Autophagy in pulmonary hypertension: Emerging roles and therapeutic implications

Autophagy is an important mechanism for cellular self-digestion and basal homeostasis. This gene- and modulator-regulated pathway is conserved in cells. Recently, several studies have shown that autophagic dysfunction is associated with pulmonary hypertension (PH). However, the relationship between autophagy and PH remains controversial. In this review, we mainly introduce the effects of autophagy-related genes and some regulatory molecules on PH and the relationship between autophagy and PH under the conditions of hypoxia, monocrotaline injection, thromboembolic stress, oxidative stress, and other drugs and toxins. The effects of other autophagy-related drugs, such as chloroquine, 3-methyladenine, rapamycin, and other potential therapeutic drugs and targets, in PH are also described.     

Recombinant Human Interferon Alpha 2b Prevents and Reverses Experimental Pulmonary Hypertension

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles. In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.     

Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.     

Eosinophils are necessary for pulmonary arterial remodeling in a mouse model of eosinophilic inflammation-induced pulmonary hypertension

There is increasing evidence that inflammation plays a pivotal role in the pathogenesis of some forms of pulmonary hypertension (PH). We recently demonstrated that deficiency of adiponectin (APN) in a mouse model of PH induced by eosinophilic inflammation increases pulmonary arterial remodeling, pulmonary pressures, and the accumulation of eosinophils in the lung. Based on these data, we hypothesized that APN deficiency exacerbates PH indirectly by increasing eosinophil recruitment. Herein, we examined the role of eosinophils in the development of inflammation-induced PH. Elimination of eosinophils in APN-deficient mice by treatment with anti-interleukin-5 antibody attenuated pulmonary arterial muscularization and PH. In addition, we observed that transgenic mice that are devoid of eosinophils also do not develop pulmonary arterial muscularization in eosinophilic inflammation-induced PH. To investigate the mechanism by which APN deficiency increased eosinophil accumulation in response to an allergic inflammatory stimulus, we measured expression levels of the eosinophil-specific chemokines in alveolar macrophages isolated from the lungs of mice with eosinophilic inflammation-induced PH. In these experiments, the levels of CCL11 and CCL24 were higher in macrophages isolated from APN-deficient mice than in macrophages from wild-type mice. Finally, we demonstrate that the extracts of eosinophil granules promoted the proliferation of pulmonary arterial smooth muscle cells in vitro. These data suggest that APN deficiency may exacerbate PH, in part, by increasing eosinophil recruitment into the lung and that eosinophils could play an important role in the pathogenesis of inflammation-induced PH. These results may have implications for the pathogenesis and treatment of PH caused by vascular inflammation.     

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.     

Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.     

Therapeutic Efficacy of Valproic Acid in a Combined Monocrotaline and Chronic Hypoxia Rat Model of Severe Pulmonary Hypertension

Background

Pulmonary hypertension (PH) is a serious disease with poor prognosis. Reports show that cells in remodeled pulmonary arteries of PH patients have similar characteristics to cancer cells, such as exuberant inflammation, increased proliferation, and decreased apoptosis. An ideal strategy for developing PH therapies is to directly target pulmonary vascular remodeling. High levels of histone deacetylase (HDAC) expression and activity are found in certain cancers, and research has shown the potential of HDAC inhibitors in repressing tumor growth via anti-inflammatory and anti-proliferative effects. To date, little is known about the effectiveness of HDAC inhibitors against pulmonary vascular remodeling in severe PH.

Objective

To investigate whether class I HDAC inhibitors suppress or reverse the development of severe PH in rats.

