rats.
These novel data suggest that IL-1beta has rapid effects on vascular smooth muscle from hypertensive rats to produce constriction and to enhance phenylephrine-induced constriction. The COX and Src-kinase pathways appear to be involved in this response.
This study was designed to study the influence of Sacoglottis gabonensis stem bark extract on the metabolic and cytotoxic side effects of 2,4-dinitrophenyl hydrazine (2,4-DNPH) on the brain and blood using male weaving rats as the experimental model. This was after the effect of the bark extract and bergenin, its isolate, on membrane lipid peroxidation and tissue natural antioxidant defences was reported. Lipid peroxidation was induced experimentally with a single intraperitoneal phenylhydrazine (2,4-DNPH) administration at the end of 3 days exposure to the bark extract or bergenin in drinking water. Three hours later, the brain, liver and red blood cells of the experimental animals were analysed for glucose level and the blood was analysed for selected key indices of oxidative stress: red blood cell (RBC) count haemoglobin (Hb), packed cell volume (PCV) and white blood cell (WBC) count (total and differential). The bark extract exhibited a protective action on brain glucose, significantly inhibiting the glucose-depleting action of both 2,4-DNPH and ethanol. It also inhibited the lowering action of DNPH and ethanol on PCV, RBC and Hb concentration of rat blood, but inhibited proliferation of white blood cells (total and differential). The data on the effect of bergenin, on the side effects of 2,4-DNPH experimental lipid peroxidation and on ethanol followed an essentially similar trend to those of the bark extract on brain glucose. Bergenin, similar to the bark extract, exerted a protective action on the brain tissue, though to a lesser extent, against the oxidants, 2,4-DNPH and ethanol. It is evident that aqueous ethanol extract of S. gabonensis stem bark has biological antioxidant properties against 2,4-DNPH and ethanol-induced tissue damage exerting its action on the haematological and metabolic side effects of the oxidants. By virtue of its essentially similar activity under the same conditions, bergenin appears to be the phytochemical constituent that is largely responsible for the observed action of the bark extract.
This study was designed to ascertain/verify whether Sacoglottis gabonensis stem bark extract has biological antioxidant activity in membrane lipid peroxidation using male weanling rats as the experimental animals and, if so, to attempt to establish/deduce the possible mechanism(s) of the antioxidant action of the bark extract. Lipid peroxidation was induced experimentally with a single intraperitoneal 2,4-dinitrophenyl hydrazine (2,4-DNPH) at the end of a 3-day administration with the bark extract in drinking water. Three hours later, the liver and red blood cells were analysed for the three primary antioxidant enzymes, namely catalase, superoxide dismutase (SOD) and glutathione peroxidase, and two nonenzymic antioxidants, namely vitamin E (alpha-tocopherol) and vitamin C (ascorbic acid) levels. Results showed that pretreatment with the bark extract exhibited divergent effects on natural antioxidant enzymes: It impaired the enzyme-inducing action of 2,4-DNPH (and of ethanol) on liver and red blood cell catalase but reduced the SOD depressing effect of the experimental oxidant (2,4-DNPH) and ethanol. Neither 2,4-DNPH nor the extract had any measurable effect on glutathione peroxidase. The bark extract also exerted a sparing effect on tissue antioxidant vitamins, ascorbic acid and vitamin E, effectively inhibiting their depletion by 2,4-DNPH or ethanol in the liver, red blood cells and brain. It is being concluded that the mechanism of antioxidant action of the bark extract against membrane peroxidation is multifactorial/multisystem, involving inhibition of catalase, enhancing the SOD capability of the liver and red blood cells and sparing tissue depletion/utilization of vitamins C (ascorbic acid) and E (alpha-tocopherol).
Although the past twenty years have seen a remarkable decline in the death rates of heart disease and stroke in the United States and several countries of western Europe, a reverse trend is occurring in other parts of the world. This is especially true in sub-Saharan Africa, India, China and Russia. World-wide, deaths from cardiovascular disease exceed those caused by cancer, infectious disease and trauma, constituting a deadly epidemic. Yet, in 1996 the Victoria Declaration stated that the world has the knowledge to eliminate cardiovascular disease as a major illness. Defeating such an initiative are other priorities such as education, housing, transportation, defense, as well as ignorance. The Earth Institute has labeled the needed effort, "A Race Against Time".
