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Introduction: Autoantibodies including anti-human protein S antibody (anti-hPS Ab) and anti-human protein C antibody (anti-hPC Ab) can be detected in patients with autoimmune diseases with hypercoagulability. The objective of the present study was to determine the effects and molecular pathways of these autoantibodies on tissue factor (TF) expression in human coronary artery endothelial cells (HCAECs)., Materials and Methods: HCAECs were treated with anti-hPS Ab or anti-hPC Ab for 3 hours. TF expression was measured by real-time PCR and Western blot. TF-mediated procoagulant activity was determined by a commercial kit. MAPK phosphorylation was analyzed by Bio-Plex luminex immunoassay and Western blot. The potential proteins interacting with anti-hPS Ab were studied by immunoprecipitation, mass spectrometry and in vitro pull-down assay., Results: Anti-hPS Ab, but not anti-hPC Ab, specifically induced TF expression and TF-mediated procoagulant activity in HCAECs in a concentration-dependent manner. This effect was confirmed in human umbilical endothelial cells (HUVECs). ERK1/2 phosphorylation was induced by anti-hPS Ab treatment, while inhibition of ERK1/2 by U0216 partially blocked anti-hPS Ab-induced TF upregulation (P<0.05). In addition, anti-hPS Ab specifically cross-interacted with platelet phosphofructokinase (PFKP) in HCAECs. Anti-hPS Ab was able to directly inhibit PFKP activities in HCAECs. Furthermore, silencing of PFKP by PFKP shRNA resulted in TF upregulation in HCAECs, while activation of PFKP by fructose-6-phosphate partially blocked the effect of anti-hPS Ab on TF upregulation (P<0.05)., Conclusions: Anti-hPS Ab induces TF expression through a direct interaction with PFKP and ERK1/2 activation in HCAECs. Anti-hPS Ab may directly contribute to vascular thrombosis in the patient with autoimmune disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Resistin has been linked to obesity, insulin resistance, atherosclerosis, and the development of cardiovascular disease. Nevertheless, the effects and the molecular mechanisms of resistin on endothelial permeability, a key event in the development of atherosclerosis, inflammation, and vascular disease, are largely unknown. In order to determine the effect of resistin on endothelial permeability, human coronary artery endothelial cells (HCAECs) were treated with clinically relevant concentrations of resistin and the endothelial permeability was measured using the Transwell system with a Texas-Red-labeled dextran tracer. The permeability of HCAEC monolayers treated with resistin (80 ng/mL) was 51% higher than the permeability of control monolayers (P<0.05). The mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin in resistin-treated cells were 37% and 42% lower, respectively, than the corresponding levels in untreated cells. The protein levels of these molecules in resistin-treated cells were significantly reduced by 35% and 37%, respectively (P<0.05), as shown by flow cytometry and Western blot analysis. Superoxide dismutase (SOD) mimetic MnTBAP effectively blocked the resistin-mediated reduction of ZO-1 and occludin levels in HCAECs. In addition, superoxide anion production was increased from 21% (untreated cells) to 55% (cells treated with 40 ng/mL resistin), and 64% (resistin, 80 mg/mL) (P<0.05). The natural antioxidant Ginkgolide A effectively inhibited resistin-induced increase in permeability and the increase in superoxide anion production in HCAECs. Furthermore, resistin treatment significantly activated p38 MAPK, but not ERK1/2. Pretreatment of HCAECs with a p38 inhibitor effectively blocked resistin-induced permeability. These results provide new evidence that resistin may contribute to the vascular lesion formation via increasing endothelial permeability through the mechanism of oxidative stress and the activation of p38 MAPK.

Aim: The objective of this study was to determine the effects of lysophosphatidic acid (LPA) on endothelial functions and molecular alternations in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs)., Methods and Results: The vessel rings and HCAECs were treated with clinically relevant concentrations of LPA for different times. Vasomotor reactivity was studied with a myograph tension system. LPA (10 and 50 μM) treatment for the vessel rings significantly reduced endothelium-dependent vasorelaxation in response to bradykinin (×10(-5)M) by 32% and 49%, respectively, compared with the control (P<0.05). LPA decreased endothelial nitric oxide synthase (eNOS) mRNA and immunoreactivity levels in the vessel rings. In HCAECs, LPA reduced eNOS mRNA, phospho-eNOS and total eNOS protein levels. In addition, superoxide anion levels in LPA-treated vessel rings and HCAECs were significantly increased by lucegenin-enhanced chemiluminescence assay and dihydroethidium staining, respectively. Mitochondrial membrane potential and ATP content in LPA-treated HCAECs were substantially decreased. The mRNA levels of reactive oxygen species generating enzymes NOX4 and p40(phox) were increased, while endogenous antioxidant enzyme superoxide dismutase 1 was decreased in response to LPA treatment in HCAECs. Furthermore, exogenous antioxidant molecule selenomethionine (SeMet) effectively reversed these LPA-induced effects in both porcine coronary arteries and HCAECs., Conclusions: LPA causes endothelial dysfunction by a mechanism associated with decreased eNOS expression and increased oxidative stress in porcine coronary arteries and HCAECs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Resistin is a newly discovered adipocyte-derived cytokine that may play an important role in insulin resistance, diabetes, adipogenesis, inflammation, and cardiovascular disease. However, it is largely unknown whether resistin impairs endothelial functions by affecting the endothelial nitric oxide synthase (eNOS) system. In this study, we determined the effect of human recombinant resistin protein on eNOS expression and regulation in human coronary artery endothelial cells (HCAECs). When cells were treated with clinically relevant concentrations of resistin (40 or 80 ng/ml) for 24 h, the levels of eNOS mRNA, protein, and activity and eNOS mRNA stability were significantly reduced. Cellular nitric oxide levels were also decreased. In addition, the cellular levels of reactive oxygen species (ROS), including superoxide anion, were significantly increased in resistin-treated HCAECs. Mitochondrial membrane potential and the activities of catalase and superoxide dismutase were reduced. Three antioxidants, seleno-L-methionine, ginsenoside Rb1, and MnTBAP (superoxide dismutase mimetic), effectively blocked resistin-induced eNOS downregulation. Meanwhile, resistin activated the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase (JNK), and the specific p38 inhibitor SB-239063 effectively blocked resistin-induced ROS production and eNOS downregulation. Furthermore, immunoreactivity of resistin was increased in atherosclerotic regions of human aorta and carotid arteries. Thus resistin directly induces eNOS downregulation through overproduction of ROS and activation of p38 and JNK in HCAECs. Resistin-induced mitochondrial dysfunction and imbalance in cellular redox enzymes may be the underlying mechanisms of oxidative stress.

