Adeno-Associated Virus-Mediated Gene Transfer of Inducible Nitric Oxide Synthase to an Animal Model of Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by progressive obstruction of pulmonary arteries owing to inflammatory processes, cellular proliferation, and extracellular matrix deposition and vasoconstriction. As treatment options are limited, we studied gene transfer of an inducible nitric oxide synthase (iNOS) using adeno-associated virus (AAV) vectors specifically targeted at endothelial cells of pulmonary vessels in a murine model of PH. Adult mice were intravenously injected with AAV vectors expressing iNOS. Mice were subjected to hypoxia for 3 weeks and killed afterward. We found elevated levels of iNOS both in lung tissue and pulmonary endothelial cells in hypoxic controls that could be further increased by AAV-mediated iNOS gene transfer. This additional increase in iNOS was associated with decreased wall thickness of pulmonary vessels, less macrophage infiltration, and reduced molecular markers of fibrosis. Taken together, using a tissue-targeted approach, we show that AAV-mediated iNOS overexpression in endothelial cells of the pulmonary vasculature significantly decreases vascular remodeling in a murine model of PH, suggesting upregulation of iNOS as promising target for treatment of PH.

Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.

G protein-mediated signaling plays a decisive role in blood pressure regulation and the phenotype of vascular smooth muscle cells (VSMCs); however, the relevance of proteins that restrict G protein activity is not well characterized in this context. Here, we investigated the influence of regulator of G protein signaling 5 (RGS5), an inhibitor of Gαq/11 and Gαi/o activity, on blood pressure and the VSMC phenotype during experimental hypertension. In mice, loss of RGS5 did not affect baseline blood pressure, but prevented hypertension-induced structural remodeling. RGS5-deficient arterial VSMCs did not acquire a synthetic phenotype as evidenced by their inability to decrease the abundance of contractile markers-α-smooth muscle actin and smooth muscle-myosin heavy chain-or to proliferate under these conditions. Mechanistically, hypertensive pressure levels or biomechanical stretch are sufficient to increase the expression of RGS5. Loss of RGS5 severely impairs the activation of RhoA and stress fiber formation. In stretch-exposed VSMCs, RhoA activity was amplified upon inhibition of PKC, which mimics the downstream effects evoked by RGS5-mediated inhibition of Gαq/11 signaling. Collectively, our findings underline that RhoA activation may depend on the restriction of G protein activity and identify RGS5 as a mechanosensitive regulatory protein that is required to promote the synthetic VSMC phenotype as a prerequisite for structural renovation of the arterial wall during hypertension.-Arnold, C., Demirel, E., Feldner, A., Genové, G., Zhang, H., Sticht, C., Wieland, T., Hecker, M., Heximer, S., Korff, T. Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.

Dose finding of 3'deoxyadenosine and deoxycoformycin for the treatment of Trypanosoma evansi infection: An effective and nontoxic dose.

The aim of this study was to evaluate the therapeutic efficacy and safety of using 3'deoxyadenosine (Cordycepin - adenosine analogue) combined with deoxycoformycin (Pentostatin - an adenosine deaminase inhibitor) in mice infected with Trypanosoma evansi. We show that the combination of Cordycepin (2.0 mg kg(-1)) and Pentostatin (0.2, 0.5, 1.0, 2.0 mg kg(1)) is effective in the clearance of T. evansi, although at the higher concentrations of Pentostatin 2 mg kg(-1) some toxicity was observed in the liver and kidney. Since the Cordycepin 2.0 mg kg(-1) and Pentostatin 0.2 mg kg(-1) combination was effective and had low toxicity, we recommend this as a therapeutic option for a T. evansi mouse model.

PD-L1 expression on tolerogenic APCs is controlled by STAT-3.

During infection, TLR agonists are released and trigger mature as well as differentiating innate immune cells. Early encounter with TLR agonists (R848; LPS) blocks conventional differentiation of CD14(+) monocytes into immature dendritic cells (iDCs) resulting in a deviated phenotype. We and others characterized these APCs (TLR-APC) by a retained expression of CD14 and a lack of CD1a. Here, we show in addition, expression of programmed death ligand-1 (PD-L1). TLR-APCs failed to induce T-cell proliferation and furthermore were able to induce CD25(+) Foxp3(+) T regulatory cells (Tregs). Since PD-L1 is described as a key negative regulator and inducer of tolerance, we further analyzed its regulation. PD-L1 expression was regulated in a MAPK/cytokine/STAT-3-dependent manner: high levels of IL-6 and IL-10 that signal via STAT-3 were produced by TLR-APCs. Blocking of STAT-3 activation prevented PD-L1 expression. Moreover, chromatin immunoprecipitation revealed direct binding of STAT-3 to the PD-L1 promoter. Those findings indicate a pivotal role of STAT-3 in regulating PD-L1 expression. MAPKs were indirectly engaged, as blocking of p38 and p44/42 MAPKs decreased IL-6 and IL-10 thus reducing STAT-3 activation and subsequent PD-L1 expression. Hence, during DC differentiation TLR agonists induce a STAT-3-mediated expression of PD-L1 and favor the development of tolerogenic APCs.