Introduction
Pulmonary hypertension (PH) is a complex and multifactorial disease which leads to overload of the right ventricle (RV) and right heart failure. Pulmonary vasoconstriction, endothelial cell (EC) dysfunction, vascular thickening, inflammation and thrombosis contribute to disease progression in idiopathic and other forms of PH [
3,
21].
The cardiac hormone atrial natriuretic peptide (ANP), via its cyclic GMP (cGMP)-synthesizing transmembrane guanylyl cyclase A (GC-A) receptor, has critical functions in the maintenance of systemic arterial blood pressure [
6,
42] and also regulates pulmonary arterial blood pressure. Hence, global inactivation of the genes encoding ANP or GC-A increased resting pulmonary arterial pressure in mice [
28,
29] or the susceptibility to hypoxia-induced PH [
58]. Conversely, infusion of synthetic ANP attenuated hypoxia-induced experimental PH [
56] and lowered pulmonary pressure in patients with high-altitude disease [
32]. Together, these experimental and clinical studies indicate that endogenous ANP plays a physiological role in maintaining pulmonary arterial pressure homeostasis. And, furthermore, that enhancement of endogenous ANP/GC-A/cGMP signalling, for instance with drugs inhibiting ANP or cGMP degradation, may have therapeutical implications [
5‐
7].
Pulmonary arterial remodelling in PH involves multiple vascular (EC and smooth muscle cells (SMC), adventitial fibroblasts) and nonvascular cell types (leucocytes, mast cells, platelets) [
3,
21]. With the exception of platelets and leucocytes, all these cell types express the GC-A receptor [
30]. Because synthetic ANP prevented acute hypoxia-induced pulmonary vasoconstriction [
26,
58] and exerted direct cGMP-mediated anti-proliferative effects in cultured pulmonary arterial SMCs [
24], the protective role of the ANP/GC-A/cGMP pathway in the lung circulation has mainly been attributed to its effects on pulmonary SMC. However, as shown in the present study, the GC-A receptor is also expressed at high levels in lung EC. Whereas endothelial dysfunction is central to all forms of PH [
3,
21], it is unknown whether this involves impaired ANP/GC-A/cGMP signalling and how this could contribute to the progression of this disease. Therefore, the goals of this study were (1) to analyze the expression and activity of GC-A in lung endothelial cells and the impact of hypoxia; (2) to dissect the role of endothelial cells in mediating the effect of ANP in the chronic regulation of pulmonary arterial pressure by studying mice with selective disruption of the GC-A-encoding gene (
Npr1) in endothelial cells; and (3) to elucidate the impact of endothelial ANP/GC-A dysfunction on EC inflammatory activation as well as the pulmonary levels of endothelin-1 (ET-1) and Angiotensin II (Ang II). It is known that these hormones are activated and contribute to cardiopulmonary remodelling in patients with PH [
21]. On the other hand, it was shown that ANP/GC-A signalling diminishes endothelial ET-1 secretion [
55] and the (inter)actions of ET-1 and Ang II in the heart and systemic circulation [
19]. However, the relevance of this functional antagonism between ANP and ET-1/Ang II expression and action in the pulmonary circulation is unknown.
Discussion
Together with previous reports [
32,
56‐
58], our experimental studies demonstrate that ANP, via its GC-A receptor, plays an important physiological role in the moderation of pulmonary arterial pressure and lung vascular remodelling under normoxic and hypoxic conditions. The major novel findings are (1) EC are a major expression site of the GC-A receptor in the lung; (2) hypoxia impairs pulmonary endothelial GC-A expression and signaling; (3) genetic inactivation of the endothelial GC-A receptor in mice (EC GC-A KO) provokes PH, pulmonary vascular remodeling and subtle perivascular inflammatory infiltration already under normoxic conditions; (4) peak RVSP values in EC GC-A KO mice were similar to the levels of mice with deletion of GC-A in all cell types (GC-A
−/−), indicating that the endothelial effects of ANP are critically involved in the chronic moderation of pulmonary arterial pressure and vascular homeostasis by this hormone, at least in the murine system; and (5) enhanced local ACE/Ang II signaling contributes to the pulmonary vascular alterations in mice with endothelial GC-A dysfunction.
