Human immunodeficiency virus-1 transgene expression increases pulmonary vascular resistance and exacerbates hypoxia-induced pulmonary hypertension development

Abstract

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary arterial resistance and vessel remodeling. Patients living with human immunodeficiency virus-1 (HIV-1) have an increased susceptibility to develop severe pulmonary hypertension (PH) irrespective of their CD4+ lymphocyte counts. While the underlying cause of HIV-PAH remains unknown, the interaction of HIV-1 proteins with the vascular endothelium may play a critical role in HIV-PAH development. Hypoxia promotes PH in experimental models and in humans, but the impact of HIV-1 proteins on hypoxia-induced pulmonary vascular dysfunction and PAH has not been examined. Therefore, we hypothesize that the presence of HIV-1 proteins and hypoxia synergistically augment the development of pulmonary vascular dysfunction and PH. We examined the effect of HIV-1 proteins on pulmonary vascular resistance by measuring pressure-volume relationships in isolated lungs from wild-type (WT) and HIV-1 Transgenic (Tg) rats. WT and HIV-1 Tg rats were exposed to 10% O2 for four weeks to induce experimental pulmonary hypertension to assess whether HIV-1 protein expression would impact the development of hypoxia-induced PH. Our results demonstrate that HIV-1 protein expression significantly increased pulmonary vascular resistance (PVR). HIV-1 Tg mice demonstrated exaggerated pulmonary vascular responses to hypoxia as evidenced by greater increases in right ventricular systolic pressures, right ventricular hypertrophy and vessel muscularization when compared to wild-type controls. This enhanced PH was associated with enhanced expression of HIF-1α and PCNA. In addition, in vitro studies reveal that medium from HIV-infected monocyte derived macrophages (MDM) potentiates hypoxia-induced pulmonary artery endothelial proliferation. These results indicate that the presence of HIV-1 proteins likely impact pulmonary vascular resistance and exacerbate hypoxia-induced PH.

Keywords: chronic hypoxia; human immunodeficiency virus; pulmonary hypertension.

Figure 1

HIV-1 Tg expression (dotted line) increases pulmonary vascular resistance when compared to WT rats (solid line) (n = 4-5). Rats were anesthetized with isoflourane, mechanically ventilated and the pulmonary artery was cannulated with a 14G cannula connected to a pressure transducer. Pressure/volume relationships were generated using a calibrated peristaltic pump at flow rates of 7, 16, 26 and 35 ml/min. ^*denotes P < 0.0001 when compared to pulmonary arteries of wild-type controls.

Figure 2

Hypoxia exposure induces greater HIF-1alpha expression in HIV-1 Tg animals (n = 3). Wild-type and HIV-1 Tg rats were housed in either normoxic or hypoxic conditions for four weeks. Lung homogenates were subjected to SDS-PAGE, transferred to nitrocellulose membranes and exposed to anti-HIF-1alpha antibodies overnight at 4°C, rinsed and incubated in anti-rabbit fluorescent antibody solution. Hypoxia exposure stimulates HIF- 1alpha protein expression in rat lung homogenates when analyzed by Western blot analysis (A). Lung homogenates from HIV-1 Tg animals exhibit a 2-fold increase in HIF-1alpha protein expression following hypoxia exposure (B). ^*denotes P < 0.01 when compared to normoxic groups. ^**denotes P < 0.05 when compared to hypoxic wild-types.

Figure 3

HIV-1 transgene expression exacerbates PH development. Hypoxic HIV-1 rats have significantly greater RVSP (A) (n = 6-11) and right ventricular hypertrophy (B) (n = 7-9) than normoxic controls and hypoxic wild-types. Wild-type and HIV-1 Tg rats were housed in either normoxic or hypoxic conditions for four weeks. Rats were anesthetized with isoflourane and right ventricular pressure was monitored using a microtip pressure transducer. ^* denotes P < 0.0001 when compared to normoxic groups. ^** denotes a P < 0.01 when compared to hypoxic wild-types.

Figure 4

HIV-1 Tg expression increases hypoxia-induced vessel muscularization (A) (n = 3). Rats were exposed to normoxia or hypoxia for four weeks. Rat lungs were isolated, pressure perfused, inflated and immersed with 4% paraformaldehyde and embedded in paraffin. The percentage of smooth muscle within the media was determined by staining with antibodies to smooth muscle α-actin (α-SMA).Scale bar in each image = 50 μm. Hypoxia exposure increases HIV-1 Tg rat vessel thickness (B). ^**denotes P < 0.0001 when compared to normoxic groups. ^**denotes P < 0.01 when compared to hypoxic controls.

Figure 5

HIV-1 protein exposure exacerbates pulmonary cellular proliferation. Lung homogenates from HIV-1 Tg rats express significantly greater PCNA than normoxic WT or HIV-1 Tg rats (A) (n = 4). Following four weeks of normoxic or hypoxic conditions, rats were sacrificed and lungs removed for protein expression analysis. Lung homogenates were subjected to SDS-PAGE, transferred to nitrocellulose membranes and stained with anti-PCNA antibodies. ^*denotes P < 0.05 when compared to normoxic controls. ^** denotes P < when compared to all other groups. Incubation of HPAEC treated with media from HIV-infected Monocyte derived Macrophages exacerbates hypoxia-induced proliferation of human pulmonary artery endothelial cells (B) (n = 4). Exosome-containing fractions of HIV-MDM (MDM+) potentiate hypoxia-induced HPAEC proliferation when compared to HIV-MDM devoid of exosomes (MDM-) (C) (n = 4). HPAEC were exposed to 1% oxygen for 72 hours in the presence or absence of HIV-MDM media. Following exposure to normoxic or hypoxic conditions, cells were subjected to MTT assay to assess cell proliferation. ^*denotes P < 0.0001 when compared to treated and untreated normoxic groups. ^**denotes P < 0.01 when compared to untreated hypoxic groups.