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: BackgroundHeterogeneity 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-α stimulation.: Materials and methodsCommercial 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-α (1 ng/ml) stimulation for 16 h was also studied by flow cytometry analysis with proper gating and fluorescence labeling of antibodies.: ResultsTwo 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-α 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-α stimulation.: ConclusionsThese data demonstrate that commercial HCAECs have two distinguished populations with different gene expression patterns and different responses following TNF-α stimulation. This study indicates that the heterogeneity of HCAECs may have biologic or pathologic significances in coronary arteries. [Copyright &y& Elsevier]

Abstract: Objective of this study was to develop a novel in vitro artery culture system to study vascular smooth muscle cell (SMC) proliferation of porcine carotid arteries in response to injury, basic fibroblast growth factor (FGF2), and FGF2 conjugated with cytotoxin saporin (SAP). Perfusion-cultured porcine carotid arteries remained contractile in response to norepinephrine and relaxant to acetylcholine for up to 96h. SMC proliferation of cultured arteries was detected by bromodeoxyuridine incorporation in both non-injured and balloon-injured arteries. In the inner layer of the vessel wall near the lumen, SMC proliferation were less than 10% in uninjured vessels, 66% in injured vessels, 80% in injured vessels with FGF2 treatment, and 5% in injured vessels with treatment of FGF2-SAP. Thus, the cultured porcine carotid arteries were viable; and the injury stimulated SMC proliferation, which was significantly enhanced by FGF2 and inhibited by FGF2-SAP. [Copyright &y& Elsevier]

: IntroductionBypass graft failure due to acute thrombosis and intimal hyperplasia remains a major challenge in small-diameter vascular prosthetic graft reconstruction. Heparin has been shown to prevent thrombus formation and inhibit intimal antithrombotic in animal studies. In this study, we evaluated the effect of small-caliber heparin-coated expanded polytetrafluoroethylene (ePTFE) grafts on platelet deposition and intimal hyperplasia in a canine model of femoral artery bypass grafting.: MethodsNine adult greyhound dogs underwent placement of bilateral femorofemoral artery bypass grafts with ePTFE grafts (4 mm diameter and 7 cm long). In each animal, a heparin-coated ePTFE graft was placed on one side while a noncoated graft was placed on the contralateral side which served as the control. Platelet deposition was measured by autologous 111indium-labeling and scintillation camera imaging analysis in 24 h. The graft patency was assessed at 4 weeks following the bypass. The effect of intimal hyperplasia was assessed with histological and morphometric analysis.: ResultsPlatelet deposition on the heparin-coated grafts at 24 h was significantly reduced by 72% as compared to controls (P = 0.001). The patency rate was 44% in control grafts and 89% in heparin-coated grafts. There was a significant reduction of graft intimal hyperplasia at both proximal (0.38 ± 0.21 mm2) and distal (0.19 ± 0.06 mm2) anastomoses in the heparin-coated grafts as compared with proximal (1.01 ± 0.28 mm2) and distal (0.42 ± 0.01 mm2) anastomoses in the untreated control grafts, respectively (P < 0.05). Heparin coating significantly reduced graft neointimal hyperplasia at patent graft anastomoses by 55–72% as compared to controls.: ConclusionsThese data demonstrate that heparin coating of ePTFE significantly reduced early platelet deposition and inhibited anastomotic neointimal hyperplasia. Moreover, small-caliber heparin-coated ePTFE graft significantly increased graft patency in a canine femoral artery bypass model. This may represent a promising treatment strategy for improving the clinical performance of small-caliber prosthetic vascular grafts. [Copyright &y& Elsevier]

