Fig 1.
Effect of RTV on cardiac function and plasma TGF-β1.
Cardiac output (A), stroke volume (B), and ejection fraction (C) were measured by echocardiography. Plasma TGF-β1 levels were measured by ELISA. Cardiac output, stroke volume, and ejection fraction were depressed by exposure to RTV, 10mg/kg daily over 8 weeks, vs. mice administered vehicle (DMSO). Plasma TGF-β1 levels were measured by ELISA (D). RTV exposure elevated TGF-β1 levels in wt mice but not in platelet TGF-β1 deficient PF4Cre Tgfb1flox/flox mice (D). Cardiac output (E) and stroke volume (F) were negatively correlated with TGF-β1 levels.
Fig 2.
PF4CreTgfb1flox/flox mice are partially protected from RTV-mediated cardiac dysfunction.
Wt and platelet-deficient TGF-β1 PF4CreTGFb1flox/flox mice were treated with RTV or vehicle (DMSO) for 8 weeks. Hearts were harvested and sections stained with Masson trichrome to evaluate for fibrosis (A), with staining quantified by ImageJ analysis (B). RTV exposure led to a marked increase in cardiac fibrosis in wt but not platelet-TGFβ1 deficient mice. Cardiac fibrosis correlated with plasma levels of TGF-β1 (C). These effects were paralleled by changes in collagen type-1 α1 and αSMA, as shown in representative cardiac sections (D) and by ImageJ analysis (E).
Fig 3.
CO suppressed RTV-induced cardiac fibrosis and TGF-β1 signaling.
Wt mice were exposed to RTV and inhaled CO (250ppm) or ambient air for 4 hrs daily for 8 weeks. CO markedly reduced RTV-induced fibrosis, assessed as described in (A). CO also reduced phospho-Smad2 (B) and phospho-p38 (p-p38) (C) staining, which was elevated over controls by RTV exposure, indicating effects on both canonical and non-canonical TGF-β1 signaling pathways, respectively.
Fig 4.
Autophagy deficient mice are resistant to the ability of CO to suppress RTV-associated cardiac fibrosis and TGF-β1 signaling.
LC3-/- mice were treated with RTV or RTV+CO for 8 weeks. (A) Cardiac fibrosis was evaluated by Masson’s trichrome staining; representative images of heart sections are shown. Quantification of interstitial fibrosis showed that CO had no effect on RTV-induced fibrosis in these mice (n = 5). (B) Phosph-Smad2 staining (red) of DAPI+ nuclei (blue). Merged images show phospho-Smad2 and DAPI double-positive nuclei (purple). Phospho-Smad2 positive nuclei were counted using the ImageJ program, which showed no decrease in phospho-Smad2 in the CO/RTV group compared to mice exposed to RTV alone (n = 5).
Fig 5.
CO modulates macrophage polarization in RTV-treated heart muscle.
(A) Total F4/80+ CD11b+ macrophages, expressed as a percentage of CD45+ leukocytes, were elevated by RTV exposure and reduced to near basal levels by CO. (B) RTV had little effect on the pro-inflammatory F4/80+ I-A/E+ M1 subset, which was reduced to basal levels by CO. (C) RTV increased the number of F4/80+ CD206+ M2 cells as a proportion of total macrophages, an effect markedly augmented by CO. The CO-mediated alteration in M2 cells predominantly involved augmentation of regulatory, anti-inflammatory F4/80+ B7-H4+ M2c cells (D).
Table 1.
Plasma hyaluronic acid levels in HIV+ postmenopausal women receiving no, or various, anti-retroviral regimens, vs. HIV- postmenopausal controls.