Cinaciguat (BAY-582667) Modifies Cardiopulmonary and Systemic Circulation in Chronically Hypoxic and Pulmonary Hypertensive Neonatal Lambs in the Alto Andino

Abstract

Neonatal pulmonary hypertension (NPHT) is produced by sustained pulmonary vasoconstriction and increased vascular remodeling. Soluble guanylyl cyclase (sGC) participates in signaling pathways that induce vascular vasodilation and reduce vascular remodeling. However, when sGC is oxidized and/or loses its heme group, it does not respond to nitric oxide (NO), losing its vasodilating effects. sGC protein expression and function is reduced in hypertensive neonatal lambs. Currently, NPHT is treated with NO inhalation therapy; however, new treatments are needed for improved outcomes. We used Cinaciguat (BAY-582667), which activates oxidized and/or without heme group sGC in pulmonary hypertensive lambs studied at 3,600 m. Our study included 6 Cinaciguat-treated (35 ug kg^-1 day^-1 x 7 days) and 6 Control neonates. We measured acute and chronic basal cardiovascular variables in pulmonary and systemic circulation, cardiovascular variables during a superimposed episode of acute hypoxia, remodeling of pulmonary arteries and changes in the right ventricle weight, vasoactive functions in small pulmonary arteries, and expression of NO-sGC-cGMP signaling pathway proteins involved in vasodilation. We observed a decrease in pulmonary arterial pressure and vascular resistance during the acute treatment. In contrast, the pulmonary pressure did not change in the chronic study due to increased cardiac output, resulting in lower pulmonary vascular resistance in the last 2 days of chronic study. The latter may have had a role in decreasing right ventricular hypertrophy, although the direct effect of Cinaciguat on the heart should also be considered. During acute hypoxia, the pulmonary vascular resistance remained low compared to the Control lambs. We observed a higher lung artery density, accompanied by reduced smooth muscle and adventitia layers in the pulmonary arteries. Additionally, vasodilator function was increased, and vasoconstrictor function was decreased, with modifications in the expression of proteins linked to pulmonary vasodilation, consistent with low pulmonary vascular resistance. In summary, Cinaciguat, an activator of sGC, induces cardiopulmonary modifications in chronically hypoxic and pulmonary hypertensive newborn lambs. Therefore, Cinaciguat is a potential therapeutic tool for reducing pulmonary vascular remodeling and/or right ventricular hypertrophy in pulmonary arterial hypertension syndrome.

Keywords: Cinaciguat; high altitude; hypoxia; newborn; pulmonary hypertension.

FIGURE 1

Acute effects of a 3 min infusion of Cinaciguat on the cardiopulmonary and systemic circulations of 6 chronically hypoxic and pulmonary hypertensive neonatal lambs. Infusions were performed in 7 successive days and results are means ± SEM of each variable at indicated times. Open circles: Basal values (B); Black circles, values after Cinaciguat infusion. Mean pulmonary arterial pressure [(mPAP, (A)], mean systemic arterial pressure [mSAP, (B)]; pulmonary vascular resistance [PVR, (C)]; systemic vascular resistance [SVR, (D)]; cardiac output [CO, (E)], and heart rate [HR, (F)] were recorded for 60 min. In the X axis, B, basal; I, infusion. Significant differences (p ≤ 0.05): a vs. basal period.

FIGURE 2

Time-course of basal cardiopulmonary and systemic circulation variables in chronically hypoxic and pulmonary hypertensive neonatal lambs treated with a daily bolus of Cinaciguat. Mean pulmonary arterial pressure [mPAP, (A)]; mean systemic arterial pressure [mSAP, (B)]; pulmonary vascular resistance [PVR, (C)]; systemic vascular resistance [SVR, (D)]; cardiac output [CO, (E)], and heart rate [HR, (F)]. Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant differences (p ≤ 0.05): a vs. basal period, b vs. Control group.

FIGURE 3

Effect of Cinaciguat treatment on cardiopulmonary and systemic circulation following a superimposed episode of acute hypoxia in chronically hypoxic and pulmonary hypertensive neonatal lambs. Mean pulmonary arterial pressure [mPAP, (A)], mean systemic arterial pressure [mSAP, (B)], pulmonary vascular resistance [PVR, (C)], systemic vascular resistance [SVR, (D)], cardiac output [CO, (E)], and heart rate [HR, (F)]. Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant differences (p ≤ 0.05): a vs. basal period, b vs. Control group; c, hypoxia vs. basal and recovery periods.