Background: Neointimal formation is mediated by phenotypic changes in vascular smooth muscle cells (SMC) and is an important mediator of restenosis following arterial reconstruction. We conjugated antioxidant ginkgolide A (GA) to gold nanoparticles (GNP) to determine the effect of GA delivery on neointimal formation. Materials and Methods: GA was conjugated to 80 nm GNP in an overnight incubation. Mouse P53LMAC01 vascular SMC were treated with various doses of GA-GNP, GA alone, GNP alone, and no treatment control. Cell proliferation and migration were analyzed, and superoxide anion levels and the phosphorylation status of ERK1/2 were determined. Mice underwent ligation of the common carotid artery along with local treatment with GNP (control) or GA-GNP. The carotid artery was harvested and subjected to immunohistochemical analysis. Results: GA-GNP treatment significantly inhibited SMC proliferation and migration in vitro in comparison to GNP treatment alone, and the effect persisted for up to 72 h after treatment. Treatment with GA-GNP also reduced superoxide anion levels in vitro. PDGF-BB substantially induced ERK1/2 phosphorylation in GNP control cells; this PDGE-BB induced ERK1/2 phosphorylation was significantly inhibited in GA-GNP-treated cells compared with GNP only. GA-GNP significantly reduced neointimal hyperplasia after injury in mice, and proliferating cell nuclear antigen (PCNA) staining was reduced substantially in the arteries of mice treated with GA-GNP. Conclusions: GA-GNP reduce vascular SMC proliferation and migration in vitro through reduced activation of ERK1/2. Local treatment with GA-GNP in areas of arterial injury reduced neointimal hyperplasia and subsequent stenosis. [Copyright &y& Elsevier]

Abstract: Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MΦ) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MΦ derived from monocyte subsets and CD11c+ DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c+ DCs, monocyte-derived DCs and MΦ, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses. [Copyright &y& Elsevier]

Background: Infection of human immunodeficiency virus (HIV) has been associated with several chronic diseases, including pulmonary artery hypertension and atherosclerosis. However, the underlying mechanisms of these vascular complications are largely unknown. The objective of this study was to test a novel hypothesis that HIV Nef, an accessory HIV protein, may directly affect endothelial functions and gene expression in pulmonary arteries. Methods: Fresh porcine pulmonary artery rings and human pulmonary artery endothelial cells (HPAECs) were treated with HIV Nef for 24 h. With a myograph device, vasomotor function was determined with thromboxane A2 analog, U46619, for contraction, bradykinin, and sodium nitroprusside for relaxation. The expression of endothelial nitric oxide synthase (eNOS) was determined with real-time PCR and immunohistochemistry. Nitric oxide (NO) production was determined by Calorimetric Nitric Oxide Assay kit. Superoxide anion levels were detected with lucigenin-enhanced chemiluminescence assay and dihydroethidium (DHE) staining. Results: The endothelium-dependent vasorelaxation in response to bradykinin was significantly reduced in HIV Nef-treated porcine pulmonary artery rings in a concentration-dependent manner. In response to bradykinin (10−8 mol/L), HIV Nef (10 ng/mL) significantly reduced vasorelaxation by 32% compared with untreated controls (P < 0.05). In addition, HIV Nef significantly decreased eNOS expression in the vessels and HPAECs. HIV Nef at 10 ng/mL significantly decreased NO production in HPAECs by 21% compared with controls (P < 0.05). Furthermore, HIV Nef significantly increased superoxide anion production in porcine pulmonary arteries and HPAECs compared with controls (P < 0.05). Consequently, Mn (III) tetrakis porphyrin, a superoxide dismutase mimic, effectively blocked HIV Nef-induced vasomotor dysfunction and superoxide anion production. The specificity of HIV Nef action was confirmed by anti-Nef antibody blocking and Nef heat inactivation. Conclusions: HIV Nef protein significantly decreases endothelium-dependent vasorelaxation in porcine pulmonary arteries. It also reduces eNOS expression and induces oxidative stress in both porcine pulmonary arteries and HPAECs. This study demonstrates a new mechanism of HIV Nef, which causes endothelial dysfunction and may contribute to the human pulmonary artery disease in HIV-infected patients. [Copyright &y& Elsevier]