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 microg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P<0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IkappaBalpha, thereby increasing NFkappaB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkappaBalpha adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFkappaB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Objective: The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs)., Methods and Results: Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red-labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-kappaB in HCAECs., Conclusions: Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

Aims: Clinically, Chlamydia pneumoniae infection and its heat shock protein 60 (cHSP60) may contribute to atherogenesis; however, its underlying mechanisms are largely unknown. The objective of this study was to determine whether cHSP60 could cause endothelial dysfunction in human coronary artery endothelial cells (HCAECs) and porcine coronary arteries., Methods and Results: When HCAECs were treated with recombinant cHSP60, endothelial nitric oxide synthase (eNOS) mRNA and protein levels, enzyme activities, cellular NO levels, mRNA stability, and promoter activities were significantly decreased. Superoxide anion production was significantly increased due to the inhibition of mitochondrial membrane potential and catalase and superoxide dismutase (SOD) activities as well as activation of NADPH oxidase. Antioxidant seleno-l-methionine (SeMet) or SOD mimetic MnTBAP effectively blocked cHSP60-induced eNOS downregulation. In addition, cHSP60 activated mitogen-activated protein kinases (MAPKs) including p38, c-Jun-N-terminal kinase/stress-activated protein kinase, and extracellular signal-regulated kinases. Specific chemical inhibitors or their dominant-negative mutant forms of these MAPKs effectively blocked cHSP60-induced eNOS downregulation. cHSP60-induced eNOS downregulation and oxidative stress were also demonstrated in porcine coronary artery rings in vitro. Functionally, endothelium-dependent vasorelaxation was significantly reduced in cHSP60-treated vessels., Conclusion: cHSP60 directly induces eNOS downregulation through oxidative stress and MAPK activation in both HCAECs and porcine coronary arteries, thereby causing endothelial dysfunction.

Background: Lactosylceramide (LacCer) is a member of the glycosphingolipid family, which has been implicated in the atherogenic process. The goal of this study was to determine the effects and molecular mechanisms of LacCer on endothelial functions in porcine coronary arteries and human coronary endothelial cells (HCAECs)., Material/methods: The vessel rings and HCAECs were treated with different concentrations of LacCer for 24 hours. Vasomotor function was studied using a myograph tension system in response to thromboxane A2 analog U46619, bradykinin and sodium nitroprusside (SNP). Superoxide anion production was determined using lucigenin-enhanced chemiluminescence. The expression of endothelial nitric oxide synthase (eNOS), NADPH oxidase subunit NOX4 and catalase was determined by real-time PCR., Results: LacCer (0.1, 1 and 10 microM) significantly decreased endothelium-dependent vasorelaxation (bradykinin) in porcine coronary artery rings in a concentration-dependent manner compared with untreated controls (P<0.05). High concentration of LacCer (10 microM) also reduced endothelium-independent vasorelaxation (SNP). However, LacCer did not affect vessel contraction (U46619). Antioxidant selenomethionine (SeMet) effectively reversed LacCer-induced endothelial dysfunction in the vessel rings. Furthermore, LacCer significantly increased superoxide anion production in the vessel rings in a concentration-dependent manner compared with untreated controls (P<0.05). In response to LacCer treatment, NOX4 mRNA levels were significantly increased, while the expression of catalase and eNOS was significantly decreased in HCAECs compared with controls (P<0.05)., Conclusions: LacCer causes endothelial dysfunction with potential mechanisms of the down-regulation of eNOS and increase of oxidative stress due to the activation of NADPH oxidase and inhibition of internal antioxidant catalase. This study suggests that LacCer may represent a risk factor to the vascular system and antioxidant SeMet may have clinical applications for prevention of vascular disease.

Objective: Ghrelin, a novel growth hormone-releasing peptide, is implicated to play a protective role in cardiovascular tissues. However, it is not clear whether ghrelin protects vascular tissues from injury secondary to risk factors such as homocysteine (Hcy). This study investigated the effect and potential mechanisms of ghrelin on Hcy-induced endothelial dysfunction., Methods: Porcine coronary artery rings were incubated for 24 hours with ghrelin (100 ng/mL), Hcy (50 microM), or ghrelin plus Hcy. Endothelial vasomotor function was evaluated using the myograph tension model. The response to the thromboxane A(2)analog U46619, bradykinin, and sodium nitroprusside was analyzed. Endothelial nitric oxide synthase (eNOS) expression was determined using real-time polymerase chain reaction and immunohistochemistry staining, and superoxide anion production was documented lucigenin-enhanced chemiluminescence analysis. Human coronary artery endothelial cells (HCAECs) were treated with different concentrations of Hcy, ghrelin, or antighrelin receptor antibody for 24 hours, and eNOS protein levels were determined by Western blot analysis., Results: Maximal contraction with U46619 and endothelium-independent vasorelaxation with sodium nitroprusside were not different among the four groups. However, endothelium-dependent vasorelaxation with bradykinin (10(-6) M) was significantly reduced by 34% with Hcy compared with controls (P < .05). The addition of ghrelin to Hcy had a protective effect, with 61.6% relaxation, which was similar to controls (64.7%). Homocysteine significantly reduced eNOS expression, whereas ghrelin cotreatment effectively restored eNOS expression to the control levels. Superoxide anion levels, which were increased by 100% with Hcy, returned to control levels with ghrelin cotreatment. Ghrelin also effectively blocked the Hcy-induced decrease of eNOS protein levels in HCAECs in a concentration-dependent manner. Antighrelin receptor antibody effectively inhibited the effect of ghrelin., Conclusion: Ghrelin has a protective effect in the porcine coronary artery by blocking Hcy-induced endothelial dysfunction, improving eNOS expression, and reducing oxidative stress. Ghrelin also shows a protective effect on HCACEs from the Hcy-induced decrease in eNOS protein levels. The effect of ghrelin is receptor-dependent. Thus, ghrelin administration may have beneficial effects in the treatment of vascular disease in patients with hyperhomocysteinemia.

