No abstract

The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.

Background and Purpose Hydrogen sulfide (H2S), an endogenous volatile mediator with pleiotropic functions, promotes vasorelaxation, exerts anti‐inflammatory actions and regulates angiogenesis. Previously, the SH‐containing angiotensin‐converting enzyme inhibitor (ACEI), zofenopril, was identified as being effective in preserving endothelial function and inducing angiogenesis among ACEIs. Based on the H2S donor property of its active metabolite zofenoprilat, the objective of this study was to evaluate whether zofenoprilat‐induced angiogenesis was due to increased H2S availability. Experimental Approach HUVECs were used for in vitro studies of angiogenesis, whereas the Matrigel plug assay was used for in vivo assessments. Key Results Zofenoprilat‐treated HUVECs showed an increase in all functional features of the angiogenic process in vitro. As zofenoprilat induced the expression of CSE (cystathionine‐γ‐lyase) and the continuous production of H2S, CSE inhibition or silencing blocked the ability of zofenoprilat to induce angiogenesis, both in vitro and in vivo. The molecular mechanisms underlying H2S/zofenoprilat‐induced angiogenesis were dependent on Akt, eNOS and ERK1/2 cascades. ATP‐sensitive potassium (KATP) channels, the molecular target that mediates part of the vascular functions of H2S, were shown to be involved in the upstream activation of Akt and ERK1/2. Moreover, the up‐regulation of fibroblast growth factor‐2 was dependent on CSE‐derived H2S response to H2S and KATP activation. Conclusions and Implications Zofenoprilat induced a constant production of H2S that stimulated the angiogenic process through a KATP channel/Akt/eNOS/ERK1/2 pathway. Thus, zofenopril can be considered as a pro‐angiogenic drug acting through H2S release and production, useful in cardiovascular pathologies where vascular functions need to be re‐established and functional angiogenesis induced.

No abstract

Peroxynitrite, via post-translational modifications to target proteins, contributes to cardiovascular injury and cancer. Since tissue inhibitor of metalloproteinase-4 (TIMP-4), the activity of which is impaired in both pathological conditions, has several amino acid residues susceptible to peroxynitrite, we investigated its role as a potential target of peroxynitrite. Peroxynitrite-induced nitration and oligomerization of TIMP-4 attenuated its inhibitory activity against MMP-2 activity and endothelial or tumor cell invasiveness. Moreover, cell treatment with peroxynitrite promoted the nitration of endogenous TIMP-4. HPLC/ESI-MS/MS analysis of peroxynitrite-treated TIMP-4 showed modifications at Y114, Y195, Y188 and Y190. In conclusion, TIMP-4 nitration might be a potential mechanism contributing to cardiovascular disease and cancer.

No abstract

Src tyrosine kinase family cooperates with activated growth factor receptors to regulate growth, invasion and metastasis. The authors examined the influence of a novel c-Src inhibitor, 1l, derived from 4-amino-substituted-pyrazolo-pyrimidines, on tumor angiogenesis and on the angiogenic output of squamous carcinoma cells, A431 and SCC-4. The effect of 1l was assessed on growth and microvessel density in A431 tumors and its effect compared with the established c-Src inhibitor PP-1. The effects of c-Src inhibition were investigated on vascular endothelial growth factor (VEGF) expression and activity in tumor cells grown in vivo and in vitro, as well as on VEGF mediated signaling and on endothelial cell functions. Nanomolar concentrations of 1l decreased tumor volume promoted by A431 implanted in nude mice, without affecting in vitro cell tumor survival. This effect was related to 1l inhibition of VEGF production, and secondary to an effect on tumor microvessel density. The rabbit cornea assay confirmed that 1l markedly decreased neovessel growth induced by VEGF. In cultured endothelial cells, 1l inhibited the VEGF-induced phosphorylation on tyr416 of c-Src, resulting in a reduced cell proliferation and invasion. Consistently, 1l dowregulated endothelial nitric oxide synthase, MAPK-extracellular receptor kinase 1-2 (ERK1-2) activity and matrix metalloproteinases (MMP-2/MMP-9), while the tissue inhibitors of metalloproteinases (TIMP2/TIMP-1) were upregulated. These results demonstrate that nM concentrations of c-Src kinase inhibitors (1l and PP-1), by reducing the production of VEGF released by tumor cell and its endothelial cell responses, have a highly selective antiangiogenesis effect, which might be useful in combination therapies. ' 2006 Wiley-Liss, Inc.

Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2−/− endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.