C-type natriuretic peptide does not attenuate the development of pulmonary hypertension caused by hypoxia and VEGF receptor blockade

Abstract

Aims: C-type natriuretic peptide (CNP) is a local regulator of vascular tone and remodeling in many vascular beds. However, the role of CNP in modulating pulmonary arterial hypertensive and vascular remodeling responses is unclear. The purpose of this study was to determine if CNP is capable of preventing the development of pulmonary hypertension (PH).

Main methods: We used animal models of PH caused by chronic hypoxia alone or in combination with the vascular endothelial growth factor (VEGF) receptor blocker SU5416. We measured pulmonary hemodynamics, right ventricular hypertrophy and vascular remodeling effects in response to a continuous infusion of low dose or high dose CNP or vehicle placebo.

Key findings: Right ventricular hypertrophy and a marked elevation in right ventricular systolic pressure (RVSP) were seen in both models of PH. Rats treated with the combination of SU5416 and chronic hypoxia also developed pulmonary endothelial hyperproliferative lesions. Continuous intravenous infusion of CNP at either dose did not attenuate the development of PH, right ventricular hypertrophy or vascular remodeling in either of the models of PH despite a three-fold increase in serum CNP levels.

Significance: CNP does not prevent the development of PH in the chronic hypoxia or SU5416 plus hypoxia models of pulmonary hypertension suggesting that CNP may not play an important modulatory role in human PH.

Figure 1. CNP does not prevent the development severe pulmonary arterial hypertension

Animals were implanted with pumps that delivered i.v. infusion of 5% dextrose (Vehicle), low dose (0.75μg/hr) or high dose (2.25μg/hr) CNP, and placed in either normoxia or hypoxia (FiO2-10%) for 3 wks after receiving a s.c. injection of diluent or 20 mg/kg of SU5416, a VEGF receptor blocker. Panel A: Representative RV pressure tracings of animals exposed to 3 weeks of Normoxia/Vehicle and SU5416 + Hypoxia/ Vehicle. Panel B: RV systolic pressure in various treatment groups. (Mean ± SEM. n=4-11.* p<0.05 compared to Diluent + Normoxia treated with vehicle. ND: Diluent +Normoxia, SUN: SU5416 + Normoxia, HD: Diluent +Hypoxia, SUH: SU5416 + Hypoxia, D5W: 5% Dextrose)

Figure 2. CNP does not prevent right ventricular hypertrophy in PH

Right ventricular weight normalized to LV + Septum (RV/LV+S) weight in rats implanted with subcutaneous pumps that delivered continuous i.v. infusion of 5% dextrose (vehicle), low dose (0.75 μg/hr) or high dose (2.25 μg/hr) CNP, and exposed to normoxia or hypoxia for 3 wks after receiving a s.c. injection of diluent or 20 mg/kg of SU5416, a VEGF receptor blocker. (n=4-11, Mean ± SEM. * p <0.05 compared to Diluent + Normoxia treated with vehicle. ND: Diluent +Normoxia, SUN: SU5416 + Normoxia, HD: Diluent +Hypoxia, SUH: SU5416 + Hypoxia, D5W: 5% Dextrose)

Figure 3. CNP does not inhibit microvascular remodeling in severe PH

Panel A Representative images of H&E stained lung sections of animals exposed to Normoxia, Hypoxia, SU5416 + Hypoxia and SU5416 + Hypoxia/ CNP showing vessels <150 μM in diameter at 40X magnification. Panel B. Medial thickness in <150 μM vessels expressed as percent of vessel diameter in animals delivered continuous i.v. infusion of 5% dextrose or 0.75 μg/hr CNP, and exposed to normoxia or hypoxia for 3 wks after receiving a s.c. injection of diluent or 20 mg/kg of SU5416. (n=3, Mean ± SEM. * p <0.05 compared to Diluent + Normoxia treated with vehicle. ND: Diluent +Normoxia, SUN: SU5416 + Normoxia, HD: Diluent +Hypoxia, SUH: SU5416 + Hypoxia, D5W: 5% Dextrose)

Figure 4. Effect of CNP on endothelial proliferative lesions in severe PH

Representative images of immunoperoxisae stating using anti-vWF antibody showing endothelial cells in microvasculature of lung sections from animals exposed to (a) diluent + normoxia treated with vehicle, (b) Diluent + Hypoxia treated with vehicle. Representative images of endothelial proliferative lesions resulting in vessel occlusion noted in animals exposed to (c) Diluent + Hypoxia treated with CNP, (d) SU5416 and Hypoxia treated with vehicle or (e) SU5416 and Hypoxia treated with CNP. (Scale bar= 50 μm) Magnification 40X. Endothelial cells are stained brown. ND: Diluent +Normoxia, SUN: SU5416 + Normoxia, HD: Diluent +Hypoxia, SUH: SU5416 + Hypoxia, D5W: 5% Dextrose

Figure 5. Diminished NPR-B expression in SU5416 + hypoxia animals infused with CNP

Representative immunblot of the NPR-B receptor expression in the whole lung lysates of SU5416 + hypoxia (SUH) animals treated with 3 weeks of CNP (2.25μg/hr) or vehicle. Equal amount of protein lysates from homogenized lungs were resolved on SDS PAGE and probed for NPR-B. The membranes were subsequently stripped and reprobed for vinculin. (n=3)