The objective of this study was to determine the effects and mechanisms of serum amyloid A (SAA) on coronary endothelial function. Porcine coronary arteries and human coronary arterial endothelial cells (HCAECs) were treated with SAA (0, 1, 10, or 25 microg/ml). Vasomotor reactivity was studied using a myograph tension system. SAA significantly reduced endothelium-dependent vasorelaxation of porcine coronary arteries in response to bradykinin in a concentration-dependent manner. SAA significantly decreased endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein levels as well as NO bioavailability, whereas it increased ROS in both artery rings and HCAECs. In addition, the activities of internal antioxidant enzymes catalase and SOD were decreased in SAA-treated HCAECs. Bio-plex immunoassay analysis showed the activation of JNK, ERK2, and IkappaB-alpha after SAA treatment. Consequently, the antioxidants seleno-l-methionine and Mn(III) tetrakis-(4-benzoic acid)porphyrin and specific inhibitors for JNK and ERK1/2 effectively blocked the SAA-induced eNOS mRNA decrease and SAA-induced decrease in endothelium-dependent vasorelaxation in porcine coronary arteries. Thus, SAA at clinically relevant concentrations causes endothelial dysfunction in both porcine coronary arteries and HCAECs through molecular mechanisms involving eNOS downregulation, oxidative stress, and activation of JNK and ERK1/2 as well as NF-kappaB. These findings suggest that SAA may contribute to the progress of coronary artery disease.

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

Objective: Our previous studies revealed upregulation of stanniocalcin-1 (STC1) in cardiac vessels in dilated cardiomyopathy. However, the functional significance of STC1 is unknown. The objective of this study was to determine the effects of STC1 on TNF-alpha-induced monolayer permeability of human coronary artery endothelial cells (HCAECs)., Methods and Results: Cells were pretreated with STC1 for 30 minutes followed by treatment with TNF-alpha (2 ng/mL) for 24 hours. Monolayer permeability was studied using a transwell system. STC1 pretreatment significantly blocked TNF-alpha-induced monolayer permeability in a concentration- and time-dependent manner. STC1 effectively blocked TNF-alpha-induced downregulation of endothelial tight junction proteins zonula occluden-1 and claudin-1 at both mRNA and protein levels. STC1 also significantly decreased TNF-alpha-induced superoxide anion production. The inhibitory effect of STC1 was specific to TNF-alpha, as it failed to inhibit VEGF-induced endothelial permeability. Furthermore, STC1 partially blocked NF-kappaB and JNK activation in TNF-alpha-treated endothelial cells. JNK inhibitor and antioxidant also effectively blocked TNF-alpha-induced NF-kappaB activation and monolayer permeability in HCAECs., Conclusions: STC1 maintains endothelial permeability in TNF-alpha-treated HCAECs through preservation of tight junction protein expression, suppression of superoxide anion production, and inhibition of the activation of NFkappaB and JNK, suggesting an important role for STC1 in regulating endothelial functions during cardiovascular inflammation.

Background: Homocysteine is an independent risk factor for atherosclerosis. The objective of this study was to investigate whether ginkgolide A (GA), a major constituent of Ginkgo biloba, could block homocysteine-induced endothelial dysfunction in porcine coronary arteries., Methods: Porcine coronary artery rings were assigned to six treatment groups: control; homocysteine (50 micromol/L); low-dose (50 micromol/L) or high-dose (100 micromol/L) GA; and homocysteine plus low-dose or high-dose GA. After 24 hours' incubation, the rings were analyzed for vasomotor function in response to a thromboxane A2 analogue (U46619), bradykinin, and sodium nitroprusside. Endothelial nitric oxide synthase (eNOS) was studied by using real-time polymerase chain reaction and immunohistochemistry analysis. Superoxide anion production was assessed by chemoluminescence analysis., Results: Endothelium-dependent relaxation (bradykinin) was significantly reduced in ring segments treated with homocysteine as compared with the control (P < .05). When homocysteine was combined with either low-dose or high-dose GA, endothelium-dependent relaxation was markedly recovered. There was no significant difference in maximal contraction (U46619) or endothelium-independent relaxation (sodium nitroprusside) among all groups. In addition, superoxide anion production was increased by 113% in the homocysteine-treated group, whereas there was no statistically significant difference between the control and GA/homocysteine groups. Furthermore, eNOS messenger RNA and protein levels were substantially reduced in the homocysteine-treated group (P < .05), but not in the GA/homocysteine combined groups., Conclusions: Homocysteine significantly impairs endothelium-dependent vasorelaxation through oxidative stress and downregulation of eNOS in porcine coronary arteries. GA effectively prevents homocysteine-induced endothelial dysfunction and molecular changes in porcine coronary arteries. This study underscores the potential clinical benefits and applications of GA in controlling homocysteine-associated vascular injury and cardiovascular disease., Clinical Relevance: Homocysteine is an independent risk factor for atherosclerosis. This study showed that ginkgolide A, a major constituent of Ginkgo biloba, effectively prevents homocysteine-induced endothelial dysfunction and molecular changes in porcine coronary arteries. This study underscores potential clinical benefits and applications of ginkgolide A in controlling homocysteine-associated vascular injury and cardiovascular disease.

Background: Secretoneurin (SN), a novel neuropeptide, may play a role in inflammation in the vascular system. However, the interaction between SN and endothelial cells is largely unknown. This study's objective is to investigate the effects of SN on endothelial permeability and its associated molecular mechanisms in human coronary artery endothelial cells (HCAECs)., Methods: HCAECs were treated with SN. Monolayer permeability was assayed with a transwell system and a Texas Red-labeled dextran tracer. The mRNA and protein levels of endothelial junctional molecules were determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Superoxide anion was determined by fluorescent dye dihydroethidium-based flow cytometry detection. Mitogen-activated protein kinase (MAPK) activation was determined by Bio-Plex immunoassay., Results: HCAECs were treated with SN (15, 30, and 60 ng/ml) for 24 hours and showed a significant increase of monolayer permeability by 12%, 33%, and 47%, respectively, compared with controls (P < 0.05). SN-treated cells showed a significant reduction of zonula occludens-1 (ZO-1) and occludin at both mRNA and protein levels (P < 0.05). In addition, SN significantly increased superoxide anion in HCAECs (P < 0.05). Furthermore, SN activated MAPKs (JNK and ERK1/2) but not p38. Both antioxidant seleno-L-methionine (SeMet) and specific inhibitors of JNK and ERK1/2 effectively blocked SN-induced monolayer permeability increase in HCAECs., Conclusions: SN significantly increases monolayer permeability and reduces the expression of ZO-1 and occludin through oxidative stress and the activation of JNK and ERK1/2 in HCAECs. This study provides direct evidence that SN impairs endothelial barrier function.