The increases in RVSP and the extent of pulmonary vascular remodeling in mice with global ANP or GC-A inactivation [
28,
29], or endothelial-restricted GC-A ablation are very consistent. In fact, less pronounced and more variable changes were observed in other disease-relevant genetic mouse models. For instance, wide ranges of RVSP were observed in mice with endothelial deletion of the BMPR2 gene (20.7–56.3 mmHg; median, 27 mmHg) compared with control mice (19.9–26.7 mmHg; median 23 mmHg), and only a subset of BMPR2-deficient mice with RVSP >30 mmHg exhibited RV hypertrophy and pulmonary vascular remodeling [
23]. Even more, exposure of wild type rats or mice to chronic hypoxia (as accepted experimental model of PH) increases RVSP by 7–10 mmHg [
15,
28,
29]. Hence, in general the functional and morphological pulmonary alterations in experimental PH are much less pronounced as in the clinical setting, emphasizing that patients have a multifactorial disease whereas experimental studies attempt to dissect the contribution of specific genes or mechanisms. The present experimental study suggests that endothelial ANP/GC-A dysfunction could be one aspect of the complex neurohumoral imbalance accompanying and aggravating PH, in particular hypoxia-induced PH in chronic high-altitude disease. Our observations may stimulate clinical studies to follow this possibility.
Experimental and clinical studies showed that during chronic hypoxia, right heart ANP and BNP synthesis and circulating NP levels increase, possibly in response to the RV pressure overload provoked by pulmonary vasoconstriction [
9‐
11,
44]. Because synthetic ANP counterregulates hypoxic pulmonary vasoconstriction [
8,
22,
26] and limits the interaction of endothelial and inflammatory cells [
25,
39] and the proliferation of cultured vascular SMC [
24], it was proposed that enhanced endogenous ANP/BNP release helps to mitigate the development of hypoxic PH [
9,
10]. However, as shown here, hypoxia can decrease lung GC-A levels and endothelial GC-A/cGMP responses to ANP, which will attenuate these protective ANP (and BNP) effects. The inhibition of ANP/GC-A signaling by hypoxia has also been observed in coronary EC [
2] but the molecular mechanism is presently unknown and requires further study.
Endothelial GC-A dysfunction might cause PH in mice by provoking chronic increases in pulmonary arteriolar tone and/or vascular remodelling. Hence, we hypothesized that ANP physiologically regulates the endothelial release or (in) activation of factors locally modulating these processes, such as ET-1, Ang II or bradykinin. And, conversely, that this effect of ANP is abolished in EC GC-A KO mice. Interestingly, whereas ET-1 mRNA and protein levels were unaltered, ACE mRNA levels were increased in GC-A-deficient MLEC and in lungs from EC GC-A KO mice. Concomitantly, pulmonary Ang II levels were greater in the mutants whereas bradykinin levels tended to be diminished. It is well known that Ang II, via AT
1 signalling, not only causes vasoconstriction, but also migration and proliferation of SMC as well as recruitment of inflammatory cells [
16,
54]. Specifically, inhibition of ACE decreased the cellular inflammatory response in experimental models of lung inflammation [
4]. Indeed, in the present study AT
1-receptor blockade with losartan largely reversed PH, pulmonary vascular remodelling and inflammation in normoxic EC GC-A KO mice. Even more, losartan significantly attenuated the exacerbation of these cardiovascular changes in response to hypoxia. Together these observations indicate that enhanced ACE-dependent local Ang II formation contributes to these phenotypical alterations. In line with our results, several experimental and clinical studies have implicated the involvement of the RAAS in the pathogenesis of PH [
35]. All components, including renin, angiotensinogen, ACE and both subtypes of Ang II receptors, are expressed in the lung [
38,
43]. Increased ACE expression and activity in the endothelium of peripheral pulmonary arteries have been found in animal models of PH and, importantly, in patients with various forms of PAH [
38,
43]. However, the pathophysiological mechanism(s) remain(s) unclear. Our studies add a novel piece of information showing that pulmonary endothelial ANP/GC-A/cGMP-dysfunction is associated with enhanced ACE expression and activity. The inhibition of ACE levels by ANP was also observed by others [
52] and we will try to clarify the mechanism in our future investigations.