Methods

Male Sprague-Dawley rats were injected with a single, subcutaneous dose of monocrotaline (60mg/kg), and were exposed to chronic hypoxia to induce severe PH. Valproic acid, a class I HDAC inhibitor, was administered to rats daily via gastric gavage (300mg/kg) in a PH prevention study (during the first 3 weeks) or a PH reversal study (from 3 to 5 weeks). At the end of experiment, hemodynamic indices were measured, ventricular hypertrophy indices were calculated and vascular remodeling phenotypes were analyzed. Results: After 3 weeks exposure to a combined stimulation of monocrotaline and chronic hypoxia, rats exhibited a reduced body weight, elevated right ventricular systolic pressure, an increased Fulton index, right ventricle weight ratio, medial wall thickness and muscularized peripheral pulmonary arteries. These parameters for PH evaluation were exacerbated from 3 to 5 weeks. Daily administration of valproic acid therapy prevented and partially reversed the development of severe PH in rats, and decreased inflammation and proliferation in remodeled pulmonary arteries.

Conclusion

These data show that class I HDAC inhibitors may be effective for treating severe PH.     

Pulmonary vascular development goes awry in congenital lung abnormalities

Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease. Birth Defects Research (Part C) 102:343–358, 2014. © 2014 Wiley Periodicals, Inc.     

The endothelin system in pulmonary hypertension

Pulmonary hypertension (PH) may result from numerous clinical entities affecting the pulmonary circulation primarily or secondarily. It is recognized that vascular endothelial dysfunction contributes to the development and perpetuation of PH by creating an imbalance between vasodilating and antiproliferative forces and between vasoconstrictive and proliferative forces. In that context, endothelin-1 (ET-1) overproduction was rapidly targeted as a plausible contributor to the pathogenesis of PH. The lung is recognized as the major site for ET production and clearance. In all animal models of PH studied, circulating plasma ET-1 levels are elevated, accompanied by an increase in lung tissue expression of the peptide. The use of selective ETA and dual ETA-ETB receptor antagonists in these models both in prevention and in therapeutic studies have confirmed the contribution of ET-1 to the rise in pulmonary vascular tone, pulmonary medial hypertrophy, and right ventricular hypertrophy. This is found consistently in models affecting the pulmonary circulation primarily or producing PH secondarily. Recent clinical trials in patients with pulmonary arterial hypertension have confirmed the therapeutic effectiveness of ET-receptor antagonists in humans. We offer a systematic review of the pathogenic role of the ET system in the development of PH as well as the rationale behind the preclinical and ongoing clinical trials with this new class of agents.     

Low dose 100 cGy irradiation as a potential therapy for pulmonary hypertension

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling and ultimately death. Two rodent models of PH include treatment with monocrotaline or exposure to a vascular endothelial growth factor receptor inhibitor and hypoxia. Studies in these models indicated that damaged lung cells evolve extracellular vesicles which induce production of progenitors that travel back to the lung and induce PH. A study in patients with pulmonary myelofibrosis and PH indicated that 100 cGy lung irradiation could remit both diseases. Previous studies indicated that murine progenitors were radiosensitive at very low doses, suggesting that 100 cGy treatment of mice with induced PH might be an effective PH therapy. Our hypothesis is that the elimination of the PH-inducing marrow cells by low dose irradiation would remove the cellular influences creating PH. Here we show that low dose whole-body irradiation can both prevent and reverse established PH in both rodent models of PH.     

Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension

Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR-II) and dysfunction of BMPR-II have been implicated in patients with primary pulmonary hypertension (PH). To clarify the possible involvement of BMP and BMPR-II in the development of hypoxic PH, the expression of BMP-2, BMPR-II, and their downstream signals were investigated in rat lung under normal and hypoxic conditions by RT-PCR, immunoblot, and immunohistochemical methods. In rats under normal conditions, BMP-2 is localized in the endothelium of the pulmonary artery, whereas BMPR-II is abundantly expressed in the endothelium, smooth muscle cells, and adventitial fibroblasts. After 0.5 and 3 days of exposure to hypoxia, upregulation of BMP-2 was observed in the intrapulmonary arteries. The change was accompanied by activation of its downstream signaling, p38 MAPK, and Erk1/2 MAPK, and the apoptotic process, measured by caspase-3 activity and TdT-mediated dUTP nick end labeling-positive cells. In contrast, a significant decrease in the expression of BMPR-II and inactivation of p38 MAPK and caspase-3 were observed in the pulmonary vasculature after 7-21 days of hypoxia exposure. Because BMP-2 is known to inhibit proliferation of vascular smooth muscle cells and promote cellular apoptosis, disruption of BMP signaling pathway through downregulation of BMPR-II in chronic hypoxia may result in pulmonary vascular remodeling due to the failure of critical antiproliferative/differentiation programs in the pulmonary vasculature. These results suggest abrogation of BMP signaling may be a common molecular pathogenesis in the development of PH with various pathophysiological events, including primary and hypoxic PH.     

Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus

Platelet-derived growth factor (PDGF) is a potent smooth muscle cell mitogen that may contribute to smooth muscle hyperplasia during the development of chronic pulmonary hypertension (PH). We studied changes in PDGFalpha- and beta-receptor and ligand expression in lambs with chronic intrauterine PH induced by partial ligation of the ductus arteriosus (DA) at gestational age 124-128 days (term = 147 days). Western blot analysis performed on whole lung homogenates from PH animals after 8 days of DA ligation showed a twofold increase in PDGFalpha- and beta-receptor proteins compared with age-matched controls (P < 0.05). Lung PDGF-A and -B mRNA expression did not differ between PH and control animals. We treated PH animals with NX1975, an aptamer that selectively inhibits PDGF-B, by infusion into the left pulmonary artery for 7 days after DA ligation. NX1975 reduced the development of muscular thickening of small pulmonary arteries by 47% (P < 0.05) and right ventricular hypertrophy (RVH) by 66% (P < 0.02). Lung PDGFalpha- and beta-receptor expression is increased in perinatal PH, and NX1975 reduces the increase in wall thickness of small pulmonary arteries and RVH in this model. We speculate that PDGF signaling contributes to structural vascular remodeling in perinatal PH and that selective PDGF inhibition may provide a novel therapeutic strategy for the treatment of chronic PH.     

自噬与肺部疾病研究进展

自噬(autophagy)是广泛存在于真核细胞中的基本生命现象,是细胞适应环境变化、防御病原微生物侵袭、维持内环境稳定的重要机制．多种肺部疾病中存在自噬活性的变化,自噬与肺部疾病的发生、发展密切相关．自噬在慢性阻塞性肺病、肺气肿、肺癌、肺结核等许多肺部疾病中发生,且发挥重要作用．现从自噬与多种肺部疾病的关系角度进行综述,有助于了解自噬在肺部疾病中发挥的作用,以便进一步研究自噬的调节,为肺部疾病的治疗提供新思路．     

Inactivation of p53 Is Sufficient to Induce Development of Pulmonary Hypertension in Rats

Objective

Pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterial hypertension (PAH) show similarities to cancer cells. Due to the growth-suppressive and pro-apoptotic effects of p53 and its inactivation in cancer, we hypothesized that the p53 pathway could be altered in PAH. We therefore explored the involvement of p53 in the monocrotaline (MCT) rat model of pulmonary hypertension (PH) and the pathophysiological consequences of p53 inactivation in response to animal treatment with pifithrin-α (PFT, an inhibitor of p53 activity).

Methods and Results

PH development was assessed by pulmonary arterial pressure, right ventricular hypertrophy and arterial wall thickness. The effect of MCT and PFT on lung p53 pathway expression was evaluated by western blot. Fourteen days of daily PFT treatment (2.2 mg/kg/day), similar to a single injection of MCT (60 mg/kg), induced PH and aggravated MCT-induced PH. In the first week after MCT administration and prior to PH development, p53, p21 and MDM2 protein levels were significantly reduced; whereas PFT administration effectively altered the protein level of p53 targets. Anti-apoptotic and pro-proliferative effects of PFT were revealed by TUNEL and MTT assays on cultured human PA-SMCs treated with 50 μM PFT.