Background: Recently, CD40 ligand (CD40L) and its receptor CD40 have been implicated in atherosclerosis. Clinical data showed that elevated CD40L levels are associated with a high risk of cardiovascular events. The aim of this study was to investigate whether CD40L could affect the expression of its membrane receptor CD40 as a feedback mechanism by which CD40L could enhance its functions in human coronary artery endothelial cells (HCAECs)., Methods: The HCAECs were treated with human soluble CD40L, and the messenger RNA (mRNA) and protein levels of CD40 were determined by real-time polymerase chain reaction and Western blot analysis, respectively. The specific effect of CD40L was confirmed by a blocking experiment with antibody against CD40L. Involvements of oxidative stress and mitogen-activated protein kinases (MAPKs) were also studied with antioxidant seleno-L-methionine (SeMet) and MAPK inhibitors such as extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor., Results: When HCAECs were cultured with CD40L (5 microg/mL) for 24 hours, CD40 mRNA levels were increased by 79% compared with controls (P < .05). Similarly, Western blot analysis showed an 80% increase in CD40 protein levels (P < .05). The CD40L-induced increase in CD40 mRNA levels were blocked specifically by anti-CD40L antibody. Antioxidant SeMet and specific ERK1/2 inhibitor (PD98059) also effectively blocked CD40L-induced CD40 mRNA increase., Conclusions: These data demonstrate that clinically relevant concentration of CD40L increased the expression of its receptor CD40 in HCAECs. The CD40L-induced upregulation of CD40 may be mediated by oxidative stress and ERK1/2 activation. This study suggests a new mechanism by which CD40L could enhance its biologic functions in the vascular system and contribute to endothelial dysfunction and vascular disease.

Objective: Resistin may be associated with obesity and cardiovascular diseases. However, it is unknown whether resistin directly contributes to angiogenesis. In the present study, we evaluated the effects of resistin on angiogenic potential, including endothelial cell proliferation, migration, and capillary-like tube formation., Methods: Human coronary artery endothelial cells (HCAECs) were treated with resistin. Cell proliferation was evaluated by [3H]thymidine incorporation and MTS assays. Cell migration was assessed by a modified Boyden chamber assay. Capillary-like tube formation was studied with a Matrigel model. Several gene expression levels were determined by real-time PCR. Activation of mitogen-activated protein kinases (MAPKs) was determined by Bio-Plex luminex analyzer. Basic fibroblast growth factor (bFGF) was used as a control. Human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVEC-L) were also included., Results: Resistin induced both endothelial proliferation and migration in a dose- and time-dependent manner with the maximal effect at 40 ng/ml. Both resistin-induced cell proliferation and migration could be effectively blocked by a resistin-neutralization antibody. In addition, resistin promoted capillary-like tube formation of HCAECs on Matrigel. Resistin also significantly upregulated the mRNA expression of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) and matrix metalloproteinases (MMP-1 and MMP-2) at both mRNA and protein levels. Furthermore, transient phosphorylation of ERK1/2 and p38 was observed after the addition of resistin to HCAECs. The resistin-induced cell proliferation and migration were both completely blocked by specific ERK1/2 and p38 inhibitors., Conclusions: Resistin induces human endothelial cell proliferation and migration, promotes capillary-like tube formation, upregulates the expression of VEGFRs and MMPs, and activates ERK1/2 and p38 pathways. Thus, resistin may play an important role in angiogenesis-associated vascular disorders.

Thymosin beta10 is a cytoplasm G-actin sequestering protein whose functions are largely unknown. To determine the direct effects of exogenous thymosin beta10 on angiogenic potentials as endothelial cell migration and capillary-like tube formation, human coronary artery endothelial cells (HCAECs) were incubated with increasing doses of thymosin beta10 (25-100 ng/ml). By using a modified Boyden chamber assay, thymosin beta10 inhibited cell migration in a dose- and time-dependent manner with the maximal effect being a 36% reduction at 100 ng/ml as compared to controls (P < 0.01). In addition, thymosin beta10 (100 ng/ml) significantly inhibited the capillary-like tube-formation of HCAECs on Matrigel, showing a 21% reduction of the total tube length as compared to negative controls (P < 0.01). Furthermore, by using real time PCR analysis, thymosin beta10 significantly decreased mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1) and integrin alphaV after 24 h treatment in HCAECs. By contrast, thymosin beta4 significantly increased HCAEC migration. These results indicate that thymosin beta10, but not thymosin beta4, have direct inhibitive effects on endothelial migration and tube formation that might be mediated via downregulation of VEGF, VEGFR-1 and integrin alphaV in HCAECs. This study suggests a potential therapeutic application of thymosin beta10 to the diseases with excessive angiogenesis such as cancer., (Copyright 2006 Wiley-Liss, Inc.)

Background: Defensins are cysteine-rich cationic polypeptides released from neutrophils that exhibit powerful antimicrobial activities. Because inflammation, including neutrophil infiltration and release of defensins, may play an important role in atherosclerosis and other vascular diseases, we determined whether alpha-defensin could cause endothelial dysfunction, a major initial event of atherosclerosis, in porcine coronary arteries., Methods: Porcine coronary arteries were sliced into 5-mm rings and treated with different concentrations of human recombinant alpha-defensin for 24 hours. Vasomotor reactivity was studied by using a myograph system. Levels of superoxide anion were detected by the lucigenin-enhanced chemiluminescence method. Endothelial nitric oxide synthase (eNOS) messenger RNA (mRNA) and protein levels were determined by real-time polymerase chain reaction and immunohistochemistry analysis, respectively., Results: Endothelium-dependent relaxation in response to bradykinin was significantly reduced by 40% for the rings treated with 1500 nM of alpha-defensin compared with controls (P< .05). Vessel contractility in response to the thromboxane A2 analogue U46619 and endothelium-independent relaxation in response to sodium nitroprusside were not affected with defensin treatment. In addition, the superoxide anion level at the endothelial layer of porcine coronary artery rings was significantly increased by 80% in the defensin-treated (1500 nM) vessels compared with controls (P< .05). Furthermore, the eNOS mRNA levels in endothelial cells isolated from the cultured rings treated with defensin (1500 nM) were significantly decreased by 27% compared with controls (P< .05). Immunoreactivity of eNOS in the defensin-treated vessel rings was also substantially reduced., Conclusions: Defensin reduces the endothelium-dependent vasorelaxation. This effect is associated with increased superoxide radical production and decreased eNOS expression in porcine coronary arteries., Clinical Relevance: Inflammation is an important mechanism of atherosclerosis and other vascular diseases. The roles and interactions of biomediators released from inflammatory cells are not fully understood, however. This study provides new information about effects and potential molecular mechanisms of a major neutrophil releasing factor, alpha-defensin, on endothelial dysfunction of porcine coronary arteries. Thus, targeting alpha-defensin and its associated molecular mechanisms may become a new strategy to prevent vascular diseases.

