Jozef Dulak

Nitric Oxide Induces the Synthesis of Vascular Endothelial Growth Factor by Rat Vascular Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1beta (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with L-arginine (10 mmol/L) or tetrahydrobiopterin (BH(4); 100 micromol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 micromol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1beta induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1beta-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-arginine or BH(4) increased NO generation by L-NAME- or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH(4). The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 micromol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.

Prostaglandin-J2 induces synthesis of interleukin-8 by endothelial cells in a PPARγ-independent manner

PPARgamma is a transcription factor of nuclear receptor superfamily, involved in the regulation of inflammation. We investigated the influence of PPARgamma-ligands, 15-deoxy-delta12,14 prostaglandin-J2 (15d-PGJ2), and ciglitazone, on the generation of interleukin-8 (IL-8) by the human microvascular endothelial cell line (HMEC- 1). Expression of PPARgamma in HMEC-1 was confirmed by RT-PCR. Both PPARgamma-ligands tested induced the activation of PPAR, but the potency of ciglitazone was higher, as evidenced by luciferase assay. Resting HMEC-1 released about 150 pg/ml of IL-8 protein. Treatment with LPS increased the IL-8 secretion up to 1 ng/ml. 15d-PGJ2 potently and dose-dependently increased both the steady-state and LPS-induced generation of IL-8 mRNA and IL-8 protein. In contrast, neither basal nor LPS-elicited expression of IL-8 was influenced by ciglitazone. We conclude, that 15d-PGJ2 is a potent inducer of IL-8 production and can be a mediator of inflammatory response, but this effect is independent of PPARgamma activation.

Gene transfer of nitric oxide synthase: Effects on endothelial biology

OBJECTIVES The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA. BACKGROUND Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC. METHODS Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence. RESULTS The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = -0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha. CONCLUSIONS Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.

Atorvastatin decreases vascular endothelial growth factor in patients with coronary artery disease.

OBJECTIVES The aim of this study was to test a possible influence of atorvastatin on the production of vascular endothelial growth factor (VEGF) in patients with coronary artery disease (CAD) and in vitro. BACKGROUND Vascular endothelial growth factor is suggested to be involved in the growth of atherosclerotic plaque by inducing its neovascularization. Hepatic hydroxymethyl glutaryl-coenzyme A reductase inhibitors (statins) are known to have atheroprotective effects beyond lipid lowering. METHODS Blood was collected from 14 male hypercholesterolemic patients with angiographically confirmed CAD at baseline and after two months of atorvastatin therapy (20 mg/d) and from eight male control patients. In an ex vivo assay, human coronary artery smooth muscle cells (HCASMC) were incubated with the patient plasma collected before and after atorvastatin therapy. To test the direct effect of atorvastatin on VEGF synthesis in vitro, HCASMC were treated with atorvastatin (1, 3 and 10 microM). The VEGF concentration was measured by enzyme-linked immunosorbent assay. RESULTS Atorvastatin therapy reduced VEGF plasma levels in CAD patients (from 31.1 +/- 6.1 to 19.0 +/- 3.6 pg/ml; p < 0.05). The VEGF plasma concentration tended to be higher in CAD patients before treatment compared to control patients (31.1 +/- 6.1 vs. 23.4 +/- 3.6 pg/ml; p = NS). Plasma collected before therapy induced significantly more VEGF in HCASMC compared to the plasma collected after treatment and compared to control cells. In vitro, atorvastatin decreased both the basal and the interleukin-1beta-induced VEGF release in HCASMC. CONCLUSIONS These data suggest that atorvastatin may lower the plasma level of VEGF in CAD patients, which could represent a novel beneficial effect of this and perhaps other statins.

Vascular endothelial growth factor (VEGF) plasma concentrations in coronary artery disease

Vascular endothelial growth factor (VEGF) has been associated with atherosclerosis progression and lesion destabilisation. Despite a beneficial effect of local VEGF administration in myocardial and peripheral ischaemia,1 recent evidence suggests a pro-atherosclerotic role of VEGF2 through its ability to enhance plaque inflammatory infiltration and neovascularisation. Despite these results, coming mostly from animal studies, there have been few investigations on the relation between VEGF and human coronary artery disease (CAD). These studies yielded conflicting results regarding VEGF concentrations and gene expression in CAD patients compared to controls.3,4 To elucidate the association of VEGF and CAD further, we performed a prospective study in consecutive patients with chest pain undergoing coronary angiography to compare the coronary status with VEGF plasma concentrations. Written informed consent was obtained from 178 patients. Plasma was collected from a femoral artery access immediately before diagnostic coronary angiography. Exclusion criteria were prior myocardial infarction (< 1 month before inclusion), tumour disease, peripheral arterial occlusive disease, ejection fraction < 30%, acute infections, chronic rheumatoid diseases, and chronic obstructive pulmonary disease. Prior statin use was defined as statin treatment for more than 10 days. VEGF plasma concentrations were determined by enzyme linked immunosorbent assay (ELISA, R&D System, Abingdon, UK). Coronary angiograms were scored visually by a blinded observer: a severity score (0–3) defined the number of vessels with a luminal stenosis ⩾ 50% (for right, left anterior descending, and …

Vascular endothelial growth factor: angiogenesis, atherogenesis or both?

Vascular endothelial growth factor (VEGF), a specific mitogen for endothelial cells, was initially regarded to be a remedy for impaired reendothelialization of arteries in patients treated with balloon angioplasty. Supplementation with VEGF was also expected to induce the formation of blood vessels

Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells.

The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 iso-forms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, b-galactosidase gene, was about 50% in COS- 7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121 and pSG5-VEGF165 plasmids can be used for therapeutic application.

Mean free path and peak dispersion in the geometration motion in gel electrophoresis

The concept of the mean free path, i.e., the mean distance between subsequent collisions of DNA molecules with gel fibers, is introduced to the model description of the geometration effect. A new formula is derived for the correction to the velocity v of long molecules in gel due to the geometration process: v = vr /

Gene Transfer of Naked VEGF Plasmid Induces the Formation of Microvessels but not Mature Collaterals in Ischaemic Limb Muscles

(1 \!\!+\!{{3{s} } \over {4{b} }})

Role of peroxisome proliferator-activated receptorγ ligands in the vessel wall

, where vr is the velocity without geometration, s is the molecule length and b is the mean free path of molecules. The peak dispersion σx is evaluated with the same model approach. We get the contribution to the bandwidth from the geometration effect. For b = s the bandwidth is about 2(sx)1/2, where x is the length of the path of the molecules in gel. The results are compared with experimental data on the linearized plasmid pKecNOS in 4% agarose gel.