FIGURE 4

Effect of Cinaciguat treatment on heart ventricles of chronically hypoxic and pulmonary hypertensive neonatal lambs. Percentages of right ventricular hypertrophy index [(right ventricle/(left ventricle + septum) ×100] (A); [(right ventricle weight/newborn weight) x 100] (B); [(left ventricle weight/newborn weight) x 100] (C), and [(septum weight/newborn weight) ×100] (D). Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs. Control group.

FIGURE 5

Effect of Cinaciguat treatment on pulmonary vascular density and small pulmonary artery remodeling in chronically hypoxic and pulmonary hypertensive neonatal lambs. Total lung vascular density (arteries/mm²) (A). Representative micrograph of small pulmonary arteries, between 100 and 150 μm of luminal diameter, from vehicle group (B) and + Cinaciguat group (C), van Gieson staining. Brown area: muscular layer; pink area surrounding muscle: adventitia layer. Bar: 100 μm. Magnification: × 40. Percentage of muscle area (D), percentage of adventitia area (E). Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs Control group.

FIGURE 6

Effect of Cinaciguat treatment on muscle layer proliferation (Ki67 expression) and cellular density in small pulmonary arteries of chronically hypoxic and pulmonary hypertensive neonatal lambs. Representative micrograph of small pulmonary arteries, between 100 and 150 μm of luminal diameter, from Control (A) and Cinaciguat-treated lambs (B). Ki67+ cells are shown with an arrow. Bar: 100 μm. Magnification: ×40. Percentage of Ki67+ cells of small pulmonary arteries (C). Control (Open circles, n = 4) and Cinaciguat-treated lambs (Black circles, n = 4). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs. Control group. Cellular density in muscle layer (cells/µm²) ×100 (D). Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs. Control group.

FIGURE 7

Effect of Cinaciguat treatment on pulmonary protein expression of cell proliferation markers in chronically hypoxic and pulmonary hypertensive neonatal lambs. WB of p21/β-Actin (A), Cyclin D1/β-Actin (B), and Cyclin D1/p21 ratio (C). The β-Actin image was re-used for illustrative purposes. Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs. Control group.

FIGURE 8

Effect of Cinaciguat treatment on pulmonary expression of vasodilatory pathway proteins and cGMP content in lung tissue in chronically hypoxic and pulmonary hypertensive neonatal lambs. WB of eNOS/β-Actin (A), HO-1/β-Actin (B), and cGMP (pmol/mg proteins) (C). The β-Actin image was re-used for illustrative purposes. Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant difference (p ≤ 0.05): b vs. Control group.

FIGURE 9

Effect of Cinaciguat treatment on the vasodilator and vasoconstrictor function in isolated small pulmonary arteries and lung protein expression of PDE5 in chronically hypoxic and pulmonary hypertensive neonatal lamb. Responses to SNP and sildenafil of small pulmonary arteries. Y-axis, percent relaxation (%); X-axis, log SNP and sildenafil molar concentration (M) [(A,C), respectively]. Histograms show maximal relaxation (%) and sensitivity (pD2) [(B,D), respectively]. Responses to KCl, Y-axis tension (N/m), X-axis KCl concentration (mM) (E). Western Blot of PDE5/β-Actin (F). The β-Actin image was re-used for illustrative purposes. For SNP experiments, Control (Open circles, n = 5) and Cinaciguat-treated lambs (Black circles, n = 5). For sildenafil experiments, Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 5). For KCl experiments, Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). For PDE5 WB experiments, Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM for all experiments. Significant difference (p ≤ 0.05): b vs. Control group.

FIGURE 10

Effect of Cinaciguat treatment on vasodilator function in isolated small pulmonary arteries and lung tissue BK_Ca protein expression in chronically hypoxic and pulmonary hypertensive neonatal lambs. Responses to NS1619, BK_Ca channel stimulator, in small pulmonary arteries, Y-axis show relaxation (%), X-axis log NS1619 M concentration (M) (A). Histogram show maximal relaxation (%) (B). WB of BK_Ca/β-Actin (C). The β-Actin image was re-used for illustrative purposes. For NS1619 studies, Control (Open circles, n = 5) and Cinaciguat-treated lambs (Black circles, n = 6). For BKCa WB, Control (Open circles, n = 6) and Cinaciguat-treated lambs (Black circles, n = 6). Values are means ± SEM. Significant differences (p ≤ 0.05): b vs. Control group.