Leptin deficiency recapitulates the histological features of pulmonary arterial hypertension in mice

Abstract

Leptin is a neuroendocrine peptide released by adipose tissue that enhances metabolism and acts on the hypothalamus to suppress appetite. Leptin also regulates aspects of cardiovascular function and low serum leptin has been associated with increased mortality in humans. We hypothesized that leptin deficiency alters the structure and function of the pulmonary vasculature.

Methods: We examined two groups of C57BL/6 male mice aged 12 weeks: five ob/ob (B6.VLepob/ob) leptin-deficient and five wild type (WT) (C57BL/6) control mice. As expected, weight was significantly greater in ob/ob mice relative to WT mice [weight (g), Mean±SD): ob/ob 52±2.5 g, wild type 30±2.5 g; p<0.001]. The pulmonary vasculature of ob/ob mice and WT control animals was examined by histology, immunohistochemistry and immunofluorescence staining.

Results: Pulmonary arterial wall thickness was significantly increased in ob/ob mice relative to WT littermates [median (interquartile range) distance in pixels: ob/ob 0.13 (0.05-0.18), wild type 0.03 (0.02-0.04); p=0.001]. The ob/ob mice also exhibited significant right ventricular hypertrophy in comparison to control animals [RV thickness (Mean±SD): ob/ob 0.75±0.19, wild type; 0.58±0.13 p<0.001]. We observed substantial macrophage infiltration and abundant proliferation of myofibroblasts and fibroblasts in histological sections of pulmonary arterioles of ob/ob mice. In addition, we noted increased hyaluronan deposition, colocalizing with SMC-actin in the pulmonary vasculature of ob/ob mice relative to WT controls.

Conclusions: The pulmonary pathology of leptin deficiency in ob/ob mice recapitulates many of the histological features of pulmonary vascular diseases, including pulmonary hypertension, suggesting that leptin deficiency is associated to the pathogenesis of pulmonary vascular disease.

Keywords: Leptin; obesity; pulmonary hypertension; vasculature.

Figure 1

Lung tissues from ob/ob (A) and control (B) lungs were stained using hematoxylin and eosin (H&E). H&E staining demonstrates thickening smooth muscle cell wall and hyperplasia in the ob/ob arteriole (arrow in A) that was not observed in control animals. The p value for the difference in thickness of the arterial wall (distance in pixels) and standard errors were calculated from three different slides by using 10 arteries in each slide. Ten distinct segents of the arterial wall were measured (C). Bar is 100 μm.

Figure 2

Immunohistochemical staining of frozen lung sections ffor the macrophage marker F4/80 in the lung of ob/ob mice (A) and control mice (B). F4/80 staining demonstrates accumulations of macrophages in the ob/ob arteriole (arrow in A) that was not observed in control animals. The p value for the difference in thickness of the arterial wall (distance in pixels) and standard errors were calculated from three different slides by using 10 arteries in each slide. Ten distinct segents of the arterial wall were measured (C). Bar is 100 μm.

Figure 3

Lung tissues from ob/ob (A) and control (B) animals were stained for collagen with picrosirius red. Baris 100 μm.

Figure 4

Lung tissues from ob/ob (left) and control (right) mice were stained for FSP1/S100A4 (fibroblasts). There is more intense fibroblast staining (green) in ob/ob arterioles (A) compared with the control (C). B and D are negative controls of the same sections stained by the secondary antibody only. Blue staining represents the 4,6-diamidino-2-phenylindole (DAPI) staining of the nuclei. Bar is 100 μm.

Figure 5

Lung tissues from ob/ob (left) and control (right) lungs were stained for smooth muscle cell actin (SMCs; smooth muscle actin) and HA (HA binding protein). The intensity of SMC actin (red) and HA (green) staining surrounding the ob/ob arteriole (A and B) is greater compared with the control (E and F). C and G are composite pictures of HA and SMC actin for the same sections of ob/ob and control lung tissues, respectively. D and H are negative controls of the same sections stained by the secondary antibody only. The minimal green staining here represents autofluorescence by the elastica, and the blue staining represents the 4,6-diamidino-2-phenylindole (DAPI) staining of the nuclei. Bar indicates 100 μm.

Figure 6

Transverse heart sections at the level of the left ventricular papillary muscles, showing right ventricle thickness (left-right arrows) in ob/ob (panel A) and wild type (panel B) mice. Panel C is a boxplot of the RV thickness measured in pixels, in 20 different sectors, on each slide for leptin deficient (ob/ob) and wild type (WT) mice. Bar indicates 100 μm.