Effective translation of research advances from the bench to clinical and public health practice at the bedside and in the community at large represents an important step in the health research discovery enterprise. Increasingly, the gap in translating these advances into practice is being recognized. Successfully addressing this translational gap for the prevention and control of chronic diseases will require the development of novel, innovative, and, if necessary, nontraditional approaches. Participants in the 8th International Conference on Vascular Endothelium discussed a variety of novel approaches that have significant promise. Three of these approaches-vaccine development, genomics and proteomics, and tissue engineering-are highlighted in this position statement and strategies for public health practice and research are suggested.
Nitric oxide (NO) plays an important role in cardiovascular homeostasis, particularly in the regulation of vascular tone and the reactivity of platelets and circulating cells. Soluble guanylate cyclase (sGC) acts as the principal biological target for NO and catalyses the formation of the intracellular second messenger cyclic GMP (cGMP); activation of this enzyme is thought to be responsible for the majority of cardiovascular actions of NO. In the present study, we have evaluated the antiplatelet effects of a novel non-NO-based sGC activator, BAY 41-2272, in vitro and in vivo. BAY 41-2272 produced a marked inhibition of platelet aggregation in washed platelets with a potency (IC(50) approximately 100 nM) some threefold less than the NO donor S-nitrosoglutathione. BAY 41-2272 also prevented aggregation in platelet-rich plasma (PRP), albeit with a much lower potency. Both NO and prostacyclin exhibited synergistic activity with BAY 41-2272 to inhibit platelet aggregation. In vivo, at doses of BAY 41-2272 that significantly reduced blood pressure, the compound had little effect on FeCl(3)-induced thrombosis. These data confirm that intraplatelet sGC activation results in inhibition of aggregation and suggests that novel non-NO-based sGC activators, which possess both hypotensive and antiplatelet activities, may be useful as therapeutic agents.
Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes.
The purpose of the present study was to determine whether an activator of soluble guanylyl cyclase (sGC), BAY 58-2667, inhibits platelet aggregation and to clarify its mechanism of action. Blood was collected from anesthetized WKY rats. The aggregation of washed platelet was measured and the production of cAMP and cGMP was determined. BAY 58-2667 produced a partial inhibition of the ADP- and collagen-induced platelet aggregation, but did not significantly affect thrombin-induced aggregation. In ADP-induced platelet aggregation, the inhibitory effects of BAY 58-2667 were associated with an increased level of both cGMP and cAMP while that of the prostacyclin analogue, beraprost, was correlated only with an increase in cAMP. The inhibitor of sGC, ODQ, enhanced the effects of BAY 58-2667. The presence of L-nitroarginine, an inhibitor of NO-synthase, hydroxocobalamin, a scavenger of NO, or that of three different NO-donors did not affect the anti-aggregating effect of BAY 58-2667. However, the anti-aggregating effects of beraprost were potentiated by BAY 58-2667. Therefore, the platelet inhibitory effects of BAY 58-2667 are associated with the generation of cGMP and a secondary increase in cAMP, both being totally NO-independent. When the sGC is oxidized, BAY 58-2667 becomes a relevant anti-aggregating agent, which synergizes with the cAMP-dependent pathway.
1. The blood-brain barrier is essential for the maintenance and regulation of the neural microenvironment. The main characteristic features of blood-brain barrier endothelial cells are an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. 2. Endothelial blood-brain barrier tight junctions differ from epithelial tight junctions, not only by distinct morphological and molecular properties, but also by the fact that endothelial tight junctions are more sensitive to microenvironmental than epithelial factors. 3. Many ubiquitous molecular tight junction components have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin and 7H6. Signaling pathways involved in tight junction regulation include G-proteins, serine-, threonine- and tyrosine-kinases, extra and intracellular calcium levels, cAMP levels, proteases and cytokines. Common to most of these pathways is the modulation of cytoskeletal elements and the connection of tight junction transmembrane molecules to the cytoskeleton. Additionally, crosstalk between components of the tight junction- and the cadherin-catenin system of the adherens junction suggests a close functional interdependence of the two cell-cell contact systems. 4. Important new molecular aspects of tight junction regulation were recently elucidated. This review provides an integration of these new results.