Background: Recently we reported that fluid shear stress promotes endothelial cell differentiation from a mouse embryo mesenchymal progenitor cell line C3H10T1/2. However, it is not clear whether the transforming growth factor-beta 1 (TGF-β1) system is associated with shear-induced endothelial differentiation. The purpose of this study was to determine the effect of shear stress on the expression of TGF-β1 and its signaling molecules in C3H10T1/2 cells. Methods: Murine C3H10T1/2 cells were incubated on collagen Type 1-coated dishes, and subjected to a steady fluid shear stress of 15 dyn/cm2 for 6, 12, and 24 h. The mRNA levels for TGF-β1, TGF-β receptors (TGF-βR), and Smad molecules were determined with real-time PCR analysis and normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. Results: TGF-β1 mRNA expression was down-regulated by 60% and 66% in shear stress-treated cells at 12 and 24 h, respectively, compared with static control group (P < 0.01). In addition, shear stress significantly decreased TGF-βR1 mRNA levels by 30% and 50% in shear stress-treated cells at 12 and 24 h, respectively (P < 0.01). For TGF-βR2, shear stress at 6, 12, and 24 h significantly reduced its expression by 93%, 95% and 97%, respectively, compared with static controls (P < 0.01). Furthermore, shear stress significant decreased mRNA levels of positive signaling molecules Smad2, Smad3, and Smad4 in a time-dependent manner (P < 0.01). However, shear stress significantly increased negative signaling molecule Smad7 mRNA levels by 100% at 24 h treatment compared with static control group (P < 0.01). Conclusions: Fluid shear stress significantly suppresses TGF-β1 functions through down-regulation of TGF-β1, TGF-βR, positive signaling molecules Smad2, Smad3, Smad4, and up-regulation of negative signaling molecule Smad7 in a mouse embryo mesenchymal progenitor cell line C3H10T1/2. This study suggests that the negative regulation of the TGF-β1 system may be involved in shear-induced endothelial cell differentiation in C3H10T1/2. [Copyright &y& Elsevier]

Background: Kaempferol is one of the most important constituents in ginkgo flavonoids. Recent studies indicate kaempferol may have antitumor activities. The objective of this study was to determine the effect and mechanisms of kaempferol on pancreatic cancer cell proliferation and apoptosis. Materials and methods: Pancreatic cancer cell lines MIA PaCa-2 and Panc-1 were treated with kaempferol, and the inhibitory effects of kaempferol on pancreatic cancer cell proliferation were examined by direct cell counting, 3H-thymidine incorporation, and MTS assay. Lactate dehydrogenase release from cells was determined as an index of cytotoxicity. Apoptosis was analyzed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. Results: Upon the treatment with 70 μm kaempferol for 4 days, MIA PaCa-2 cell proliferation was significantly inhibited by 79% and 45.7% as determined by direct cell counting and MTS assay, respectively, compared with control cells (P < 0.05). Similarly, the treatment with kaempferol significantly inhibited Panc-1 cell proliferation. Kaempferol treatment also significantly reduced 3H-thymidine incorporation in both MIA PaCa-2 and Panc-1 cells. Combination treatment of low concentrations of kaempferol and 5-fluorouracil showed an additive effect on the inhibition of MIA PaCa-2 cell proliferation. Furthermore, kaempferol had significantly less cytotoxicity than 5-fluorouracil in normal human pancreatic ductal epithelial cells (P = 0.029). In both MIA PaCa-2 and Panc-1 cells, apoptotic cell population was increased when treated with kaempferol in a concentration-dependent manner. Conclusions: Ginkgo biloba extract kaempferol effectively inhibits pancreatic cancer cell proliferation and induces cancer cell apoptosis, which may sensitize pancreatic tumor cells to chemotherapy. Kaempferol may have clinical applications as adjuvant therapy in the treatment of pancreatic cancer. [Copyright &y& Elsevier]

