Autoimmune disease mouse model exhibits pulmonary arterial hypertension

<div><p>Background</p><p>Pulmonary arterial hypertension is often associated with connective tissue disease. Although there are some animal models of pulmonary hypertension, an autoimmune disease-based model has not yet been reported. MRL/lpr mice, which have hypergammaglobulinemia, produce various autoimmune antibodies, and develop vasculitis and nephritis spontaneously. However, little is known about pulmonary circulation in these mice. In the present study, we examined the pulmonary arterial pressure in MRL/lpr mice.</p><p>Methods and results</p><p>We used female MRL/lpr mice aged between 12 and 14 weeks. Fluorescent immunostaining showed that there was no deposition of immunoglobulin or C3 in the lung tissue of the MRL/lpr mice. Elevation of interferon-γ and interleukin-6 was recognized in the lung tissue of the MRL/lpr mice. Right ventricular systolic pressure, Fulton index and the ratio of right ventricular weight to body weight in the MRL/lpr mice were significantly higher than those in wild type mice with same background (C57BL/6). The medial smooth muscle area and the proportion of muscularized vessels in the lung tissue of the MRL/lpr mice were larger than those of the C57BL/6 mice. Western blot analysis demonstrated markedly elevated levels of prepro-endothelin-1 and survivin as well as decreased endothelial nitric oxide synthase phosphorylation in the lung tissue of the MRL/lpr mice. Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling assay showed the resistance against apoptosis of pulmonary arterial smooth muscle cells in the MRL/lpr mice.</p><p>Conclusion</p><p>We showed that MRL/lpr mice were complicated with pulmonary hypertension. MRL/lpr mice appeared to be a useful model for studying the mechanism of pulmonary hypertension associated with connective tissue diseases.</p></div

The levels of survivin expression and apoptosis of pulmonary arterial smooth muscle cells in the lung tissue of MRL/lpr mice.

<p>Survivin expression in the lung tissues was determined by western blotting (A). Representative immunoblots (upper panels) and graph (bottom panel) are shown. Bars are mean ± S.D. of quantitative densitometric analyses; n = 5 for each experimental group; *P<0.05 vs. C57BL/6 mice. Representative microphotographs of the pulmonary arteries in C57BL/6 mice and MRL/lpr mice stained by TUNEL staining (B). TUNEL-positive cells were indicated by red arrows. The graph showed the percentage of TUNEL-positive nuclei in medial smooth muscle layer (open bar; C57BL/6 mice, solid bar; MRL/lpr mice). Results are expressed as mean ± S.D. of 5 animals. *P<0.05 vs. C57BL/6 mice.</p

Cytokines in the lung tissue of MRL/lpr mice.

<p>The levels of cytokines in the lung tissue were assessed as described in the Methods. (open bar; C57BL/6 mice, solid bar; MRL/lpr mice). Results are expressed as mean ± S.D. of 9 to 11 animals. *P<0.05 vs. C57BL/6 mice.</p

Decreased eNOS activation and increased prepro-ET-1 expression in the lung tissue of MRL/lpr mice.

<p>eNOS expression and phosphorylation (A), and prepro-ET-1 expression (B) were determined by western blotting. Representative immunoblots (upper panels) and graphs (bottom panels) are shown. Bars are mean ± S.D. of quantitative densitometric analyses; n = 5 for each experimental group; *P<0.05 vs. C57BL/6 mice.</p

Medial wall thickening and muscularization of the pulmonary arteries in MRL/lpr mice.

<p>Representative microphotographs of the pulmonary arteries in C57BL/6 mice and MRL/lpr mice stained by EM staining (A) and α-SMA (B). A: Medial wall area was assessed as described in the Methods. B: Peripheral pulmonary arteries were classified into NM (non-muscular), PM (partially muscular), and FM (fully muscular) according to the degree of muscularization (open bar; C57BL/6 mice, solid bar; MRL/lpr mice). Results are expressed as mean ± S.D. of 5 animals. *P<0.05 vs. C57BL/6 mice.</p

Schema of the signaling pathways in pulmonary artery endothelial cells and smooth muscle cells in MRL/lpr mice.

<p>Vasoconstriction predominates over vasodilatation due to NO impairment and upregulated prepro-ET-1 expression. Survivin promoted smooth muscle cells proliferation and resistance of apoptosis in MRL/lpr mice.</p

Measurement of RVSP and ventricular weight in MRL/lpr mice.

<p>RVSP in C57BL/6 and MRL/lpr mice (A). Results are expressed as mean ± S.D. of 5 to 6 animals. *P<0.05 vs. C57BL/6 mice. RV/LV+S and RV/body weight (B) in C57BL/6 and MRL/lpr mice. Results are expressed as mean ± S.D. of 8 animals. *P<0.05 vs. C57BL/6 mice.</p

Carbamoyl Pyridone HIV‑1 Integrase Inhibitors 3. A Diastereomeric Approach to Chiral Nonracemic Tricyclic Ring Systems and the Discovery of Dolutegravir (S/GSK1349572) and (S/GSK1265744)

We report herein the discovery of the human immunodeficiency virus type-1 (HIV-1) integrase inhibitors dolutegravir (S/GSK1349572) (<b>3</b>) and S/GSK1265744 (<b>4</b>). These drugs stem from a series of carbamoyl pyridone analogues designed using a two-metal chelation model of the integrase catalytic active site. Structure–activity studies evolved a tricyclic series of carbamoyl pyridines that demonstrated properties indicative of once-daily dosing and superior potency against resistant viral strains. An inherent hemiaminal ring fusion stereocenter within the tricyclic carbamoyl pyridone scaffold led to a critical substrate controlled diastereoselective synthetic strategy whereby chiral information from small readily available amino alcohols was employed to control relative and absolute stereochemistry of the final drug candidates. Modest to extremely high levels of stereochemical control were observed depending on ring size and position of the stereocenter. This approach resulted in the discovery of <b>3</b> and <b>4</b>, which are currently in clinical development