Conclusions

Pharmacological inactivation of p53 is sufficient to induce PH with a chronic treatment by PFT, an effect related to its anti-apoptotic and pro-proliferative properties. The p53 pathway was down-regulated during the first week in the rat MCT model. These in vivo experiments implicate the p53 pathway at the initiation stages of PH pathogenesis.     

Interleukin 17A inhibits autophagy through activation of PIK3CA to interrupt the GSK3B-mediated degradation of BCL2 in lung epithelial cells

We recently found that activation of IL17A signaling promotes the development and progression of acute and chronic pulmonary fibrosis, and that the blockade of IL17A activity attenuates pulmonary fibrosis by promoting the resolution of inflammation and the activation of autophagy. Although the induction of autophagy stimulating the collagen degradation in the fibrotic lung tissue has been identified as a mechanism responsible for the antifibrotic role of targeting IL17A, it remains to be clarified how IL17A signaling suppresses autophagy. Here we report that the phosphorylation of B-cell CLL/lymphoma 2 (BCL2), an apoptosis regulatory protein, was inhibited in the presence of IL17A in lung epithelial cells, and this reduction suppressed the ubiquitination degradation of BCL2, which subsequently attenuated autophagy by promoting the interaction of BCL2 and BECN1. We found that IL17A regulated the phosphorylation of BCL2 through activating the phosphoinositide 3-kinase (PI3K)-glycogen synthase kinase 3 β (GSK3B) signaling cascade. In response to IL17A stimulation, PI3K was activated and resulted in phosphorylation of GSK3B at Ser9, which subsequently attenuated the interaction of GSK3B with BCL2. Interrupting the GSK3B and BCL2 interaction precluded the phosphorylation of BCL2 at Ser70, which could trigger the ubiquitination degradation, and restrained the ubiquitination degradation of BCL2. Consequently, a decrease in the BCL2 degradation induced by IL17A resulted in a suppressed autophagy in lung epithelial cells. These findings indicate that the IL17A-PI3K-GSK3B-BCL2 signaling pathway participates in the attenuation of autophagic activity in lung epithelial cells, which is attributed to be primarily responsible for the development and progression of IL17A-induced pulmonary fibrosis.     

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.     

Autophagy and inflammation in chronic respiratory disease

Persistent inflammation within the respiratory tract underlies the pathogenesis of numerous chronic pulmonary diseases including chronic obstructive pulmonary disease, asthma and pulmonary fibrosis. Chronic inflammation in the lung may arise from a combination of genetic susceptibility and environmental influences, including exposure to microbes, particles from the atmosphere, irritants, pollutants, allergens, and toxic molecules. To this end, an immediate, strong, and highly regulated inflammatory defense mechanism is needed for the successful maintenance of homeostasis within the respiratory system. Macroautophagy/autophagy plays an essential role in the inflammatory response of the lung to infection and stress. At baseline, autophagy may be critical for inhibiting spontaneous pulmonary inflammation and fundamental for the response of pulmonary leukocytes to infection; however, when not regulated, persistent or inefficient autophagy may be detrimental to lung epithelial cells, promoting lung injury. This perspective will discuss the role of autophagy in driving and regulating inflammatory responses of the lung in chronic lung diseases with a focus on potential avenues for therapeutic targeting.

Abbreviations AR allergic rhinitis

AM alveolar macrophage

ATG autophagy-related

CF cystic fibrosis

CFTR cystic fibrosis transmembrane conductance regulator

COPD chronic obstructive pulmonary disease

CS cigarette smoke

CSE cigarette smoke extract

DC dendritic cell

IH intermittent hypoxia

IPF idiopathic pulmonary fibrosis

ILD interstitial lung disease

MAP1LC3B microtubule associated protein 1 light chain 3 beta

MTB Mycobacterium tuberculosis

MTOR mechanistic target of rapamycin kinase

NET neutrophil extracellular traps

OSA obstructive sleep apnea

PAH pulmonary arterial hypertension

PH pulmonary hypertension

ROS reactive oxygen species

TGFB1 transforming growth factor beta 1

TNF tumor necrosis factor