Abstract: Smooth muscle cells (SMCs) under shear stress may alter their gene expression patterns to adapt to a new hemodynamic environment. Their plasticity may play an important role in vascular development, healing, and remodeling as well as vascular lesion formation under abnormal environmental conditions. A mouse vascular SMC line (P53LMACO1) cultured under shear stress significantly increased the mRNA levels of endothelial cell markers including Platelet-endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF), and VE-cadherin, while significantly decreasing the mRNA levels of SMC markers including alpha-smooth muscle actin (α-SMA), calponin-1, smooth muscle myosin heavy chain (SMMHC), and transgelin as compared to static control cells. Protein levels of PECAM-1 and vWF were significantly increased, while protein levels of α-SMA were substantially decreased in the shear stress-cultured cells. In addition, shear stress-cultured cells showed an enhanced capability to form capillary-like structures on Matrigel. Thus, shear stress may promote endothelial cell transdifferentiation from SMCs. [Copyright &y& Elsevier]

The heme-containing enzyme myeloperoxidase (MPO) is both present and active in inflammatory conditions. This enzyme is potentiated by its formation of multiple inflammatory mediators. The two most common mediators are the modified tyrosines: nitrotyrosine and 3-chlorotyrosine. Along with other modified tyrosines, these molecules have been found to be elevated in atherosclerosis, lung disease, sepsis, vasculitis, and other inflammatory diseases. By treating some of these diseases, the levels of modified tyrosines have been shown to decrease. There have been a wide range of animal models designed to study the in vivo effects of these tyrosine molecules. In addition, there are also several reports in the literature of the in vitro actions of modified tyrosine molecules demonstrated by various cell-culture models. The purpose of this review is to evaluate the clinical significance and biological functions of these modified tyrosine molecules in atherosclerosis as well as a variety of other inflammatory conditions. It is timely information because of their association with diseases as well as lack of overview of their molecular actions. This review will focus on the formation, clinical significance, and animal and cell-culture models of these important molecules. [Copyright &y& Elsevier]

Background: Homocysteine (Hcy) is an independent risk factor for cardiovascular disease by its multiple effects on vascular cells and throbmosis factors, which may be involved in oxidative stress mechansims. Ginsenoside Rb1, a constituent of ginseng, bears various beneficial effects on the cardiovascular system. In the present study, we investigated the effect of Hcy on endothelial proliferation and a protective effect of ginsenoside Rb1 on the action of Hcy. Methods: We initially incubated a mouse lymph node endothelial cell line (SVEC4-10) with increasing concentrations of Hcy or for different time periods and then assessed cell proliferation by using [3H]-thymidine incorporation. We then incubated SVEC4-10 cells with Hcy (50 μM) for 24 h with or without Rb1 (10 μM) to examine its inhibitory effect on the proliferation. These experiments were repeated in human umbilical vein endothelial cells (HUVECs). To explore the underlying molecular mechanisms, we measured superoxide anion, a reactive oxygen species (ROS), by using dihydroethidium (DHE) staining. Results: SVEC4-10 cells treated with Hcy (50, 100, and 200 μM) for 24 h significantly reduced cell proliferation by 43%, 42%, and 40%, respectively, as compared with control cells (P < 0.01). SVEC4-10 cells treated with Hcy (50 μM) for 12 and 24 h showed a significant reduction of cell proliferation (P < 0.05). In HUVECs, Hcy (50 μM) significantly reduced cell proliferation by 55% as compared with control cells (P < 0.05). In the presence of Rb1, Hcy-induced inhibition of cell proliferation was effectively blocked in both SVEC4-10 and HUVECs. Furthermore, Hcy (50 μM) significantly increased superoxide anion production by 23% in SVEC4-10 as compared with control cells (P < 0.05). However, in the presence of Rb1, Hcy increased superoxide anion production by only 8%, showing that RB1 almost completely blocked the effect of Hcy. Conclusion: Hcy significantly inhibits endothelial proliferation with increased production of superoxide anion, which is effectively blocked by ginsenoside Rb1. This study provides some new aspects of Hcy-induced endothelial dysfunction, and suggests a potential role of Rb1 to block Hcy action, which may have clinical applications. [Copyright &y& Elsevier]