HIV protease inhibitors (PIs) have been implicated to cause cardiovascular complications. Previous studies demonstrated that the PI ritonavir (RTV) caused endothelial dysfunction in porcine arteries. This study investigated and compared the effects of 5 commonly used PIs on vasomotor function, endothelial nitric oxide synthase (eNOS) expression, and oxidative stress in porcine coronary arteries. Vessel rings were incubated with 15 microM of RTV, amprenavir (APV), saquinavir (SQV), indinavir (IDV), or nelfinavir (NFV) for 24 hours. Vasomotor function was studied using a myograph system. The contractility of the rings was significantly reduced for RTV and SQV. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation was significantly reduced for RTV, APV, and SQV. The eNOS mRNA levels were significantly reduced for RTV, APV, and SQV. Furthermore, the superoxide anion (O(2)(-)) levels of the vessels were significantly increased for RTV and APV. It was found that nitric oxide production was decreased, whereas the level of nitrotyrosine proteins was increased in RTV-treated vessels. Furthermore, antioxidant seleno-L-methionine (SeMet) reversed RTV-induced O(2)(-) production and vasomotor dysfunction. Thus, the HIV PIs RTV, APV, and SQV at 15 microM have more potent in vitro effects on vasomotor dysfunction, eNOS downregulation, and O(2)(-) production than IDV and NFV. The antioxidant SeMet can block these adverse effects of RTV. The results suggest that antioxidant therapy may have applications for controlling PI-associated cardiovascular complications.

Background: Lysophosphatidylcholine (LPC) is a product of phosphatidylcholine hydrolysis by phospholipase A(2) and a mediator of the lipid-induced atherosclerotic changes. In this study, we determined the effects of LPC on vasomotor functions, oxidative stress, and endothelial nitric oxide synthase (eNOS) expression in porcine coronary arteries., Methods: Porcine coronary arteries were cut into 5-mm rings and were treated with LPC or antioxidant selenomethionine (SeMet). For the vasomotor studies, we used a myograph tension system. Levels of superoxide anion (O(2)(-)) were detected by the lucigenin-enhanced chemiluminescence method. The eNOS protein level was studied by immunohistochemistry with avidin-biotin complex immunoperoxidase procedure., Results: Endothelium-dependent relaxation in response to bradykinin was reduced by 36% and 81% for the rings treated with 12.5 and 25 mum of LPC, respectively, as compared with controls (P < 0.05). Endothelium-independent relaxation in response to sodium nitroprusside also was reduced by 63% after treatment with 25 mum LPC (P < 0.05). The O(2)(-) level was increased in the porcine arteries treated with 25 mum of LPC by 41% as compared with controls (P < 0.05). The antioxidant SeMet reversed the effects of LPC on vascular relaxation and O(2)(-) production. Immunoreactivity of eNOS in LPC-treated vessel rings also was reduced substantially., Conclusions: LPC impairs endothelium-dependent and endothelium-independent vasorelaxation. This effect is associated with increased superoxide radical production and decreased eNOS activity and is practically reversed with the use of the antioxidant SeMet.

Background: HIV protease inhibitor ritonavir (RTV) is associated with many cardiovascular complications and causes vascular dysfunction through oxidative stress. In the present study, we determined the effects of RTV and curcumin (a pigment derived from turmeric) on porcine coronary arteries., Study Design: Artery rings were incubated with 15 microM concentration of RTV and curcumin (5 or 10 microM) for 24 hours. Vasomotor function was studied with a myograph tension system. Endothelial nitric oxide synthase (eNOS) mRNA and protein levels were studied using real-time polymerase chain reaction, Western blot, and immunohistochemistry. Nitric oxide was measured using Griess assay. Superoxide anion levels were determined by lucigenin enhanced chemiluminescence., Results: RTV considerably reduced vessel contraction by 71%, endothelium-dependent relaxation by 59%, and endothelium-independent relaxation by 52%, as compared with controls. Curcumin effectively blocked RTV-induced vasomotor dysfunction. RTV-treated arteries showed substantial reductions of eNOS mRNA by 77%, eNOS protein by 72%, and nitric oxide release by 37% as compared with controls. The RTV plus curcumin-treated vessels showed substantial increases of eNOS and nitric oxide levels as compared with the RTV-treated vessels. Finally, there was a 47% increase of superoxide anion production in the RTV-treated vessels as compared with controls. Again, curcumin effectively reversed this effect of RTV., Conclusions: HIV protease inhibitor RTV impairs vasomotor functions, reduces eNOS expression and nitric oxide release, and increases oxidative stress in porcine coronary arteries. Curcumin effectively blocks these detrimental effects of RTV.

Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A(2) analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10(-5) M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls (P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups (P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group (P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O(2)(-) production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O(2)(-). These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.

