Umbilical Vein Endothelial Cells Research Articles

Involvement of scavenging receptor‐B1 in NO release by insulin‐like growth factor binding protein‐3 (IGFBP3) in human endothelial and CD34 cells

We recently reported the vascular protective potential of IGFBP3 involving recruitment of circulating endothelial progenitor cells (CD34) to the sites of vascular injury. NO plays an important role in the migration of CD34 cells and therefore, we hypothesized that NO is involved in the effects of IGFBP3. NO release was quantified by DAF‐FM fluorescence in human cord blood‐derived CD34 cells, umbilical vein endothelial cells (HUVECs), pulmonary microvascular endothelial cells (HMVECs) and human retinal ganglionic cells (HRGCs). IGFBP3 (100 ng/ml) increased NO release in all four cell types. Pretreatment with scavenging receptor‐B1 (SRB1) antibody inhibited NO release in endothelial cells but not in HRGCs. HDL induced NO release in endothelial and CD34 cells but not in HRGCs in which mRNA for SRB1 was not detected. Co‐treatment with IGFBP3 and HDL caused a greater release of NO than with either individual treatment. DMS, a sphingosine kinase inhibitor blocked NO release in CD34 cells. IGFBP3 caused eNOS and vasodilator‐stimulated peptide (VASP) phosphorylation and redistribution of VASP in endothelial cells that was inhibited by L‐NAME. IGFBP3 and HDL caused relaxation of phenylephrine‐preconstricted rat mesenteric arterial segments that was inhibited by L‐NAME and combination of L‐NAME and indomethacin, respectively. These results suggest involvement of NO in the vascular protective effect of IGFBP3.

HIV protease inhibitors induce ER stress and apoptosis in human endothelial cells

Endothelial dysfunction/injury represents a key step in the early stage of atherogenesis. Recent studies have shown that HIV protease inhibitors (PIs) induce endothelial dysfunction, but the underlying molecular mechanism remains unclear. Our previous studies have shown that HIV PI‐induced ER stress and activation of the unfolded protein response (UPR) represents an important cellular mechanism by which HIV PIs disrupt lipid metabolism and induce inflammation in hepatocytes and macrophages. The aim of this study was to determine if HIV PIs also induce ER stress and apoptosis in human endothelial cells.MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with individual HIV PIs for various time periods. The mRNA and protein levels of ER stress responsive genes were measured by real‐time RT‐PCR and Western blot analysis, respectively. The apoptosis was detected by fluorescence microscopy using Annexin V‐FITC/propidium iodide. The protein levels of TNF‐α, IL‐6, and MCP‐1 were determined by ELISA.ResultsBoth lopinavir and ritonavir dose‐dependently activated the UPR, increased IL‐6 and MCP‐1 expression, and induced apoptosis in HUVECs. But amprenavir had no significant effect.ConclusionsHIV PI‐induced ER stress response and activation of the UPR may represent a critical molecular mechanism underlying HIV PI‐associated endothelial dysfunction and atherogenesis.

Unidirectional Transport Across Cultured Ovine Amniotic Epithelial Cell Monolayer

To determine whether ovine amniotic cells in monolayer culture exhibit unidirectional transport. Permeability of (14)C-inulin was measured in two directions across ovine amniotic cells grown to confluence on cell culture inserts. Permeability was 0.69 + 0.17 (SE) microl/min/cm(2) from the top of the insert where the cells were attached to the bottom and 0.11 + 0.02 microl/min/cm(2) in the reverse direction (P < 0.001). Permeability was similar in the two directions for chorionic cells and umbilical vein endothelial cells. Addition of vascular endothelial growth factor did not alter permeability. Transcellular transport in ovine amniotic monolayers is directionally specific. The transport ratio of 6.2:1 for (14)C-inulin is comparable to the in vivo ratio of 4.7:1 for the ovine intramembranous pathway and is consistent with passive diffusion in both directions and vesicular transport in one direction across the monolayer at 5.2 times the rate of passive diffusion.

