Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol.

Beneficial effects of evodiamine on P2X(4)-mediated inflammatory injury of human umbilical vein endothelial cells due to high glucose.

Evodiamine has been reported to exhibit anti-inflammatory and anti-nociceptive effects, but the underlying mechanisms remain to be defined. P2X4 receptor (P2X4R) is a subtype of ATP receptors and plays important roles in pain, inflammatory and immune responses. We aimed to investigate whether evodiamine has beneficial effects on endothelial inflammatory injury mediated by chronic high glucose condition. We found that culturing human umbilical vein endothelial cells (HUVECs) with high glucose significantly increased the expression of P2X4 receptor in HUVECs, cytosolic Ca(2+) concentrations and intracellular reactive oxygen species (ROS) while decreasing nitric oxide (NO); these effects could be reversed by evodiamine. High glucose also significantly increased the expression of the pro-inflammatory activators (NF-κB) and TNFR-ɑ, which was accompanied by the elevation of P2X4R levels. Evodiamine was able to down-regulate the elevated NF-κB, TNFR-ɑ, P2X4R and ROS, and up-regulate the decreased NO. Thus the evodiamine may exert the anti-inflammation activity on high-glucose challenge HUVEC via suppressing the P2X4R signaling pathway, exhibiting beneficial ability to protect HUVECs from glucotoxicity.

Effect of Atropa belladonna L. on skin wound healing: biomechanical and histological study in rats and in vitro study in keratinocytes, 3T3 fibroblasts, and human umbilical vein endothelial cells.

The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague-Dawley rats subjected to two parallel full-thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT-assay. Results of the in vivo experiments showed in AB-treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research.

Stimulatory effect of Cinnamomum cassia and cinnamic acid on angiogenesis through up-regulation of VEGF and Flk-1/KDR expression.

Complementary and alternative medicine, Cinnamomum cassia is one of the medicinal plants that have been used to improve various diseases caused by insufficient blood circulation. However, relatively little work has been carried out on the angiogenic responses of C. cassia and its active compound cinnamic acid (CA), despite its excellent pharmacological action. In this study, we study the effect of the ethanol extract of C. cassia (CCE) and its active compound CA, on angiogenic processes in in vitro and in vivo. In the Matrigel plug assay in vivo, CCE and CA dose dependently increased von Willebrand Factor (vWF) antigen expression, and hemoglobin contents, whose elevation paralleled the onset of angiogenesis and was considered an early indicator of endothelial activation. CCE and CA induced endothelial cells proliferation, migration and tubule-like structure in vitro. The 25-50% increase observed with CCE (at low doses of 1 or 10 ng/ml) in HUVEC and BAEC proliferation was similar to that observed with CA. The migration and tubule-like structure effect were observed with HUVEC and BAEC. However, the effect of CCE, CA and VEGF on cell proliferation, migration and tubule-like structure in HUVEC were bigger than the effect of CCE, CA and VEGF in BAEC. In addition, CCE and CA each induced 2.2-fold and 2.5-fold increases the production of VEGF, the mRNA expression of VEGF and Flk-1/KDR, the receptor involved in proliferation, migration, and tubule-like structure of endothelial cells. These data suggest that CCE and its active compound CA induce angiogenesis in vivo and in vitro, and this pathway is related with VEGF and Flk-1/KDR expression of endothelial cells.

Inhibitory effect of curcumin on corneal neovascularization in vitro and in vivo.

PURPOSE: To examine the effect of curcumin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn rat model. METHODS: HUVEC proliferation, migration, and apoptosis were examined after treatment with various concentrations of curcumin. The effect of curcumin on the activation of nuclear factor-kappaB (NF-kappaB) was measured by an electrophoretic mobility shift assay (EMSA) in vivo. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea in Sprague-Dawley rats. After topical drug treatments with curcumin, vascular endothelial growth factor (VEGF) was evaluated in the corneal tissue by reverse transcription polymerase chain reaction and by immunohistochemistry. Corneal neovascularization was evaluated by slit-lamp biomicroscopy. RESULTS: Curcumin at a concentration of 40 micromol/l for 24 h significantly inhibited the growth of HUVECs. The Boyden microchamber assay showed that curcumin dramatically inhibited the migration of HUVECs at a concentration of 40 micromol/l. When TUNEL assays were performed, the number of apoptotic cells increased after treated with curcumin. The EMSA revealed that curcumin inhibits the activation of NF-kappaB in HUVECs. The expression of VEGF in the corneal tissues was inhibited by curcumin on days 7 and 14 after alkaline burn. CONCLUSIONS: Curcumin may be useful as an angiogenic inhibitor in the treatment of corneal diseases that show neovascularization.