Cytochrome p450 (CYP) inhibitors provide protection against myocardial infarction following both global and focal cardiac ischemia and reperfusion (I/R). We hypothesized that sulfaphenazole, an inhibitor of CYP2C6 and 9, also attenuates post-ischemic endothelial dysfunction by reducing CYP-mediated superoxide generation (which scavenges nitric oxide (NO)), thereby restoring NO bioavailability and vascular tone. Rat hearts were perfused in the Langendorff mode for 20 min in the presence, or absence, of sulfaphenazole and then subjected to 30 min global no-flow ischemia followed by 15 min reperfusion. Septal coronary resistance arteries were isolated and mounted on glass cannulae for measurements of luminal diameter. Preconstricted arteries were exposed to acetylcholine to elicit endothelium-dependent, NO-mediated vasodilation. Acetylcholine caused near maximal dilation in control tissues not subjected to I/R. Following I/R, endothelium-dependent vasodilation was reduced. Pretreatment with sulfaphenazole restored endothelial sensitivity to acetylcholine. Vasoresponsiveness to endothelium-independent vasodilators, sodium nitroprusside and isoproterenol, were also reduced following I/R. However, sensitivity to endothelium-independent vasodilators was not restored by pretreatment with sulfaphenazole. I/R-induced superoxide production was assessed by dihydroethidium staining of flash frozen hearts. Sulfaphenazole treatment significantly reduced superoxide production in arterial walls following I/R injury. We conclude that sulfaphenazole restores post-ischemic endothelium-dependent, NO-mediated vasodilation by reducing superoxide production, suggesting that CYP2C9 plays a key role in post-ischemic vascular dysfunction.
Two safrole oxide derivatives, 1-methoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (MOD) and 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (EOD), were newly synthesized as promoters of apoptosis in vascular endothelial cells (VECs). The purpose of this study was to investigate the effects of these two safrole oxide derivatives on cell growth and apoptosis induced by deprivation of survival factors (serum and fibroblast growth factors, aFGF and bFGF) in VECs. Morphological changes were observed with light microscopy. Cell growth was determined by using MTT (3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenytetrazolium) method. DNA fragmentation was analyzed by agarose gel electrophoresis and fluorescence microscopy. Apoptosis rate and cell cycle distribution were analyzed by flow cytometry (FCM). The cells deprived of FGF and serum were exposed to MOD 10-40 mg l(-1) for 24 h. Cell growth was suppressed (P<.01), while detachment and DNA fragmentation of these cells were promoted (P<.01). When the cells were treated with MOD30 mg l(-1) for 24 h, apoptosis rate was 21.43% (P<.01). The fact that 66.50% of the cells were trapped in S phase of cell cycle indicated that the cell cycle was blocked at S phase. Treated with EOD 10-40 mg l(-1) for 24 h, the cells were observed; the results showed that VEC growth was inhibited and the apoptosis was triggered (P<.01). At 30 mg l(-1) concentration, EOD blocked 55.22% of the cells at S phase. The data suggested that MOD and EOD might promote apoptosis of VEC by blocking the cell cycle at S phase.
Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), stimulate the release of C-reactive protein (CRP). We investigated the association between the TNF-alpha G-308A polymorphism and plasma CRP levels.
Subjects were 456 White (225 men, 231 women) and 232 Black (83 men, 149 women) healthy adults who underwent a 20-week standardized exercise program in the HERITAGE Family Study. The TNF-alpha gene promoter polymorphism was determined using PCR amplification followed by NcoI digestion. Plasma CRP was measured using a high-sensitivity assay.
Genotype frequencies were in Hardy-Weinberg equilibrium. After adjustment for age, smoking, alcohol consumption, maximal oxygen uptake and, in women, hormone use, the AA homozygotes for the G-308A polymorphism had higher baseline CRP levels than other genotypes in White and Black men (P<0.001 and P = 0.044, respectively) and in Black women (P = 0.032). Body mass index partly explained these associations in Blacks. The exercise program results provided further evidence for an association with the polymorphism. Among those with high CRP at baseline (> or = 3.0 mg/L), regular exercise decreased CRP less in AA homozygotes than in other genotypes (P = 0.043).