Preclinical Evaluation of CRx-501, a Potent Selective A2A Agonist as a Novel Drug Candidate for the Treatment of Multiple Myeloma

We have made the surprising discovery that agonism of adenosine A2A receptors has potent and synergistic antiproliferative effects in B-cell malignancies. Here we describe the preclinical evaluation of CRx-501, a potent selective adenosine A2A receptor agonist that synergizes with established anti-MM agents resulting in enhanced efficacy in pre-clinical models of MM. CRx-501 demonstrates potent single agent inhibition of proliferation in MM cell lines with EC50s ranging from 2–20 nM and maximum effects ranging from 20–75% inhibition of proliferation in the 10 MM cell lines surveyed. While CRx-501 demonstrates single agent effects, this molecule potently synergizes with glucocorticoids (dexamethasone and prednisolone), bortezomib, lenalidomide, melphalan and doxorubicin as well as emerging drug classes including HDAC inhibitors and HSP90 inhibitors. Substantial increases in overall effect levels and 2 to 100 fold potency shifts are observed with CRx-501 combinations in a broad panel of 10 MM cell lines including those both sensitive and resistant to current MM agents. In MM.1S cells, addition of 50 nM CRx-501 to 100 nM Dexamethasone results in 95% inhibition of proliferation as compared to approximately 60 and 75% inhibition with either CRx-501 or dexamethasone alone respectively. This combination results in a 10 fold shift in the dexamethasone IC50 and a combination index of 0.2 indicating high levels of synergy. Importantly, in cells resistant to dexamethasone up to the highest concentrations tested (2 microM; EJM, ANBL-6, MM.1R, KSM-12PE, MOLP-8), addition of CRx-501 can induce synergistic inhibition of proliferation converting resistance into sensitivity. Similarly, in combination with lenalidomide, CRx-501 results in a >50 fold shift in IC50, an enhancement of efficacy from 50% to 90% inhibition of proliferation and activity against a lenolidamide resistant cell line (MOLP-8). Synergy is also observed with bortezomib resulting in a 2-fold shift in the IC50 of bortezomib in the presence of CRx-501. Importantly, CRx-501 is highly selective for B-cell malignancies and demonstrates no single agent activity or synergy in normal primary human cell types including peripheral blood mononuclear cells, umbilical vein endothelial cells, aortic smooth muscle cells, coronary artery endothelial cells and solid tumor cell lines at concentrations 2–3 orders of magnitude greater than the IC50 in MM cell lines. These antiproliferative effects occur through apoptosis. Treatment with CRx-501 in combination with dexamethasone causes a rapid and synergistic induction of Annexin V expression as compared to either single agent alone. Surprisingly, the potency and efficacy of CRx-501 is enhanced in the presence of 10 ng/mL interleukin-6 and HS-5 human bone marrow stromal cells. Concentrations of CRx-501 that are effective in vitro are achievable in mice with a 3 mg/kg s.c. dose resulting in a mean plasma AUC0–24 of 1228 ng*hr/mL. The synergistic antiproliferative effects of CRx-501 translate into xenograft models of MM with no significant body weight loss. Mice bearing subcutaneous H929 tumors show a 73% reduction in tumor volume after treatment with the combination of dexamethasone (1 mg/kg, s.c QD) and CRx-501 (3 mg/kg s.c. QD) for 34 days compared to a 36% or 37% decrease after treatment with either CRx-501or dexamethasone respectively. In summary, we report the preclinical evaluation of CRx-501, a potent and highly synergistic A2A agonist as a novel, selective, synergistic drug candidate for the treatment of MM. Our preclinical data provides compelling evidence in support of the further development of CRx-501 for use in multi-drug combination therapy of MM.