Abstract: HIV protease inhibitors (PIs) are often associated with metabolic and cardiovascular complications although they are effective anti-HIV drugs. In this study, we determined whether HIV PI ritonavir could increase endothelial permeability, one of the important mechanisms of vascular lesion formation. Human dermal microvascular endothelial cells (HMECs) treated with ritonavir showed a significant increase of endothelial permeability in a dose- and time-dependent manner assayed with a transwell system. Ritonavir significantly reduced the mRNA levels of tight junction proteins zonula occluden-1, occludin, and claudin-1 by 40–60% as compared to controls (P &#60;0.05) by real-time PCR analysis. Protein levels of these tight junction molecules were also substantially reduced in the ritonavir-treated cells. In addition, HMECs treated with ritonavir (7.5, 15, and 30μM) showed a substantial increase of superoxide anion production by 10%, 32%, and 65%, respectively, as compared to controls. Antioxidants (EGCG and SeMet) effectively reduced ritonavir-induced endothelial permeability. Furthermore, ritonavir activated ERK1/2 (phosphorylation), but not P38 and JNK. Specific ERK1/2 inhibitor, PD89059, significantly abolished ritonavir-induced endothelial permeability by 92%. Thus, HIV PI ritonavir increases endothelial permeability, decreases levels of tight junction proteins, and increases superoxide anion production. ERK1/2 activation is involved in the signal transduction pathway of ritonavir-induced endothelial permeability. [Copyright &y& Elsevier]

Abstract: C-reactive protein (CRP) is associated with cardiovascular disease. However, its biological functions for the vascular system are largely unknown. The objective of this study was to determine whether CRP could affect endothelial cell proliferation and expression of VEGF receptors (VEGFRs) and/or neuropilins. Human coronary artery endothelial cells (HCAECs) treated with CRP showed a significant reduction of mRNA levels of VEGFR-2, VEGFR-3, NRP-1, and NRP-2 by 34%, 63%, 41%, and 43%, respectively, as compared to untreated control cells (p &#60;0.05) by real-time PCR analysis. In addition, VEGF165-induced cell proliferation was determined by [3H]thymidine incorporation and MTS assay as well as capillary-like tube formation on Matrigel. HCAECs pretreated with CRP significantly decreased VEGF165-induced [3H]thymidine incorporation by 73%, MTS absorbance by 44%, and capillary-like tube formation by 54% as compared to CRP-untreated cells (p &#60;0.05). These data demonstrate that CRP significantly attenuates VEGF165-induced HCAEC proliferation and capillary-like tube formation through downregulation of expression of VEGFRs and NRPs. This study suggests a new molecular mechanism underlying the adverse effect of CRP on the vascular system. [Copyright &y& Elsevier]

Background: Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein as well as a secreted protein and has recently been reported to be an immunomodulatory molecule. The objective of this study was to determine the effect of CypA on dendritic cell (DC) differentiation, activation, and functional maturation. The role of p38 MAP kinase in DC functions was also investigated. Materials and methods: KG1 cells (CD34+ human myeloblastic cell line) were treated with cytokines (GM-CSF+IL-4) and/or CypA and expression of cell surface markers was analyzed by FACS analysis. The antigen-uptake capacity of different DCs was determined by FITC-dextran uptake assay. Antigen-presentation capacity of DCs was determined by allogeneic mixed lymphocyte reaction (MLR) by [3H] thymidine incorporation assay. To assess the T cell polarization stimulated by KG1 derived DCs, various Th1 and Th2 cytokines secreted by allostimulated CD4+ and CD8+ T cells were determined by Bioplex cytokine assay. Total and phosphorylated p38 MAPK activity in CypA treated DCs was detected by Bioplex p38 total and phosphoprotein assay. Results: During the differentiation of KG1 cells to immature DCs, cell surface expression of CD11b was increased by 30.6% for CypA alone, 55% for CypA plus cytokines, and 44% for cytokines alone. Similarly, CypA alone increased the cell surface expression of CD11c by 59% as compared to CypA plus cytokines (68%) and cytokines alone (50%). CypA up-regulated the antigen uptake capacity of the immature DCs to a greater extent (5 times) as compared to cytokines alone (2.5 times). Moreover, CypA augmented the capacity of DCs to present antigens to allogenic CD8+ T cells, and also increased the secretion of Th1 type cytokines TNF-α and IFN-γ from the allogenic CD4+ T cells. Furthermore, CypA induced the phosphorylation and hence activation of MAP kinase p38. Pre-treatment with SB-203580, a p38 inhibitor, significantly reduced MLR stimulatory capacity of CypA-induced DCs in both CD8+ and CD4+ T cells (P &#60; 0.05). Conclusions: CypA enhances DC differentiation and maturation by up-regulating CD11b and CD11c expression. CypA can also augment DC antigen uptake and antigen presentation, which may be mediated by the p38 signaling pathway. [Copyright &y& Elsevier]