Cell viability and prostacyclin release in cultured human umbilical vein endothelial cells.

Construction of efficient substitutes of human blood vessels is strongly dependent on the use of viable and fully functional cultured endothelial cells (ECs). However, very few reports have been published to date focused on the evaluation of cell viability of cultured ECs. In this work, we have determined cell viability, von Willebrand factor, and prostacyclin (PGI(2)) activity in primary cell cultures of human umbilical vein ECs, to identify the specific cell passage that is more appropriate for the development of artificial organs by tissue engineering. Cell viability was determined by quantification of the intracellular concentration of several ions by highly sensitive electron probe X-ray microanalysis, whereas von Willebrand was assayed by immunohistochemistry and PGI(2) release was quantified by radioimmunoassay. The results of our analyses demonstrate that the K/Na ratio was different for each cell passage (4.72 for the first passage, 4.55 for the second passage, and 7.82 for the third passage), suggesting that the highest cell viability corresponds to the third passage. In contrast, PGI(2) production was higher at the first two cell passages, with a significant decrease at the third passage (6.46 +/- 0.10, 5.98 +/- 0.08, and 1.62 +/- 0.05 ng/mL of supernatant for the first, second, and third passages, respectively), whereas von Willebrand expression was similar among the three cell passages analyzed in this work (64.12%, 66.66%, 65.93% of positive cells, respectively). These data suggest that cells corresponding to the second cell passage show the best ratio of viability to functionality and should therefore be used for tissue engineering protocols.

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells.

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O(2)(.-)) production in women recruited to the "Vitamins in Preeclampsia" (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14-20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O(2)(.-) was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.

Protective effect of tanshinone IIA on human umbilical vein endothelial cell injured by hydrogen peroxide and its mechanism.

Tanshinone IIA (Tan IIA) is isolated from Salvia miltiorrhiza, the root of which is widely used as a traditional Chinese medicine to treat atherosclerosis. The aim of the present study was to evaluate the putative protective effect of Tan IIA in a human umbilical vein endothelial cell line (ECV-304) injured by hydrogen peroxide in vitro and the mechanism of its protection. The percentage of cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The endothelial cell apoptosis and expression of cluster of differentiation 40 (CD40) were detected by flow cytometric analysis. Preincubation with Tan IIA significantly increased the viability of ECV-304 cell injured by hydrogen peroxide, which was accompanied with the increased nitric oxide level and superoxide dismutase activity in a dose-dependent manner. Moreover, cell apoptosis and CD40 expression were decreased in a dose-dependent manner. In conclusion, our data suggests that Tan IIA protects ECV-304 cell damage induced by hydrogen peroxide through its anti-oxidant effect and CD40 anti-inflammatory approach.

[Effect of icariin on hypoxia induced vascular endothelial cells injury].

OBJECTIVE: To study the effect of icariin on vascular endothelial cells (VECs) injury induced by hypoxia. METHODS: The hypoxia-ischemia model was established. The effect of icariin on injury of VECs activity induced by hypoxia was determined by MTT assay. The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and lactate dehydrogenase (LDH) activity in cell homogenate were measured with corresponding kit. Effect of icariin on cells apoptosis induced by hypoxia was determined by Hoechst 33342 fluorescent staining, cell ultrastructure observation under transmission electron microscopy and analysis on gene fragmentation by flow cytometry and DNA gel electrophoresis. RESULTS: ICA could inhibit the hypoxia induced VECs reduction, suppress LDH activity, reduce the MDA production, and enhance SOD activity under hypoxia. Hypoxia could induce VECs apoptosis, revealed chromation condensed in nuclei with the fragments arranged along the nuclear membrane. DNA gel electrophoresis showed typical ladder strands of DNA. Cells displayed a typical sub-diploid peak in flow cytometry. ICA could significantly inhibit the hypoxia induced apoptosis of VECs. CONCLUSION: ICA has the protective effect on hypoxia injured VECs, which may be related to its effect of anti-apoptosis, anti-lipid peroxidation and SOD activity enhancing.

Enhanced gene transfer activity of peptide-targeted gene-delivery vectors.

We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.

Inhibition of mitogen-mediated proliferation of rat vascular smooth muscle cells by labedipinedilol-A through PKC and ERK 1/2 pathway.