Effects of double targeting gene therapy by suicide gene combined with endostatin gene in treatment of bladder cancer: an experimental study

To investigate the effect of double targeting gene therapy by using recombinant adeno-associated virus-thymidine kinase (TK)-internal ribosome entry site (IRES)-endostatin (ES) (rAAV-TIE). Bladder cancer cells of the line T24 were cultured and transfected with rAAV-ES, rAAV-MCS (blank virus), and rAAV-TIE respectively. 72 hours later the levels of ES in the supernatants were measured by ELISA and annexin V apoptosis test kit was used to examine the apoptosis. Human umbilical vein endothelial cells (HUVECs) were transfected with rAAV-ES, rAAV-TIE, and rAAV-MCS respectively. MTT method and flow cytometry were used to detect the apoptosis of the HUVECs. Balb/c nude rats were inoculated subcutaneously with T24 cells. Twenty rats with tumor were randomly divided into 4 equal groups to be treated by rAAV-MCS, rAAV-TK, rAAV-ES, or rAAV-TIE, and 5 rats were used as control group. Four weeks later, blood samples were collected to detect the ES level by ELISA. The tumors were taken out to undergo microscopy to calculate the microvessel density(MVD). 72 h after transfection, ES could be detected in the supernatants of the T24 cells transfected with rAAV-ES, and rAAV-TIE. The apoptotic rates of the T24 cells transfected with rAAV-TK and rAAV-TIE were 34.12% and 36.91% respectively, significantly higher than those of the T24 cells transfected with rAAV-MCS and of the control group (3.08% and 0.84%, all P < 0.05). Transfection of rAAV-ES and rAAV-TIE increased the apoptotic rate of the HUVECs time-dependently. Nine days after the transfection rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumes of the rAAV-ES, rAAV-TK, and rAAV-TIE groups were (0.75 +/- 0.08), (0.71 +/- 0.11), and (0.52 +/- 0.09) cm(3) respectively, all significantly lower than those of the rAAV-MCS group and control group [(1.27 +/- 0.13) and (1.24 +/- 0.17) cm(3) respectively, all P < 0.05]. rAAV-TIE effectively inhibits the tumorigenesis and angiogenesis in bladder cancer. Double targeting gene therapy against bladder cancer can be achieved by using rAAV.

Angiotensin inhibition stimulates PPARγ and the release of visfatin

Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARB) exhibit beneficial antidiabetic effects in patients with type 2 diabetes independent of their blood pressure-lowering effects. Some antidiabetic properties of ARB and ACE-I might by exerted by activation of peroxisome proliferator-activated receptor gamma (PPARgamma). However, it is not clear whether this action is drug specific. The binding affinity of telmisartan, valsartan, lisinopril, rosiglitazone and angiotensin II to PPARgamma was assessed in a cell-free assay system. PPARgamma signalling was studied in isolated skeletal muscle cells using Western blot analysis of phosphorylated protein kinase B (pAKT) and phosphorylated insulin like growth factor-1 receptor (pILGF-1R). Further, the ability of the drugs under study to stimulate the release of the adipocytokine visfatin was investigated in isolated human adipocytes, skeletal muscle cells, and umbilical vein endothelial cells (HUVEC). The binding affinity to PPARgamma was highest for telmisartan with a half-maximal effective concentration of 463 nM, followed by lisinopril (2.9 microM) and valsartan (6.2 microM). In skeletal muscle cells phosphorylation of ILGF-1R was 2-fold increased after incubation with telmisartan or valsartan and 1.7-fold with lisinopril. pAKT expression was enhanced after incubation with telmisartan, valsartan and with lisinopril. The release of visfatin from adipocytes was 1.6-fold increased after treatment with lisinopril and about 2.0-fold increased with telmisartan and valsartan. Similar results were obtained in skeletal muscle cells and HUVEC. Our data confirm agonism of telmisartan, valsartan and lisinopril on PPARgamma. Pharmacokinetic differences may explain different potencies of PPARgamma stimulation by drugs acting on the renin-angiotensin system in clinical settings.