The AA genotype of the TNF-alpha G-308A polymorphism is associated with higher plasma CRP levels and less favorable CRP response to regular exercise.
The mast cell-derived serine protease chymase is importantly involved not only in degradation, but in synthesis of bioactive peptides as well. Several studies suggest that chymase is the predominant enzyme in the production of angiotensin II (Ang II) from angiotensin-I in interstitial tissues. Interestingly, chymase has also been suggested to mature endothelin-1 (ET-1) from its precursor, big-ET-1 in vitro. The lack of availability of specific chymase inhibitors, beyond the chymotrypsin-like inhibitor chymostatin, currently hampers the investigation of the chymase/ET-1/Ang II paradigm in physiology and cardiovascular diseases. Nonetheless, the recent advent of highly selective chymase inhibitors is shedding new light on the role of this enzymatic pathway in the several inflammatory prone vascular diseases as summarized in the present review. Considering increasing evidence towards significant interactions between Ang II and ET-1 in cardiovascular diseases, the present review will address the role of chymase in the production of those two peptides. Whether chymase-dependent production of ET-1 plays an important role in cardiovascular pathologies will also be discussed.
Alterations in vascular responses to beta-adrenoceptor agonists in normotensive pregnancy and pre-eclampsia are not fully understood. Thus, we studied changes in vasodilator responses to beta(2)-adrenoceptor agonist formoterol and beta(3)-adrenoceptor agonist BRL 37344 on umbilical arteries isolated from normotensive (n=12) and pre-eclamptic (n=12) pregnant women. Changes in the relaxant effect of formoterol and BRL 37344 were investigated by measuring isometric tensions in endothelium-denuded strips of umbilical arteries in the presence or absence of metoprolol, ICI 118.551 and SR 59230A (beta(1), beta(2), beta(3)-adrenoceptor antagonists, respectively, 10(-6) mol/L). Effects of formoterol and BRL 37344 on cAMP levels of umbilical arteries were evaluated by radioimmunoassay kits. Formoterol (10(-10)-10(-4) mol/L) and BRL 37344 (10(-10)-10(-4) mol/L) caused concentration-dependent relaxation of the contraction induced by phenylephrine (10(-5) mol/L) in umbilical artery strips isolated from both groups. E(max) values of formoterol and BRL 37344 (for normotensive pregnant women: 87.33+/-0.87 and 53.25+/-1.17 vs. for pre-eclampsia: 73.68+/-1.58 and 43.64+/-1.19, n=12, P>0.05, respectively) were significantly smaller in strips from pre-eclamptic women (P<0.05), with no significant change in pD(2) values. E(max) values of formoterol were significantly higher than those of BRL 37344 in both tissue (P<0.05). ICI 118.551 and SR 59230A, but not metoprolol, antagonized the relaxant effects of formoterol and of BRL 37344 on umbilical artery strips isolated from normotensive and pre-eclamptic pregnant women. Formoterol and BRL 37344 increased cAMP levels in both groups, but less significant in pre-eclamptic strips (P<0.05). These results suggest that the relaxation caused in human umbilical arteries by formoterol and BRL 37344 is mediated by a mixed population of beta(2)- and beta(3)-adrenoceptor subtypes, with contribution of cAMP. Umbilical arteries from subjects with pre-eclampsia showed a weaker beta(2)- and beta(3)-receptor-mediated relaxation to formoterol and BRL 37344, suggesting that the reduced action of formoterol and BRL 37344 may be partly due to a decreased effect of cAMP.
This study was designed to explore the role of simvastatin and its effects on the Akt/GSK3beta survival signal and apoptosis pathway after experimental subarachnoid hemorrhage (SAH). SAH was induced by blood injection into the cisterna magna in New Zealand white rabbits. Increased expression of phospho-Akt and phospho-GSK3beta was observed in brain tissue after SAH. Apoptosis and related proteins, including P53, apoptosis-inducing factor (AIF), cytochrome C, and cleaved caspase-3, were also activated. Simvastatin, at both low dose (10 mg/kg) and high dose (40 mg/kg), further increased expression of phospho-Akt and phospho-GSK3beta, decreased activation of caspase-3, and inhibited apoptosis. Preserved blood-brain barrier and attenuated brain edema were observed following simvastatin treatment. In addition, the neuroprotective effects of simvastatin were blocked by wortmannin (2.5 microg/kg/min), an irreversible PIK3 inhibitor. P53, AIF, and cytochrome C were not affected by simvastatin treatment. Findings from the present study suggest that simvastatin ameliorates acute brain injury after SAH. The potential mechanisms of action include activation of the Akt/GSK3beta survival signal and inhibition of caspase-dependent apoptosis pathway.
