Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation

Abstract

Background: Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2 +/ - mice. Accordingly, 5-LO and its metabolite, leukotriene B 4 , are candidates for the second hit. The purpose of this study was to determine how 5-LO–mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Methods: Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO–expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2 -mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO–mediated inflammation and were assessed for phenotypic and transcriptomic changes. Results: Lung inflammation, induced by intratracheal delivery of 5-LO–expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2 +/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2 +/- rats gained endogenous 5-LO expression associated with elevated leukotriene B 4 biosynthesis. Bmpr2 -mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO–mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B 4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. Conclusions: In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2 +/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B 4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

Endothelial BMPR2 Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling

“… 41 Although valuable as preclinical research tools, these models do not reproduce the near-complete BMPR-II deficiency that is observed in many patients with PAH. Future studies exploring the impact of BMP9 administration on the pathophysiology of PAH in the Bmpr2 EC −/− conditional knockout, either at baseline or in response to PAH-inducing stimuli like chronic hypoxia or 5-lipoxygenase overexpression, 42 , 43 will help to establish the functional relevance of our current findings to this subset of BMPR2 -deficient PAH patients.…”

Hemolysis Derived Products Toxicity and Endothelium: Model of the Second Hit

“…A two-hit model is also proposed in hereditary pulmonary arterial hypertension (hPAH), in which Bmpr2 mutations is a strong susceptibility factor but where only 20% of carriers develop the disease. Pulmonary inflammation, generated by overexpressing 5-lipoxygenase (a condition observed in hPAH patients), was associated with severe PAH in Bmpr2 +/− rats, while neither the single ‘hit’ of Bmpr2 haploinsufficiency nor 5-LO-specific pulmonary inflammation alone initiated the disease—their combined presence was required to develop a lethal PAH [200].…”

Perivascular Inflammation in Pulmonary Arterial Hypertension

“…In spite of this trial failing to reach its primary endpoint, promising preclinical studies still implicate LTB4 in PAH pathogenesis in specific subpopulations. A recent study demonstrated that in BMPR2 haploinsufficient rats, the viral transduction of 5-lipoxygenase (5-LO), the enzyme that produces LTB4, results in the development of severe PAH [ 56 ]. Of note, BMPR2 mutations are among the most common human mutations found in hereditary PAH, albeit with low penetrance [ 57 ].…”

“…The transduction of 5-LO resulted in the development of PAH in these rats with similar frequency to humans with BMPR2 mutations. Additionally, the neointimal cells in these animals developed a spontaneous, endogenous expression of non-viral 5-LO, which is a finding that was also seen in patient tissue [ 56 ]. Together, these data demonstrate a fundamental interplay between leukotrienes, transforming growth factor (TGF)-β / bone morphogenic protein (BMP) signaling, and the development of PAH.…”