KRIT1 MODULATES β1-INTEGRIN-MEDIATED ENDOTHELIAL CELL PROLIFERATION

Using ribonucleic acid interference on cultured cell lines, we examined the role of Krev interaction trapped 1 (krit1) and integrin cytoplasmic domain-associated protein-1 alpha (icap1alpha) in beta1-integrin-mediated cell proliferation. Upon depletion of either krit1 or icap1alpha in the HeLa cells, umbilical vein endothelial cells, and microvascular endothelial cells, we examined the cell number and proliferation changes in the cells, followed by the evaluation of beta1-integrin-mediated mitogen-activated protein kinase signal pathway and microscopic study. Depletion of krit1 reduces cell number and decreases endothelial cell proliferation. Examination of beta1-integrin signaling downstream of focal adhesion kinase reveals decreased phosphorylation along the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Depletion of icap1alpha, a protein known to interact with krit1, has similar effects, suggesting synergistic function. We also show that krit1 colocalizes with icap1alpha in both the nucleus and the cytoplasm; however, most of icap1alpha is found in the nucleus and most of krit1 is found in the cytoplasm at steady state. On depletion of krit1, icap1alpha decreases in the cytoplasm and is no longer detected in the nucleus. Both krit1 and icap1alpha act concordantly to play a critical role in beta1-integrin-mediated cell proliferation. Our data further suggest that krit1 both stabilizes and shuttles icap1alpha and thus modulates its regulation of beta1-integrin-mediated signal transduction.

Endothelium-specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice

Hazardous environmental and genetic factors can damage endothelial cells to induce atherosclerotic vascular disease. Recent studies suggest that class III deacetylase SIRT1 may promote cell survival via novel antioxidative mechanisms. The current study tested the hypothesis that SIRT1, specifically overexpressed in the endothelium, is atheroprotective. Human umbilical vein endothelial cells (HUVECs) were used to study the effects of oxidized low-density lipoprotein (LDL) on SIRT1 expression. Endothelial cell-specific SIRT1 transgenic (SIRT1-Tg) mice were used to study the effects of SIRT1 on aortic vascular tone. SIRT1-Tg mice were crossed with apolipoprotein E null (apoE(-/-)) mice to obtain SIRT1-Tg/apoE(-/-) mice for the analysis of atherogenesis in the presence of endothelial overexpression of SIRT1. SIRT1 expression in HUVECs was increased by the treatment with oxidative LDL. Adenoviral-mediated overexpression of SIRT1 was protective of apoptosis of HUVECs. Calorie restriction increased, whereas high-fat diet decreased, the SIRT1 expression in mouse aortas. In SIRT1-Tg mice, high fat-induced impairment in endothelium-dependent vasorelaxation was improved compared with that of wild-type littermates. This was accompanied by an upregualtion of aortic endothelial nitric oxide synthase expression in the SIRT1-Tg mice. The SIRT1-Tg/apoE(-/-) mice had less atherosclerotic lesions compared with apoE(-/-) controls, without affecting blood lipids and glucose levels. These results suggest that endothelium-specific SIRT1 overexpression likely suppresses atherogenesis via improving endothelial cell survival and function.

Anti-endothelial cell antibodies in insulin-dependent diabetes mellitus.

The prevalence of IgG class antibodies to endothelial cells (AECA) was studied in 136 young patients with insulin-dependent diabetes mellitus by an enzyme immunoassay using human umbilical cord vein endothelial cells. The patients were divided into four groups according to the time between diagnosis and study and their results were compared with those in control children and blood donors. AECA became progressively more frequent with the duration of diabetes, being 4% in diabetics tested within 2 weeks of diagnosis and reaching 34% after an average disease duration of 11.2 years. They were not more common in patients with neuropathy, retinopathy or nephropathy than in patients without these complications, but were associated with co-existing thyroid disease and IgA deficiency. The results suggest that in insulin-dependent diabetes mellitus AECA are associated with co-existing autoimmune disorders but not with diabetic microvascular disease.

Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms

Activation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-1 (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM-1, recognized by a monoclonal antibody (MoAb) ENA1, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab')2 fragments of MoAb ENA1 up to 60%. An additive inhibition of the adhesion was established by pre-incubation of the PMN with anti-CD18 MoAb and/or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENA1, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD18 MoAb and/or LAI reduced the adhesion observed if intact ENA1 were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD18 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a role in graft rejection and in diseases where antibodies directed against endothelium are involved.