Labedipinedilol-A is a novel 1, 4-dihydropyridine type calcium antagonist with alpha-receptor blocking activity. This study investigates the effects of labedipinedilol-A on mitogen-induced proliferation of rat vascular smooth muscle cells (VSMCs). Labedipinedilol-A's inhibition on cell proliferation was measured by the tetrazolium salt (XTT) test. Labedipinedilol-A dose-dependently inhibited mitogen-induced DNA synthesis, determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Labedipinedilol-A was also found capable of inhibiting the migration of VSMCs induced by PDGF-BB with an IC50 value of 5.6 microM. In accordance with these findings, labedipinedilol-A revealed blocking of the FBS-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Labedipinedilol-A appeared to cause inhibition of mitogens-induced PKC translocation, suggesting the probable involvement of protein kinase C (PKC) in this cellular response. Labedipinedilol-A reduced both intracellular Ca and the phosphorylation of extracellular signal-regulated protein kinase 1/2 in PDGF-BB-stimulated VSMCs. It also suppressed the levels of proliferative cell nuclear antigen (PCNA) in VSMCs both time- and dose-dependently. These results indicate that labedipinedilol-A may inhibit cell proliferation by attenuating activation of the ERK 1/2 pathway, which is regulated by PKC and Ca, suggesting that it may have great potential in the prevention of progressive atherosclerosis.

[Protection of artesunate on activation and injury of vascular endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the protective effects and mechanism of artesunate (AR) on the activation and injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). METHODS: After HUVECs were cultured and turned to fusion manner, LPS and different concentration of AR (0.04 mg/L, 0.2 mg/L, 1 mg/L, 5 mg/L and 20 mg/L) were added respectively and co-incubated for 24 hrs. The expression of von Willebrand factor (vWF) in the conditioned media was tested by ELISA, the expression of intercellular adhesion molecule (ICAM-1) protein was determined by Western blot method and the expression of tumor necrosis factor alpha (TNFalpha) mRNA was determined by in situ hybridization. RESULTS: After being exposed to 1 microg/ml LPS, vWF and ICAM-1 expression were higher than those in the control group. AR could significantly down-regulate the increased expressions concentration-dependently, significant difference showed as the concentration of AR reached 1 mg/L (P < 0.05). In situ hybridization showed that AR in 0.2 mg/L and 1 mg/L could markedly down-regulate the TNFalpha mRNA expression, showing significant difference as compared with that in LPS group (P < 0.05, P < 0.01). CONCLUSION: AR has protective effect on LPS induced HUVECs activation and injury, which might be related with its inhibition on TNFalpha mRNA expression.

Phytoestrogens increase the capacity of serum to stimulate prostacyclin release in human endothelial cells.

BACKGROUND: Both the estrogen receptor (ER) alpha and beta isoforms are expressed in the endothelium. The ER beta has been assigned a crucial role in normal vascular wall function. Prostacyclin has been ascribed a beneficial effect on vessel wall physiology. Isoflavones bind with higher affinity to ER beta. We investigated the hypothesis that their administration to postmenopausal women can promote endothelial prostacyclin production. METHODS: Twenty-five healthy postmenopausal women with mild climacteric symptoms received capsules containing 55 mg/day isoflavones derived from soy and red clover for 6 months. Cultured human umbilical vein endothelial cells (HUVECs) were exposed for 24 h to serum collected before the initiation of therapy and then after 3 and 6 months of continuous therapy. Prostaglandin production was measured in culture medium. RESULTS: In the presence of serum obtained after isoflavone treatment, the prostacyclin production increased significantly from 2.7 +/- 0.5 ng/mg protein at baseline to 3.4 +/- 0.7 ng/mg protein at 3 months (p < or = 0.05), and to 3.8 +/- 0.7 ng/mg protein at 6 months (p < or = 0.05 vs. baseline and 3 months' treatment). CONCLUSIONS: Serum obtained from postmenopausal women treated with isoflavones stimulates the capacity to produce prostacyclin by HUVECs in culture, an effect that could contribute to a beneficial cardiovascular effect of phytoestrogens.

Targeting angiogenic processes by combination rofecoxib and ionizing radiation.