Notably, in the present study losartan did not clearly ameliorate hypoxic pulmonary hypertension in control mice. The increase in RVSP was only partly prevented, whereas RV hypertrophy and lung vascular remodelling were not at all attenuated by the drug. Hence, mechanisms independent of the AT
1 receptor seem to predominate. In line with our observations, blockade of the AT
1 receptor by olmesartan [
49] or genetic deletion of ACE [
53] also failed to ameliorate hypoxic PH and RV hypertrophy in mice. In contrast, AT
1 antagonists (GR138950C, olmesartan) reversed hypoxia-induced cardiopulmonary remodelling in rats [
40,
57]. The discrepancy between these results remains unexplained; species differences might be involved.
Beside increased Ang II diminished bradykinin levels may contribute to PH and lung perivascular inflammation of EC GC-A KO mice. The small nine amino-acid vasoactive peptide bradykinin has dual roles by exerting pathophysiological as well as beneficial physiological effects, mainly by stimulation of bradykinin B2 receptors. Specifically in the lung, inhibition of bradykinin metabolic breakdown by ACE inhibitors or exogenous administration of B2 receptor agonists exerted protective effects, reducing pulmonary arterial pressure in experimental hypertension [
50] and neutrophil recruitment by lipopolysaccharide [
4]. These protective effects of bradykinin involve the endothelial release of NO, prostacyclin and tissue-type plasminogen activator [
4]. Hence, we hypothesize that PH and perivascular inflammation in EC GC-A KO mice is mediated through both a local increase in Ang II and a decrease in bradykinin mediated signalling.
In general, experimental and clinical studies emphasize that a compromised endothelial barrier plays a central role in the pathogenesis of PH [
3,
45]. In fact, both acute and chronic hypoxia in mice and rats induce subtle but significant inflammation in the lung
prior to the onset of structural changes in the vessel wall [
34,
36]. On the other hand, numerous studies in vitro/in vivo indicated that ANP exerts pulmonary endothelial barrier-protecting actions. Synthetic ANP reduced hypoxia, TNF-α, thrombin, or bacterial endotoxin (PepG)-induced paracellular hyperpermeability of pulmonary microvascular and macrovascular endothelial cells cultured on permeable supports and acute PepG-induced lung injury in mice [
30,
51]. Conversely, enhanced PepG-induced lung injury, ICAM-1/VCAM-1 expression and vascular leak were observed in ANP
−/− mice [
51]. We did not observe macroscopic signs of pulmonary edema in EC GC-A KO mice under normoxic or hypoxic conditions. However, we found increased pulmonary levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Together with the imbalance between Ang II and bradykinin signalling these changes possibly contribute to enhanced pulmonary neutrophil infiltration and PH in EC GC-A KO mice.
Study limitations
One limitation of the EC GC-A KO mice is that the GC-A receptor is absent not only in pulmonary but also in systemic endothelia. Unfortunately, a selective disruption of target genes within the pulmonary circulation is technically impossible so far and therefore this limitation is shared by other disease relevant genetic mouse models [
20]. Hence, because EC GC-A KO mice have mild systemic arterial hypertension and subtle LV hypertrophy, it is possible that PH was secondary to the systemic phenotype. However, invasive haemodynamic studies clearly demonstrated that cardiac output and LV function of EC GC-A KO mice are unaltered, also after chronic hypoxia. In addition there are no signs of pulmonary edema, corroborating that the PH of these mice is not secondary to left ventricular failure. Even more, we did not observe vascular thickening or inflammation in other tissues of EC GC-A KO mice. Together these observations indicate that the pulmonary vascular alterations of EC GC-A KO mice are not secondary to systemic changes.
Concordant to the mice with systemic ANP or GC-A deletion ([
28,
29] and present study), mice with EC-restricted GC-A ablation have mild PH already under baseline, normoxic conditions, which was aggravated by chronic hypoxia. However, the absolute increases in mean RVSP and in vascular remodelling in response to CH were similar in EC GC-A KO mice and in controls. Again this is consistent with previous observations in mice with global ANP or GC-A inactivation [
28,
29]. Hence, it remains impossible to definitively determine whether ANP/GC-A dysfunction aggravates hypoxic PH or merely produces normoxic PH that is then amplified by hypoxia.