Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

BACKGROUND AND PURPOSE

ApolipoproteinE-deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (−/−) mice.

EXPERIMENTAL APPROACH

ApoE (−/−) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg⁻¹·h⁻¹) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg⁻¹·h⁻¹) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys.

KEY RESULTS

Ang-(1-7)-treated apoE (−/−) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (−/−) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (−/−) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased.

CONCLUSION AND IMPLICATIONS

Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.     

Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (alpha1-GC, alpha2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either alpha1- or alpha2-GC to dissect their biological functions. In platelets, alpha1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, alpha1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, alpha2-GC, representing only 6% of the total GC content in WT, also completely relaxed alpha1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by alpha2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.     

The efficiency of simian foamy virus vector type-1 (SFV-1) in nondividing cells and in human PBLs

Current retroviral vectors based on murine leukemia virus (MuLV) are unable to efficiently transduce nondividing cells. Lentiviruses, such as the human immunodeficiency virus 1 (HIV-1) are efficient at transducing nondividing, growth-arrested, and post-mitotic cells, but due to complex safety considerations, they may have limited potential for human clinical gene transfer. For this reason, alternatives to MuLV and HIV-1 vectors need to be explored. In this paper, we have found that simian foamy virus vector (SFV-1) containing a CMV-LacZ expression cassette is able to efficiently transduce multiple cell types of various species that include epithelial, lymphoid, and hematopoietic-derived human cell lines and fibroblast cell lines of several species. Previously it was reported that foamy virus replication is cell cycle dependent (P. D. Bieniasz, R. A. Weiss, and M. O. McClure, 1995. J. Virol. 69, 7295-7299). However, others studies demonstrated nuclear import of viral DNA in arrested cells (A. Saibi, F. Puvion-Dutilleul, M. Schmid, J. Peries, and H. d. The 1997. J. Virol. 71, 1155-1161). Here, we show efficient LacZ transduction by SFV-1 vectors in several chemically arrested cell lines and terminally differentiated human neurons. SFV-1 vector can transduce cell lines arrested in G1/S phase of the cell cycle by aphidicolin treatment with similar efficiencies to that of dividing cells. The terminally differentiated human neural cell line, NT2N, was transduced with 30-50% efficiency, corroborating our data obtained with the arrested cell lines. To further examine whether the SFV-1 vector can efficiently deliver a gene into clinically important cells for gene therapy, we transduced primary human peripheral blood cells (PBLs) in the presence and absence of phytohemagglutanin (PHA) stimulation. We observed 81% transduction efficiency in non-stimulated PBLs and 87% in PHA-stimulated PBLs with vector infection carried out twice in 8 hours intervals at a multiplicity of infection of 1. Together, these data indicate that SFV-1 based retroviral vectors may provide a safe, efficient alternative to current onco- and lentiviral vectors for gene transfer in cells from a broad spectrum of lineages across species boundaries.