Tumor growth and angiogenesis are interdependent. Cyclooxygenase (COX) catalyzes the synthesis of prostaglandins from arachidonic acid. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit COX-mediated synthesis of prostaglandins. COX-1 is constitutively expressed in a wide range of tissues, whereas COX-2 is cytokine inducible. Enhanced COX-2 expression has been attributed a key role in the development of inflammation and related processes observed in pathologically altered disease states. Two specific COX-2 inhibitors, namely rofecoxib (Vioxx) and celecoxib (Celebrex), both oral agents and U.S. Food and Drug Administration approved, have been shown preclinically and clinically to have efficacy comparable to that of NSAIDs for relief of pain and inflammation in osteoarthritis, with decreased risk of gastrointestinal damage. Little is known about how angiogenesis is affected by the combination of rofecoxib and radiation. We have evaluated the combination of rofecoxib, at various concentrations, and radiation on cytokine-induced angiogenesis in vitro. We have found that rofecoxib inhibited endothelial cell proliferation, migration, and tube formation (differentiation) at clinically relevant doses. In combination with radiation, inhibition of endothelial cell function further increased twofold. The combination of rofecoxib and radiation suggests a complementary strategy with clinical ramifications to target angiogenesis-dependent malignancies.

[Protective effect of Angelica on ECV(304) from injury induced by hyperlipidemic serum in vitro].

The aim of this article was to examine the protective effect of Chinese traditional medicine Angelica on human umbilical vein endothelial cells (HUVECs, ECV(304)) from injury induced by hyperlipidemic serum (HLS) and to study the underlying mechanisms. Microstructures of HUVECs were observed by a scanning electron microscope. Spectrophotometer and immunocytochemical methods were used to detect the content of NO in the suspension and expression of ICAM-1, TGFbeta(1), bFGF on the cell surface, respectively. After being incubated with HLS for 24 hours, HUVECs exhibited pronounced morphological changes, such as disappearance of microvilli on the endothelial cell (EC) surface, rupture of cell membranes, etc. Expression of ICAM-l and bFGF in ECs was significantly increased, while expression of TGFbeta(1) and the release of NO from ECs were significantly decreased. All these effects of HLS on ECs can be reversed by Angelica significantly. The above effects of Angelica may be related to its anti-atherosclerotic action.

Antioxidants may contribute in the fight against ageing: an in vitro model.

Elderly humans have altered cellular redox levels and dysregulated immune responses, both of which are key events underlying the progression of chronic degenerative diseases of ageing, such as atherosclerosis and Alzeimer's disease. Poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NFkB and AP-1. These factors are co-ordinately responsible for a huge range of extracellular signalling molecules responsible for inflammation, tissue remodelling, oncogenesis and apoptosis, progessess that orchestrate many of the degenerative processess associated with ageing. It is now clear that levels of endogenous anti-oxidants such as GSH decrease with age. This study aimed to investigate the potential of exogenous anti-oxidants to influence inflammatory responses and the ageing process itself. We investigated the potential of the dietary antioxidant, quercetin, to reverse the age related influences of GSH depletion and oxidative stress using in vitro human umbilical vein endothelial cells (HUVEC) and human skin fibroblast (HSF) cell models. Oxidative stress-induced inflammatory responses were investigated in a GSH depletion and a Phorbol 12-myristate 13-acetate (PMA)-induced stress model. As measured with a sensitive HPLC fluorescence method, GSH in HUVEC was depleted by the addition of L-buthionine-[S,R]-sulfoxiniine (BSO), a gamma-glutamylcysteine synthetase inhibitor, to the culture medium at a concentration of 0.25 mM. Time course studies revealed that the GSH half-life was 4.6 h in HUVEC. GSH depletion by BSO for 24 h led to a slight increase in intracellular adhesion molecule - 1 (ICAM1) expression and prostaglandin E2 (PGE2) secretion in both types of cells. However, GSH depletion markedly enhanced PMA-induced ICAM and PGE2 production in HUVEC. Responses were progressively elevated following prolonged BSO treatment. Inhibition studies showed that 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, not only abolished most of PMA-induced ICAM-1 expression and PGE2, production, but also eliminated GSH depletion-enhanced PMA stimulation. This enhancement was also inhibited by supplementation with quercetin. The results clearly demonstrate that GSH depletion increased the susceptibility of vascular endothelial cells and fibroblasts to oxidative stress associated inflammatory stimuli. This increased in vitro susceptibility may be extrapolated to the in vivo situation of ageing, providing a useful model to study the influence of micronutrients on the ageing process. In conclusion, these data suggest that dietary antioxidants could play a significant role in the reduction of inflammatory responses.