Enhanced Gene Delivery to Human Primary Endothelial Cells Using Tropism-Modified Adenovirus Vectors

Endothelial cells have been noted to have relatively low expression of the native receptor for adenovirus serotype 5 (Ad5), coxsackie and adenovirus receptor (CAR), and are thus refractory to Ad5 infection. In this study, we hypothesize that increases in the infectivity of Ad5 in primary human pulmonary artery (HPAEC), coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC) can be achieved through genetic capsid modification of Ad5 to bypass CAR-dependent infection. The modifications tested in this study include incorporation of an integrin-binding RGD peptide motif (Ad5.RGD), a poly-lysine motif (Ad5.pK7), a combination of both of these peptide domains (Ad5.RGD.pK7), an adenovirus serotype 3 knob domain (Ad5/3Luc1) and canine adenovirus serotype 1 or 2 knob domains (Ad5Luc1-CK1 and Ad5Luc1-CK2). In HPAEC and HCAEC, the greatest infectivity enhancements were achieved using Ad5/3Luc1 (26-fold and 30-fold respectively). HUVEC was most readily infected by Ad5Luc1-CK1 (213-fold). These results demonstrate that gains in Ad5 infectivity in endothelial cells can be accomplished with genetic capsid modifications.

The control of cell adhesion and viability by zinc oxide nanorods

The ability to control the behavior of cells that interact with implanted biomaterials is desirable for the success of implanted devices such as biosensors or drug delivery devices. There is a need to develop materials that can limit the adhesion and viability of cells on implanted biomaterials. In this study, we investigated the use of zinc oxide (ZnO) nanorods for modulating the adhesion and viability of NIH 3T3 fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells. Cells adhered far less to ZnO nanorods than the corresponding ZnO flat substrate. The few cells that adhered to ZnO nanorods were rounded and not viable compared to the flat ZnO substrate. Cells were unable to assemble focal adhesions and stress fibers on nanorods. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Time-lapse imaging revealed that cells that initially adhered to nanorods were not able to spread. This suggests that it is the lack of initial spreading, rather than long-term exposure to ZnO that causes cell death. We conclude that ZnO nanorods are potentially useful as an adhesion-resistant biomaterial capable of reducing viability in anchorage-dependent cells.

Pioglitazone inhibits dendritic cell adhesion and transmigration through down-regulation of dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin

To investigate the effect of pioglitazone (Pio) on dendritic cell-(DC) specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) expression in DCs and explore the possible mechanism of Pio inhibiting DC adhesion and transmigration. DCs derived from human peripheral blood mononuclear cells were cultured and divided into 6 groups: blank control group, Pio 0.1 micromol/L group, Pio 1.0 micromol/L group, Pio 10 micromol/L group, GW9662, a peroxisome proliferator activated receptor (PPAR)-gamma antagonist, 10 micromol/L group, and GW9662 10 micromol/L + Pio 10 micromol/L group. Western blotting was used to detect the protein expression of DC-SIGN 24 h later. Human umbilical vein endothelial cells (HUVECs) were obtained and co-cultured with the DCs undergoing different treatments. Immunofluorescence test was used to detect the protein expression of DC-SIGN. DCs labeled with 5-chloromethylfluorescein diacetate (CMFDA) were added into the monocellular layer of fused ECs. Blank DCs and DCs pretreated with anti-DC-SIGN antibody were used as blank and experimental groups. Laser confocal microscopy was used to observe the adhesion ability of the DCs. HUVECs were inoculated into the upper chamber of Transwell plate and CMFDA-labeled DCs of above mentioned groups were added to the mono-cellular layer of these ECs. Serum-free culture medium with monocyte chemoattractant protein-1 was added into the lower chamber. Eight hours later the transmigration ability was observed. Western blotting showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 0.96 +/- 0.09 and 0.80 +/- 0.08 respectively, both significantly lower than that of the blank control group (1.25 +/- 0.23, P < 0.05 and P < 0.01); and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 1.10 +/- 0.12, significantly higher than that of the Pio 10 micromol/L group (P < 0.05). Immunofluorescence test showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 22.3 +/- 5.4 and 14.4 +/- 2.3 respectively, both significantly lower than that of the control group (29.5 +/- 5.1, P < 0.05 and P < 0.01), and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 24.9 +/- 4.3, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The adhesion rates of the Pio 1.0 micromol/L and Pio 10 micromol/L groups were 10.8% +/- 2.0% and 7.6% +/- 1.5% respectively, both significantly lower than that of the control group (13.4% +/- 2.1%, P < 0.05 and P < 0.01); and the adhesion rate of the GW9662 + Pio 10 micromol/L group was 12.1% +/- 1.9%, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The transmigration inhibition rate of DCs of the Pio 0.1, 1.0, and 10 micromol/L groups were 4.1%, 12.9%, and 17.2% compared with the transmigration rate of the blank control group (P < 0.05, P < 0.05, and P < 0.01). The transmigration rate of the GW9662 + Pio 10 micromol/L group was significantly higher than that of the Pio 10 micromol/L group (P < 0.05), and not significantly different from that of the blank control group (P > 0.05). The transmigration rate of the anti-DC-SIGN intervention group was lower by 17.8% than that of the control group (P < 0.01). Pio down-regulates the DC-SIGN protein expression and inhibits DC adhesion and transmigration through the pathway of PPAR-gamma.