Background: The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs). Materials and methods: PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells. Results: During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions. Conclusions: These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy. [Copyright &y& Elsevier]

Background: Lysophosphatidylcholine (LPC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 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 (O2 −) 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 μm of LPC, respectively, as compared with controls (P &#60; 0.05). Endothelium-independent relaxation in response to sodium nitroprusside also was reduced by 63% after treatment with 25 μm LPC (P &#60; 0.05). The O2 − level was increased in the porcine arteries treated with 25 μm of LPC by 41% as compared with controls (P &#60; 0.05). The antioxidant SeMet reversed the effects of LPC on vascular relaxation and O2 − 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. [Copyright &y& Elsevier]

Adipose tissue has recently emerged as an active endocrine organ that secretes a variety of metabolically important substances, collectively called adipocytokines or adipokines. In this review we summarize the effects of the adipokines leptin, adiponectin, and resistin on the vasculature and their potential role for pathogenesis of vascular disease. Leptin is associated with arterial wall thickness, decreased vessel distensibility, and elevated C reactive protein (CRP) levels. Leptin possesses procoagulant and antifibrinolytic properties, and it promotes thrombus and atheroma formation, probably through the leptin receptors by promoting vascular inflammation, proliferation, and calcification, and by increasing oxidative stress. Research for development of pharmacologic antagonism for the leptin receptor is currently under way. Adiponectin inhibits the expression of the adhesion molecules ICAM-1, VCAM-1, and P selectin. Therefore, it interferes with monocyte adherence to endothelial cells and their subsequent migration to the subendothelial space, one of the initial events in the development of atherosclerosis. Adiponectin also inhibits the transformation of macrophages to foam cells in vitro and decreases their phagocytic activity. Resistin, discovered in 2001, represents the newest of the adipokines and was named for its ability to promote insulin resistance. Resistin increases the expression of the adhesion molecules VCAM-1 and ICAM-1, up-regulates the monocyte chemoattractant chemokine-1, and promotes endothelial cell activation via ET-1 release. Although many aspects of its function need further clarification, it appears that resistin will add significantly to our knowledge of the pathophysiology of vascular disease and the metabolic syndrome. [Copyright &y& Elsevier]

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 × 10−6, 1 × 10−5, 5 × 10−5 g/L), or a combination of the two (10−8g/L estrogen, 1 × 10−5g/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 10−7M bradykinin, porcine coronary artery rings treated with 1 × 10−6, 1 × 10−5 and 5 × 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. [Copyright &y& Elsevier]