The purpose of the study was to examine whether arachidonic acid inhibits vascular Na(+)-K(+)-ATPase in pulmonary vasculature and if so, what are the mechanisms involved. Functional Na(+)-K(+)-ATPase activity was studied in terms of K(+)-induced relaxation in sheep pulmonary arterial rings contracted with K(+)-free solution and 5-HT. Arachidonic acid (10-100 μM) caused concentration-dependent inhibition of KCl-induced relaxations and also increased basal arterial tone. Cytochrome P-450 inhibitor, 17-octadecynoic acid (17-ODYA) completely reversed the arachidonic acid (30 μM)-induced inhibition of KCl relaxation. Further, in the presence of HET0016, a selective blocker of 20-hydroxyeicosatetraenoic acid (20-HETE), arachidonic acid-induced inhibition of KCl relaxation was not evident. Accordingly, 20-HETE, a cytochrome P-450 metabolite of arachidonic acid, also significantly attenuated KCl-induced relaxations. Norhydihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, however, partially restored the relaxation to K(+), impaired in the presence of arachidonic acid (30 μM). On the other hand, cyclooxygenase inhibitor indomethacin failed to reverse the inhibitory effect of arachidonic acid on KCl-induced relaxation. Staurosporin, a protein kinase C inhibitor, completely reversed the inhibitory effect of arachidonic acid and 20-HETE on K(+)-induced relaxation. In conclusion, the results suggest that 20-HETE, a cytochrome P-450 metabolite of arachidonic acid has a predominant role in the inhibition of functional Na(+)-K(+)-ATPase activity in the sheep pulmonary artery, while the lipooxygenase pathway has a secondary role. It is also evident that protein kinase C is involved in the inhibition of Na(+)-K(+)-ATPase by arachidonic acid/20-HETE in sheep pulmonary artery.
The aim of the present study was to evaluate the effects of inhibition of cytochrome P-450 (CYP) activity by 1-aminobenzotriazole (ABT) and by CoCl(2), first, on the development of hypertension when treatment was started in young male heterozygous Ren-2 transgenic rats (TGR) and, second, on blood pressure (BP) when treatment was started in adult TGR with established hypertension. Normotensive Hannover Sprague-Dawley (HanSD) rats served as controls. In addition, the renal cortical activities of omega-hydroxylase, the enzyme catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), and of epoxygenase, the enzyme responsible for epoxyeicosatrienoic acids (EETs) production, and urinary excretion of 20-HETE and EETs in TGR and HanSD rats were assessed. TGR have higher renal tissue omega-hydroxylase activity and urinary excretion of 20-HETE but have significantly lower renal epoxygenase activity and urinary excretion of EETs than HanSD rats. Treatment of young TGR with ABT and CoCl(2) attenuated the development of hypertension and cardiac hypertrophy and prevented glomerulosclerosis. Administration of ABT and CoCl(2) in adult TGR decreased BP, cardiac hypertrophy, but did not reduce glomerulosclerosis. Our data suggest that altered production and/or action of CYP-derived metabolites play a permissive role in the development and maintenance of hypertension in TGR by enhancing ANG II-induced vasoconstriction.
