Novel peptide for attenuation of hypoxia-induced pulmonary hypertension via modulation of nitric oxide release and phosphodiesterase -5 activity

Abstract

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.

Fig.1

P1-stimulates intracellular NO release in PAEC. P1 (10 μM)-stimulated NO release was monitored and measured as fluorescence change for NO-bound 4,5-diaminofluorescein diacetate (DAF-2). Panel A= representative confocal images of control (a, 0 min; c, 10min) and P1 (b, 0 min; d, 10 min) of three independent experiments. Panel B = P1-induced time-dependent NO release in PAEC (n=3 for each time point). *p<0.05 or **p <0.01 vs control.

Fig. 2

P1 selectively inhibits cGMP-specific PDE-5 activity and increases cGMP levels in PAEC. Cells were incubated with P1 (0.5 – 5 μM) or zaprinast (5 μM) for 1 hr at 37° C. After incubation, cell supernatants were used for PDE activity assay (panels A and C), whereas cell lysates were used for Western blot analysis of PDE-5 (panel B) and cGMP measurement of PDE5 (panel D). cGMP levels were measured using cGMP immunoenzyme assay kit and PDE5 expression using polyclonal rabbit anti-PDE5 antibody (1:500 dilution). Data in panels A, C, and D represent means ± SE; n= 4 for each data point. Panel B: Shows representative blot and densitometric analysis of blots from three separate experiments with identical results. **p <0.01 or *p< 0.05 vs control in panels A, C and D, respectively. #p<0.05 vs zaprinast in panel D.

Fig. 3

P1 Inhibits cGMP Binding to PDE5 in PAEC. PDE5 immunoprecipitate from control and P1 (10 μM)-stimulated PAEC were used to assess [³H]cGMP binding as described in Methods. Data represent mean ± SE, n=4 for each cGMP concentration. **p<0.01 vs control.

Fig. 4

P1 increases lung cGMP levels and PA vasorelaxation. Panel A,rats were exposed to hypoxia or normoxia for 4 weeks with or without administration of P1 (60 mg/kg/day i.p.). After exposure lung levels of cGMP were determined as described in Methods. Data represent mean ± SE; n=4 in each group.**p<0.001 vs. normoxia, *p<0.05 vs. hypoxia. Panel B, hypoxia-induced vesocontraction of PA segments were determine with or without P1 (10 μM) and in the presence of L-NAME (100 μM) or using scrambled P1 (10 μM) as described in Methods.Data represents mean ± SE; n=3 in each group. ** p<0.01 vs all other groups.

Fig. 5

P1 attenuates hypoxia-elevated PAP. The real time changes in mean PAP (mPAP) were monitored for 34 days after telemetry implanted surgery. Hypoxia exposure was initiated on day 10 with continued monitoring of PAP changes. On day 21 one group were administered P1 in saline (60 mg/kg, once per day, i.p.) and other received equal volume of saline with continued monitoring of changes in mPAP until end of experiment as described in Methods. Data represents mean ± SE, n=4 in each group. *p< 0.05 vs saline/ respective day.

Fig. 6

P1 attenuates hypoxia-induced PAP increase, RV hypertrophy, and vascular remodeling but not SAP. End stage measurements of mPAP (Panel A) and mean SAP (mSAP) (panel B) were monitored in rats exposure to hypoxia or normoxia for 4 weeks with or without administration of P1 (60 mg/kg/day, i.p.). PAP and SAP measurements were performed as described in Methods. All of rats were used for monitoring RV hypertrophy expressed as the ratio of right ventricle (RV) weight to left ventricular (LV) plus septum weight (RV/LV+S) (panel C) and vascular remodeling (panel D). Data in panels A and C represent mean ± SE, n=8 and in panel B ± SE, n=4 in each group. Panel D shows representative histographs of 4 rats in each group.