Objective: Homocysteine (Hcy) is an independent risk factor for atherosclerosis. This study investigates the effects of ginsenoside Rb1, a major constituent of ginseng, on Hcy-induced endothelial dysfunction and molecular changes in porcine coronary arteries., Methods: The coronary arteries were harvested from pig hearts and cut into 5-mm ring segments, which were then divided into six groups, including control, Hcy alone (50 microM), low-dose (1 microM) or high-dose (10 microM) Rb1 alone, and Hcy plus low-dose or high-dose Rb1. After 24-hour incubation, the rings were analyzed for vasomotor function in response to thromboxane A2 analog U46619, bradykinin, and sodium nitroprusside (SNP), respectively. In addition, superoxide anion was assessed by lucigenin-enhanced chemiluminescence analysis. Endothelial nitric oxide synthase (eNOS) was studied using real-time polymerase chain reaction and Western blot., Results: Endothelium-dependent relaxation (bradykinin) was significantly reduced in rings treated with Hcy alone as compared with the control (49.80% vs 71.77%, n = 8, P < .05), whereas neither high-dose nor low-dose Rb1 alone affected the endothelium-dependent relaxation. The low-dose Rb1-Hcy combined group had a partially improved endothelium-dependent relaxation (54.44%), whereas the high-dose Rb1-Hcy combined group showed a complete recovery of endothelium-dependent relaxation (72.89%). There was no substantial difference in maximal contraction induced by U46619 or endothelium-independent relaxation by SNP among all groups (P > .05). Furthermore, superoxide anion was markedly increased by 137% in the Hcy-treated group as compared with the control, but there were no statistically significant changes from the control in all other groups (P > .05). Lastly, eNOS mRNA and protein levels were substantially reduced in the Hcy-treated group, but not in the Rb1-Hcy combined groups., Conclusions: This is the first study to show that ginsenoside Rb1 can effectively block Hcy-induced endothelial dysfunction and superoxide anion production as well as eNOS downregulation in porcine coronary arteries. This study suggests that ginseng and its active constituents may have potential clinical applications in controlling Hcy-associated vascular injuries., Clinical Relevance: Homocysteine (Hcy) is an independent risk factor for atherosclerosis and other vascular lesions. It causes endothelial dysfunction and oxidative stress. Ginseng compounds have effects of vasorelaxation and antioxidation. The purpose of this study was to determine the effect of ginsenoside Rb1, a major constituent of ginseng, on Hcy-induced endothelial dysfunction and molecular changes in porcine coronary arteries. Our results showed that ginsenoside Rb1 can effectively block Hcy-induced dysfunction of endothelium-dependent vasorelaxation as well as superoxide anion production and eNOS downregulation. This study suggests that ginseng compounds may have potential clinical applications in controlling Hcy-associated vascular diseases and other vascular lesions.

Objective: Resistin, a novel adipocyte-derived cytokine, is involved in the development of insulin resistance and diabetes mellitus. In this study, we determined whether resistin could affect vasomotor function, oxidative stress, and endothelial nitric oxide synthase (eNOS) expression in porcine coronary arteries., Methods: Porcine coronary arteries were treated with resistin or antioxidant seleno-L-methionine (SeMet). Vasomotor function was studied by using a myograph system. Levels of superoxide anion (O 2 - ) were detected by the lucigenin-enhanced chemiluminescence method. The eNOS mRNA and protein levels were determined by real-time polymerase chain reaction and immunohistochemistry, respectively. Culture of isolated porcine coronary artery endothelial cells (PCAECs) was also included., Results: Endothelium-dependent relaxation in response to bradykinin was reduced by 15% and 30% for the rings treated with 10 and 40 ng/mL of resistin, respectively, as compared with controls ( P < .05). Endothelium-independent relaxation in response to sodium nitroprusside (SNP) was also reduced by 11% after treatment with 40 ng/mL of resistin ( P < .05). The O 2 - level was increased in the 40 ng/mL resistin-treated vessels by 88% as compared with controls ( P < .05). SeMet reversed these effects. The eNOS mRNA levels in PCAEC cultures treated with resistin (10 and 40 ng/mL) were decreased by 27% and 55%, respectively ( P < .05) and by 39% in the endothelial cells purified from porcine coronary artery rings after treatment with 40 ng/mL of resistin ( P < .05). Immunoreactivity of eNOS in the resistin-treated vessel rings was also substantially reduced., Conclusions: Resistin reduces the endothelium-dependent and endothelium-independent vasorelaxation. This effect is associated with increased superoxide radical production, decreased eNOS expression, and is effectively reversed by the antioxidant SeMet., Clinical Relevance: Obesity has been considered to be an independent risk factor for coronary artery disease and other vascular lesions. Resistin is a newly discovered adipocyte-derived cytokine, and its plasma levels are increased in obese individuals. However, it is not clear whether resistin could directly contribute to vascular disease formation. This study showed that resistin can cause endothelial dysfunction in porcine coronary arteries through oxidative stress and down-regulation of eNOS. Thus, this study may suggest a new mechanism of obesity-associated vascular disease and that antioxidants may effectively prevent vascular disease in obese individuals.

Background: The effects of hormone replacement therapy (HRT) on the vascular endothelium have been controversial. In this study, we determined the effects of HRT on endothelium-dependent relaxation in a porcine coronary artery model., Methods: Coronary artery rings harvested from female swine were incubated as controls or with estrogen (10(-9), 10(-8), 10(-7) g/L), progesterone (1 x 10(-6), 1 x 10(-5), 5 x 10(-5) g/L), or a combination of the two (10(-8)g/L estrogen, 1 x 10(-5)g/L progesterone). After 24 h in tissue culture, the rings were tested on a myograph system to measure contraction and endothelium-dependent relaxation. Myograph analysis was performed with the thromboxane A2 analogue U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Nitric oxide synthase (eNOS) levels were determined by immunohistochemistry. Levels of superoxide anion in the progesterone or estrogen treated tissues were assessed by lucigenin-enhanced chemiluminescence analysis., Results: In response to 10(-7)M bradykinin, porcine coronary artery rings treated with 1 x 10(-6), 1 x 10(-5) and 5 x 10(-5) g/L of progesterone showed a significant reduction in endothelium-dependent vasorelaxation by 36%, 45%, and 68%, respectively, as compared to controls (P <0.05). However, rings treated with estrogen showed no significant difference as compared to controls. Furthermore, estrogen treatment with progesterone reversed the effect of progesterone, showing no difference in vessel relaxation as compared to controls. There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between the control and the hormone-treated groups. The eNOS immunoreactivity was reduced in progesterone-treated coronary artery rings. Furthermore, coronary endothelium exposed to progesterone showed a 59% increase in superoxide anion production, while estrogen produced a 67% decrease when compared to controls (P <0.05 for both)., Conclusion: This data suggests that the progesterone component of HRT has a detrimental influence on endothelium-dependent relaxation. This effect appears to be related to decreased eNOS levels, as well as increased consumption of NO by superoxide anion in the endothelium of tissues exposed to progesterone. Estrogen can block progesterone-induced endothelial dysfunction and superoxide anion production in the pig coronary artery model.