Maturation of capillary-like structures in a tube-like construct in perfusion and rotation culture

In an effort to engineer capillary-like networks in vitro, different cultivation methods were compared. Five small-diameter tube-like constructs (‘vessel equivalents’) were fabricated from porcine gelatin scaffolds and seeded with human adipose tissue stromal cells and umbilical vein endothelial cells. After initial growth, the vessel equivalents were divided. One segment was exposed to pulsatile perfusion and the other was kept in rotating culture. Specimens harvested at the start of the experiments and after 16 days of rotation or perfusion were compared histomorphometrically with respect to capillary-like network formation in the vessel wall. Most capillary-like structures were found in the luminal portion of perfused vessel equivalents. Maturation of these capillary-like structures ascertained by recruitment of α-actin-positive cells also reached the highest degree in the luminal portion of perfused specimens. Perfused specimens showed significantly less apoptosis. Pulsatile perfusion promotes the development and maturation of a capillary-like network in this in-vitro approach.

Selenium Inhibits High Glucose- and High Insulin-induced Adhesion Molecule Expression in Vascular Endothelial Cells

Initiation of an atherosclerotic lesion requires endothelial expression of adhesion molecules. Selenium (Se), a biologically essential trace element, can inhibit cytokine (e.g., TNF-alpha)-induced expression of adhesion molecules. Atherosclerosis is accelerated in diabetic patients. This is at least partially caused by hyperglycemia and hyperinsulinemia increasing adhesion molecule expression. These experiments tested whether Se can also alter high glucose- and high insulin-induced expression of adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were pretreated with Se and stimulated by high glucose or high insulin. Expression of adhesion molecules was measured by Western blot. Se (100 nmol/L) significantly inhibited glucose (25 mmol/L)-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin. Moreover, Se significantly inhibited insulin (100 nmol/L)-induced VCAM-1 and ICAM-1 expression, whereas high insulin had no inducing effect on E-selectin. Se also inhibited high glucose- and high insulin-induced activation of p38 mitogen-activated protein kinase (p38), which indicated that the preventive effects of Se on adhesion molecules may be associated with p38. The important role of p38 in Se effects was further confirmed using p38 inhibitor SB203580. These results suggest that Se can inhibit high glucose- and high insulin-induced expression of adhesion molecules. Such antagonism is at least partially mediated through the modulation of p38 pathway. Therefore, Se may be considered as a potential preventive intervention for diabetes-accelerated atherosclerosis.