We used a selective EP4 receptor agonist, ONO-4819, and a human leukemic T cell line MOLT-3 cells, which express all four prostaglandin E2 (PGE2) receptors (EP1-EP4), to investigate whether the EP4 PGE2 receptor subtype is involved in regulating lymphocytic cholinergic activity. Phytohemagglutinin (PHA), a T cell activator, significantly enhanced the expression of EP4 receptor mRNA during the first 3-6 h of exposure, after which, expression gradually declined. Furthermore, PHA stimulation slightly but significantly up-regulated the expression of EP2 mRNA after 12 h and of EP3 mRNA after 6 h. By contrast, expression level of EP1 receptor mRNA was not affected by PHA. ONO-4819 (1 microM), which was added to cultures after 3 h of PHA stimulation, significantly increased cellular ACh content and release, and up-regulated ChAT mRNA expression and activity but inhibited MOLT-3 cell proliferation. These findings suggest that the activation of T lymphocytes up-regulates EP4 receptor mRNA expression and, to a lesser extent, EP2 and EP3 receptors and that PGE2 enhances nonneuronal lymphocytic cholinergic transmission in activated T cells, at least in part, via EP4 receptor-mediated pathways.
Previously, we identified DPTH, an analogue of antiepileptic drug phenytoin (5,5-diphenylhydantoin, DPT), capable of retarding the cell cycle in the human vascular endothelial cells. Our data suggest that DPTH inhibits human umbilical venous endothelial cells (HUVEC) proliferation by increasing the level of p21 protein, which in turn inhibits the activities of cyclin-dependent kinase (CDK)2 and CDK4, and finally interrupts the cell cycle. To search chemicals with more potency in anti-angiogenic activity, we designed and synthesized several chemical compounds based on the structure-activity relationship consideration. We evaluated the anti-angiogenic activity of these compounds by examining their effects on DNA synthesis, cell number, p21 induction and capillary-like tube formation. Our results showed that introduction of side chain containing an aromatic ring structure with right spatial arrangement at sulfur atom of DPTH enhanced the anti-angiogenic activity in HUVEC.
The term angiogenesis derives from the Greek words 'angeio' meaning blood vessel, and 'genesis' meaning production or birth, together referring to the creation of blood vessels within the body. This term has been used to generally indicate the growth and remodeling process of the primitive vascular network into a complex network during pre-natal development. After birth, reparative angiogenesis is activated during wound healing and in response to ischemia, while pathological angiogenesis contributes to tumor growth and metastasis, arthritis and ocular diseases, such as diabetic retinopathy. MicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that control gene expression by acting on target mRNAs for promoting either their degradation or translational repression. There is increasing evidence that miRNAs play important roles in vascular development as well as in vascular diseases. In this review, we aim at describing the role of miRNAs in angiogenesis, focusing, in particular, on post-ischemic neovascularization. First, we will describe the regulation and the expression of miRNAs in endothelial cells. Then, we will analyze the role of miRNAs in reparative and pathological angiogenesis. Finally, we will discuss the innovative strategies available to inhibit the level of pathogenic anti-angiogenic miRNAs and to increase expression of therapeutic miRNAs.
R(+)-[2,3-dihydro-5-methyl-3-[(moroholinyl)methyl] pyrrolo [1,2,3-de]-1,4benzoxazinyl]-1(1-naphthalenyl) methanone mesylate (Win 55,212-2) is a synthetic cannabinoid classically classified as a potent CB(1) and CB(2) agonist with high stereoselectivity and a slight preference for CB(2) cannabinoid receptors. Its vascular actions are not always explained by its binding to these cannabinoid receptors and new targets are being proposed. The aim of this study was to further assess the vascular actions of Win 55,212-2. Isometric tension changes in response to a cumulative concentration-response curve of Win 55,212-2 (10(-9) M-10(-4) M) were recorded in aortic rings from male Wistar rats. The involvement of the endothelium, cannabinoid receptors, vanilloid receptors, and the release of calcitonin gene related peptide (CGRP) was tested. Win 55,212-2 caused a concentration-dependent vasorelaxation in rat aorta. This vascular effect was significantly inhibited by endothelial denudation, inhibition of nitric oxide synthesis, a CB(1) receptor antagonist, a transient receptor potential vanilloid-1 antagonist, capsaicin desensibilization, and a CGRP receptor antagonist (P<0.001). CB(2) and non-CB(1)/non-CB(2) receptor antagonists only caused a slight inhibitory effect in vasorelaxation to Win 55,212-2. The present findings indicate that endothelium and nitric oxide-dependent vasorelaxation induced by Win 55,212-2 mainly involves vanilloid receptors while CB(1), CB(2) and nonCB(1)/nonCB(2) cannabinoid receptors have a minor participation in its vascular effect.