Development of coronary vessels is a complex process in developmental biology and it may have clinical implications. Although coronary vessels develop as a form of vasculogenesis followed by angiogenesis, the cells of the entire coronary system do not arise from the developing heart. The key events of the coronary system formation include the generation of primordium and proepicardial organ; formation of epicardium; generation of subepicardial mesenchymal cells, and the formation, remodeling and maturation of the final vascular plexus. These events represent a complex regulation of the cell fate determination, cellular migration, epicardial/ mesenchymal transformation, and patterning of vasculatures. Recent studies suggest that several transcription factors, adhesion molecules, growth factors and signaling molecules play essential roles in these events. This article reviews the literature on the development of coronary vessels, and discusses current advances and controversies of molecular and cellular mechanisms, thereby directing future investigations.

Background: The objective of this study was to determine the expression of somatostatin (SST) and its receptors (SSTRs) and their regulation by TNF-alpha as well as cell proliferation in response to SST in human endothelial cells., Materials and Methods: Human coronary artery endothelial cells (HCAECs) were cultured without or with TNF-alpha (0.1, 1, or 10 ng/ml) for 24 h. The mRNA levels of SST, SSTR-1-5, as well as a housekeeping gene (beta-actin) were determined by real-time RT-PCR. Expression of SSTR-2 was also demonstrated by immunofluorescence staining. Cell proliferation in response to SST treatment (0.04, 0.2, or 1 ng/ml) was performed by [3H]thymidine incorporation., Results: Without TNF-alpha treatment, HCAECs showed mRNA expression of SST, SSTR-1, SSTR-2, and SSTR-5. The mRNA of SSTR-2 was expressed at a higher level than that of SSTR-1 and SSTR-5. However, SSTR-3 and SSTR-4 were not expressed or were minimally expressed. After treatment with TNF-alpha, the mRNA levels of SST, SSTR-1, SSTR-2, and SSTR-5 were significantly reduced in a dose-dependent fashion. TNF-alpha (1 ng/ml) reduced SST, SSTR-1, SSTR-2, and SSTR-5 by 93, 51, 85, and 99%, respectively, compared to controls (P < 0.001, t test). The immunoreactivity of SSTR-2 was also reduced after TNF-alpha treatment. SST-treated cells showed a significant reduction in [3H]thymidine incorporation in a dose-dependent manner. TNF-alpha treatment decreased SST inhibitory potential in cell proliferation., Conclusions: HCAECs express SST, SSTR-1, SSTR-2, and SSTR-5, which are all decreased by TNF-alpha treatment. Furthermore, treatment with exogenous SST significantly reduces cell proliferation, and this inhibitory effect is also decreased by TNF-alpha.

Purpose: Curcumin, a yellow polyphenolic compound from the plant Curcuma ionga , is a commonly used spice and coloring agent with beneficial effects of anti-tumor, anti-inflammatory, and antioxidant activities. The objective of this study was to determine the effect of curcumin on homocysteine-induced endothelial dysfunction in a porcine coronary artery model., Methods: Porcine coronary arteries were cut into 5-mm rings, which were incubated for 24 hours either as control rings, with homocysteine (50 micromol/L), curcumin (5 micromol/L), or a combination of curcumin (5 micromol/L) and homocysteine (50 micromol/L). Myograph tension analysis was performed in response to vessel active drugs including thromboxane A2 analog U466419 (contraction), endothelium-dependent vasorelaxation (bradykinin), and endothelium-independent vasorelaxation (sodium nitroprusside). Immunohistochemical staining was performed for endothelial nitric oxide synthase (eNOS). In addition, superoxide anion production was determined by lucigenin-enhanced chemiluminescence., Results: All groups of porcine coronary artery rings showed no difference in maximal contraction after U46619 challenge. However, endothelium-dependent vasorelaxation in response to 10(-5) mol/L bradykinin was 40% in the homocysteine-treated group, as compared to 73% in the control group (P = .03). Of importance, curcumin could effectively block homocysteine-induced impairment of endothelium-dependent vasorelaxation. All groups showed no difference in endothelium-independent vasorelaxation. In addition, eNOS immunoreactivity was reduced in the homocysteine group, but the combined homocysteine and curcumin group showed eNOS levels comparable to those in the control group. Furthermore, superoxide anion levels of the endothelial layer were significantly increased by 2-fold in homocysteine-treated vessels as compared to control vessels (P = .02), whereas curcumin could block the effect of homocysteine on superoxide anion production., Conclusion: These data demonstrate that curcumin effectively reverses the endothelial dysfunction induced by homocysteine. In addition, curcumin significantly blocked homocysteine-induced superoxide anion production and eNOS down-regulation. This study suggests a therapeutic role for dietary curcumin in patients with homocysteinemia, thereby reducing cardiovascular morbidity and mortality., Clinical Relevance: Hyperhomocysteinemia is a significant clinical problem. It is an independent risk factor for cardiovascular diseases. This study provides new information for better understanding the molecular mechanisms of homocysteine-induced vascular injury. More importantly, curcumin, a natural substance, can effectively block the detrimental effect of homocysteine on the vascular system. Thus curcumin could be used in patients with hyperhomocysteinemia, and to prevent cardiovascular diseases.

Background: The objective of this study was to examine the effect of estrogen combined with homocysteine on vasomotor function and endothelial integrity in intact porcine coronary arteries., Materials and Methods: Pig coronary artery rings were incubated with estrogen, homocysteine, or estrogen and homocysteine for 24 h. Myographic analysis was performed with thromboxane A2 analog U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Endothelial nitric oxide synthase (eNOS) levels were determined by immunohistochemistry. Levels of superoxide anion were assessed by lucigenin-enhanced chemiluminescence analysis., Results: Endothelium-dependent vasorelaxation (bradykinin) for the homocysteine alone group was 62% compared with control (P < 0.05), and endothelium-dependent vasorelaxation for the estrogen alone group was 85% compared with control (P > 0.05). Endothelium-dependent vasorelaxation for the estrogen-homocysteine combined group was 79% compared with 89% for control (P > 0.05). There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between all three groups and control. In addition, the eNOS immunoreactivity was declined in the homocysteine group and had no major change in the estrogen or estrogen plus homocysteine-treated group as compared with controls. The superoxide free radical measurement showed a marked increase in the homocysteine group, no major change from controls in the estrogen group, and a much-lessened effect in the combination of estrogen and homocysteine., Conclusions: These data demonstrate that combining estrogen with homocysteine significantly blocks the effect of homocysteine on impairing endothelium-dependent vasorelaxation as well as on decreasing eNOS expression and increasing oxidative stress in porcine coronary arteries. This study suggests that estrogen may play a role in preventing homocysteine-mediated endothelial dysfunction and may be of benefit in the hyperhomocysteinemic patient.