Human recombinant erythropoietin augments serum asymmetric dimethylarginine concentrations but does not compromise nitric oxide generation in mice

Patients with advanced chronic renal disease (CRD) suffer from excessive morbidity and mortality due to complications of accelerated atherosclerosis. Recombinant human erythropoietin (EPO), which is routinely used to treat the anaemia present in approximately 90% of dialysis-dependent patients with end-stage renal disease, may induce vascular dysfunction by reducing nitric oxide (NO) availability. Pathophysiologic concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are found in patients with CRD and correlate with vascular disease and cardiovascular mortality. The aim of the current study was to investigate the effect of EPO on ADMA concentrations and NO generation in vitro and in vivo. Furthermore, we wanted to study the effect of EPO on the expression of the enzymes that regulate ADMA metabolism and NO generation. Human umbilical vein endothelial cells (HUVECs) were exposed to therapeutic concentrations of EPO. The expression and metabolic activity of dimethylarginine dimethylaminohydrolase II (DDAH II), the enzyme that degrades ADMA, was evaluated. Following subcutaneous administration of EPO to Balb/c mice for 10 weeks, serum ADMA concentrations were determined. Systolic blood pressure was measured noninvasively. Urinary nitrite and nitrate (NOx) concentrations were assessed by Griess assay. Protein expression of DDAH and NOS in livers and kidneys was measured by western blotting. EPO suppressed ADMA elaboration by HUVECs. Systolic blood pressure and serum concentrations of ADMA were significantly elevated in EPO-treated mice. The protein expression of DDAH I in the kidney and liver was upregulated while hepatic expression of DDAH II was decreased and renal DDAH II expression remained unchanged by EPO administration. However, EPO augmented urinary NOx concentrations as well as the expression of NOS 1 and NOS 2 in the kidney. In spite of elevating serum ADMA concentrations, EPO does not appear to compromise overall NO generation in Balb/c mice.

Diamond-like Carbon Film Coating for Tissue Engineering

AbstractIn this study, we focus on effect of diamond-like carbon (DLC) coating on scaffold for tissue engineering. DLC film was deposited on segmented polyurethane (SPU) scaffold sheet which consists of micro SUP fibers. Structural and compositional effects of the DLC film coating were investigated on cell growth as an investigation of biological response. The surface composition, morphology, structures, and wettability of the DLC film coating was estimated by using X-ray photoelectron spectrometer (XPS), Scanning Electron Microscope (SEM), Ar-laser Raman spectrophotometer (Raman), and contact angle measurement. And then, human umbilical vein endothelial (HUV-EC-C) cells were grown on the DLC coated scaffold sheet. The results presented here suggest that DLC film coating is promising approach to improve biological for tissue engineering.

Strikingly Different Angiogenic Properties of Endothelial Progenitor Cell Subpopulations: Insights from a Novel Human Angiogenesis Assay

Objectives An endothelial cell (EC)-specific angiogenesis assay was developed to functionally characterize angiogenic properties of 2 distinct putative endothelial progenitor cells (EPCs): early EPCs and late outgrowth endothelial cells (OECs). Background Endothelial progenitor cells promote revascularization of ischemic tissue. However, the nature of different EPCs and their role in angiogenesis remains debated. Methods Tubulogenesis was assessed by immunohistochemistry in co-cultures of differentiated ECs (including human umbilical vein, coronary artery, and microvascular ECs) or non-ECs with monolayers of human fibroblasts (MRC5). Using adaptations of the co-culture assay, early EPCs and OECs, isolated from peripheral blood mononuclear cells, were assessed by 3-dimensional immunofluorescence microscopy for their capacity for: 1) independent tubulogenesis; 2) incorporation into pre-existing vascular networks; and 3) paracrine angiogenic effects using transwell cultures. Results Branched interconnecting EC-specific tubules formed with all differentiated ECs after 72 h. Proangiogenic and antiangiogenic agents modulated tubulogenesis appropriately (vascular endothelial growth factor 10 ng: +142 ± 13%, 1 μM anti-vascular endothelial growth factor: −44 ± 7% vs. control, p Conclusions A novel EC-specific tubulogenesis assay highlights strikingly different angiogenic properties of different EPCs: late OECs directly participate in tubulogenesis, whereas early EPCs augment angiogenesis in a paracrine fashion, with implications for optimizing cell therapies for neovascularization.

Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene

Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS). This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene. Human umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0, 10, 30, 100, and 300 micromol/L) for 72 hours. The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP). The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR. The activity of DDAH2 and eNOS in cells, and the concentrations of ADMA and NO in culture medium were assayed respectively. Mild increased concentration of Hcy (10 and 30 micromol/L) induced hypomethylation, while high concentration of Hcy (100 and 300 micromol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene. The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy, and decreased in high concentration of Hcy correspondingly. The inhibition of DDAH2 activity, the increase of ADMA concentration, the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration. The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway, which showed highly relevant and dose-effect relationship. The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in DDAH2 gene.

Effect of Umbilical Cord Blood Hematopoietic Stem Cells on Formation of Angiogenic Structures.

There is a significant unmet need for novel therapeutic treatments for patients presenting with chronic ischemic conditions such as coronary artery disease and diabetes. Revascularization measures, such as infusions with endothelial progenitor cells (EPC) characterized by the expression of early hematopoietic stem cell markers, hold significant potential in treating these patients. Pre-clinical and clinical studies using transplanted EPC to restore blood flow and improve cardiac function in animal models of ischemia have proven effective. Recent studies have used bone marrow mononuclear cells while some more recent studies have focused on enriched stem cell treatments, such as purified bone marrow hematopoietic stem cell (HSC) CD34+/133+ cell populations, in patients with coronary artery ischemia. In this study, the hypothesis to be tested was that umbilical cord blood-derived hematopoietic stem cells (CD34+/CD133+) cells may augment the formation and stability of angiogenic networks of cord-like structures derived from umbilical vein endothelial cells (HUVEC) cultured in growth factor-reduced Matrigel (GFR MG) assays. Umbilical cord blood MNC were isolated with ficoll and separated into HSC CD34+/133+ and CD34−/133− fractions. Positive fractions were flow cytometry, sorted for HSC, and stained with the lipophilic fluorescent red dye CM-DiI and the HUVEC were stained with the lipophilic fluorescent green dye Oregon Green. HUVEC alone or HSC and HUVEC were then co-cultured under hypoxic conditions (1% O2) on the GFR MG in 96 well plates. Cells were photographed with a fluorescent microscope at 16, 48, and 72 hours. Transwell experiments (0.4μm pores) were also performed with HSC CD34+/133+ and CD34−/133− fractions prepared and suspended in transwells above HUVEC plated on GFR MG on bottom wells. The presence of both HSC CD34+/133+ and CD34−/133− fractions increased the numbers of nodes (branch points of structures) and allowed the structures to persist when observed over three days (a representative experiment of N =3) (Table):Day 1Day 1Day 2Day 2Day 3Day 3Node #% TotalNode #% TotalNode #% TotalHUVEC11.6 ± 4.91001.3 ± 1.29.20.33 ± 0.582.2HUVEC + HSC CD34+/133+17.3 ± 9.21006.3 ± 4.535.34.7 ± 5.521.4HUVEC + HSC CD34−/133−34 ± 13.210019.7 ± 2.561.610 ± 3.629.8The HSC CD34−/133− fraction resulted in a greater increase in node formation than the HSC CD34+/133+ and both fractions stimulated significant persistence in formed structures. In addition, CM-Dil labeled cells were localized at nodes points. Results with the transwell assay demonstrated that when either HSC CD34+/133+ or CD34−/133− fractions were suspended above HUVEC, augmentation of the formation of cord-like structures was not observed. In summary, both umbilical cord blood-derived HSC CD34+/133+ and CD34−/133− fractions possess properties that augment the formation of angiogenic structures. We observed that the number of nodes are greater in the presence of both HSC CD34+/133+ and CD34−/133− fractions than with HUVEC alone. The transwell experiment suggested that cell-to-cell interactions are necessary for augmentation of the cord structures. In future studies, we will address the mechanism of intercellular interactions that result in the augmentation of cord-like structures and which particular subpopulations within cord blood, both from HSC CD34+/133+ and CD34−/133− fractions are required for augmentation of structure formation.