Background: Cyclophilin A (CypA) is a cytosolic protein which involves many biological functions including immune modulation, cell growth, tumorigenesis, and vascular disease. The objective of this study was to determine the effect of CypA on cell proliferation and several gene expressions in human endothelial cells and vascular smooth muscle cells. Methods: Human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HMVEC-L), and human aorta smooth muscle cells (HAoSMC) were used in this study. Cells were treated with 10 nM CypA for 24 h. The cell proliferation was determined by [3H]thymidine incorporation. The mRNA levels of 13 genes including CD147 (receptor for CypA), PDGF-BB, endothelin-1 (ET-1), vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1), NRP-2, eNOS, iNOS, nNOS, ICAM-1, and PECAM-1 were semiquantitatively determined by real time RT-PCR as standardized with a house keeping gene β-actin. Results: CypA significantly increased cell proliferation of HAoSMC and HMVEC-L by 31% and 45%, respectively, as compared to controls, but had no effect on HCAEC. Blocking CD147 did not affect the mitogenic action of CypA. In addition, CypA also significantly increased the mRNA expression of CD147 by 43% and VEGFR-2 by 65% in HAoSMCs (P < 0.05, t test). HAoSMCs expressed much higher CD147 and neuropilin-1 (NRP-1) mRNA than HMVECs-L and HCAECs (P < 0.017, ANOVA). Furthermore, CypA increased ET-1 mRNA by 22% and VEGFR-1 mRNA by 23% in HMVECs-L, but had limited effects on HCAECs. HMVECs-L had much higher expressions of PDGF-BB, ET-1, VEGFR-2, VEGFR-1, VEGFR-3, and NRP-2 than HAoSMCs and HCAECs (P < 0.017, ANOVA). By contrast, HCAECs had much higher ICAM-1 mRNA levels than HMVECs-L and HAoSMCs (P < 0.017, ANOVA). Conclusions: These data demonstrate that CypA has a mitogenic effect on HAoSMCs and HMVECs-L, but not HCAECs. CD147 may not mediate the action of CypA. In addition, CypA substantially alters the mRNA levels of several key genes in human vascular cells, indicating potential multifunctional roles of CypA in vascular system. Furthermore, this study provides several new aspects of gene expressions in vascular cells. [Copyright &y& Elsevier]

Background: The objective of this study was to determine the expression of somatostatin (SST) and its receptors (SSTRs) and their regulation by TNF-α 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-α (0.1, 1, or 10 ng/ml) for 24 h. The mRNA levels of SST, SSTR-1-5, as well as a housekeeping gene (β-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-α 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-α, the mRNA levels of SST, SSTR-1, SSTR-2, and SSTR-5 were significantly reduced in a dose-dependent fashion. TNF-α (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-α treatment. SST-treated cells showed a significant reduction in [3H]thymidine incorporation in a dose-dependent manner. TNF-α 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-α treatment. Furthermore, treatment with exogenous SST significantly reduces cell proliferation, and this inhibitory effect is also decreased by TNF-α. [Copyright &y& Elsevier]

Background: Recently, neuropilins (NRPs) were identified as new VEGF receptors (VEGFRs) and VEGFR-3 was revealed to be implicated in angiogenesis besides in lymphangiogenesis. However, quantitative expression and regulation of NRPs and VEGFRs remain unclear. The objective of this study was to compare the expression of NRPs and VEGFRs and investigate their regulation by tumor necrosis factor (TNF-α) in human endothelial cells (ECs) as well as their roles in VEGF165-induced EC proliferation. Materials and methods: Human umbilical vein ECs (HUVECs) were treated with TNF-α (2 ng/ml) or PBS for 24 h. The mRNA and protein levels of NRP-1, NRP-2, VEGFR-1, VEGFR-2, and VEGFR-3 were semiquantitatively determined by real-time PCR and Western blot, respectively. Real-time PCR data were presented as the difference of reaction cycle thresholds (Ct) between β-actin and each of the genes of interest (2-ΔCt). EC proliferation in response to VEGF165 (10 ng/ml) with or without anti-VEGFR-2 neutralization antibody pretreatment was analyzed by [3H]thymidine incorporation. Results: In PBS-treated HUVECs, mRNA levels of NRPs and VEGFRs were NRP-1 (0.013), NRP-2 (0.007), VEGFR-2 (0.006), VEGFR-1 (0.0024), and VEGFR-3 (0.0009). After TNF-α treatment, mRNA levels of VEGFR-2, VEGFR-3, and NRP-1 were significantly reduced by 72, 65, and 53%, respectively (P < 0.05). The protein expression of all NRPs and VEGFRs were also detected by Western blot. TNF-α significantly reduced protein levels of VEGFR-2, VEGFR-3, and NRP-1 by 59, 35, and 22%, respectively. However, both mRNA and protein levels of VEGFR-1 and NRP-2 were not affected by TNF-α. Furthermore, TNF-α treatment significantly reduced EC proliferation in response to VEGF165 by 67%. After blocking VEGFR-2 with neutralization antibody, TNF-α treatment elicited a 30% reduction of EC proliferation in response to VEGF165. Conclusions: These data demonstrate that HUVECs express higher mRNA levels of NRP-1 and NRP-2 than those of VEGFRs, and TNF-α treatment significantly decreases the expression of VEGFR-2, VEGFR-3, and NRP-1, which may be responsible for TNF-α-induced reduction of EC proliferation in response to VEGF165. [Copyright &y& Elsevier]