Background: Heterogeneity of endothelial cells may affect angiogenesis, vascular healing, and cardiovascular disease formation. The objective of this study was to identify and characterize populations of human coronary artery endothelial cells (HCAECs), their gene expression levels, and response to TNF-alpha stimulation., Materials and Methods: Commercial HCAECs were cultured with EGM-2 complete medium. Cell population was determined by flow cytometry analysis based on size-scatter distribution. Gene expression for each population with or without TNF-alpha (1 ng/ml) stimulation for 16 h was also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies., Results: Two distinguished populations, HCAEC-I (small) and HCAEC-II (large), were identified through all passages (from 2 to 10) during cultures. Both HCAEC-I and HCAEC-II showed more than 90% positive staining for both von Willebrand factor and CD31 and were negative for CD34 and CD4 (less than 2%). HCAEC-I had substantially higher expression of cadherin-5 (71.91% versus 51.07%) than HCAEC-II. HCAEC-I also expressed much higher levels of vascular endothelial growth factor receptor-2 (VEGF-R2) (17.35% versus 0.77%), endothelial nitric oxide synthase (eNOS) (44.64% versus 2.54%), VCAM-1 (6.08% versus 1.18%), E-selectin (18.68% versus 1.42%), CXCR4 (61.05% versus 7.98%), and CCR5 (48.66% versus 1.97%) than HCAEC-II. HCAEC-II, however, had substantially higher expression of P1H12 (93.07% versus 58.73%) and ICAM-1 (94.37% versus 58.62%) than HCAEC-I. Following TNF-alpha stimulation, both populations substantially increased the expression of ICAM-1, VCAM-1, and E-selectin. HCAEC-I, however, had much higher expressions of VEGFR-2, eNOS, CXCR4, and CCR-5 than HACEC-II after TNF-alpha stimulation., Conclusions: These data demonstrate that commercial HCAECs have two distinguished populations with different gene expression patterns and different responses following TNF-alpha stimulation. This study indicates that the heterogeneity of HCAECs may have biologic or pathologic significances in coronary arteries.

Background: Hyperhomocysteinemia is an independent risk factor of coronary artery disease. Clinical studies have indicated that moderate red wine consumption is associated with a reduction of incidence of coronary artery disease. In this study, we determined the effect of red wine on homocysteine- induced endothelial dysfunction in porcine coronary arteries., Materials and Methods: Porcine coronary arteries were dissected from 6 pig hearts and cut into 5-mm ring segments, which were assigned into 4 groups (9 rings/group): blank control, homocysteine treated (50 muM), red wine treated (0.08% alcohol), and homocysteine plus red wine treated. The rings were cultured in cell culture medium with or without treatment for 24 h. Myograph analysis was performed with U46619 (10(-7) M) for contraction and cumulative bradykinin (10(-9) to 10(-5) M) for endothelium-dependent relaxation. The endothelial nitric oxide synthase (eNOS) levels were analyzed by RT-PCR, Western blot, and immunohistochemistry., Results: In response to 10(-5) M bradykinin, porcine coronary artery rings treated with homocysteine (50 muM) showed a significant reduction of endothelium-dependent vasorelaxation by 43% as compared to controls (P < 0.05). However, rings treated with red wine (0.08% alcohol) plus homocysteine showed no significant difference as compared to controls. Endothelium-dependent vasorelaxation was not different between control and red wine treated groups. Furthermore, eNOS mRNA density levels were significantly reduced by 36% in homocysteine treated group as compared to controls (P < 0.05). eNOS protein levels were also substantially reduced in the homocysteine-treated group. However, red wine treatment reversed the effect of homocysteine-induced eNOS downregulation., Conclusions: Homocysteine significantly impaired endothelial functions including endothelium-dependent vasorelaxation and eNOS mRNA and protein levels in porcine coronary arteries; and red wine effectively prevented homocysteine-induced endothelial dysfunction. This study suggests that protecting coronary endothelial cells from homocysteine damage may be an important mechanism of red wine for preventing coronary artery disease.

Purpose: Human immune deficiency virus (HIV) infection is often associated with chronic diseases, including atherosclerosis. However, the molecular mechanisms are largely unknown. We examined the effect of Tat protein, an HIV regulatory protein, on endothelial function in porcine coronary arteries., Methods: Porcine coronary arteries were dissected from nine pig hearts and cut into 5-mm ring segments, which were incubated as controls or with Tat protein (10(-7), 10(-9), 10(-11) mol/L) or Tat protein plus anti-Tat antibody, for 24 hours. Myography was performed with thromboxane A(2) analog U46619 (10 (-7) mol/L) for contraction and with graded doses of bradykinin (10(-8), 10(-7), and 10(-6) mol/L) or sodium nitroprusside (10(-5) mol/L) for relaxation. Endothelial nitric oxide synthase (eNOS) messenger RNA was determined with reverse transcriptase polymerase chain reaction (RT-PCR), and protein levels were determined with Western blot analysis. Immunoreactivity of eNOS of treated rings was also detected., Results: Endothelium-dependent vasorelaxation (10-7 mol/L of bradykinin) was significantly reduced (46.41%) in pig coronary artery rings treated with 10(-7) mol/L of Tat protein, as compared with control arteries (P <.05). Arteries treated with Tat protein plus anti-Tat antibody relaxed similarly as control arteries. There were no differences in smooth muscle contractility (U46619) or endothelium-independent vasorelaxation (sodium nitroprusside) between control and Tat protein-treated groups. RT-PCR for eNOS mRNA showed reduction in eNOS levels for Tat-treated coronary artery rings by 73%, as compared with control vessels (P <.05). Tat protein-treated vessels demonstrated substantially less eNOS protein band intensity and immunoreactivity compared with control vessels., Conclusions: Tat protein significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA and protein expression in endothelial cells of porcine coronary arteries. This study suggests that Tat protein-mediated endothelial dysfunction may be important in coronary heart disease in HIV-infected patients.