Background: Somatostatin inhibits cell proliferation and may act as a tumor suppressor by interacting with five different somatostatin receptors (SSTRs). We hypothesized that SSTR expression is down-regulated in human pancreatic cancer. In the current study, we used a powerful real-time RT-PCR technique to examine the mRNA expression levels of all five SSTR subtypes in human pancreatic cancer.Materials and methods: Total RNA was extracted from three pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and Hs 766T), three surgical specimens of pancreatic cancer, and adjacent pancreatic tissue, and a pancreatic cancer cell line transfected with the SSTR-2 gene. Specific primers were designed and mRNA levels for the five SSTRs were analyzed with real-time quantitative RT-PCR using a Bio-Rad iCycler system.Results: The pancreatic tumor specimens had a 2.5- and 4.3-fold reduction of SSTR-2 and SSTR-5 mRNA levels, respectively, as compared to their adjacent normal pancreatic tissues. SSTR-1 and SSTR-3 were also detected in both the cancer specimens and the adjacent tissues, but SSTR-4 was absent. Human pancreatic cancer cell lines also expressed SSTR-2 and SSTR-5 mRNA, but not SSTR-1, SSTR-3, and SSTR-4. Up-regulation of SSTR-2 mRNA by 2.2 × 104-fold in Panc-1 cells resulted in receptor expression and growth inhibition.Conclusion: Expression of SSTR-2 and SSTR-5 could be important in the growth inhibitory effect of somatostatin in human pancreatic cancer. Down-regulation of SSTR transcription or SSTR mRNA instability may result in loss of a tumor suppressive effect of SSTRs in human pancreatic cancer. [Copyright &y& Elsevier]

: BackgroundThe 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 methodsPig 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.: ResultsEndothelium-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.: ConclusionsThese 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. [Copyright &y& Elsevier]

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 μM), 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 μM) 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. [Copyright &y& Elsevier]

To enhance mucosal immune responses using simian/human immunodeficiency virus-like particles (SHIV VLPs), we have produced novel phenotypically mixed chimeric influenza HA/SHIV VLPs and used them to immunize C57BL/6J mice intranasally. Antibody and cytotoxic T-cell (CTL) responses as well as cytokine production in both systemic and mucosal sites were compared after immunization with SHIV VLPs or chimeric HA/SHIV VLPs. By using enzyme-linked immunosorbent assay (ELISA), the levels of serum IgG and mucosal IgA to the HIV envelope protein (Env) were found to be highest in the group immunized with chimeric HA/SHIV VLPs. Furthermore, the highest titer of serum neutralizing antibody against HIV Env was found with the group immunized with chimeric HA/SHIV VLPs. Analysis of the IgG1/IgG2a ratio indicated that a TH1-oriented immune response resulted from these VLP immunizations. HA/SHIV VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV VLP-immunized mice. Moreover, a MHC class I restricted T-cell activation ELISPOT assay showed a mixed type of TH1/TH2 cytokines in the HA/SHIV VLP-immunized mice, indicating that the chimeric VLPs can enhance both humoral and cellular immune responses to the HIV Env protein at multiple mucosal and systemic sites. The results indicate that incorporation of influenza HA into heterotypic VLPs may be highly effective for targeting vaccines to mucosal surfaces. [Copyright &y& Elsevier]