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For more than 2 years, COVID-19 has been holding the world at awe with new waves of infections, novel mutants, and still limited (albeit improved) means to combat SARS-CoV-2-induced respiratory failure, the most common and fatal presentation of severe COVID-19. In the present perspective, we draw from the successes and—mostly—failures in previous acute respiratory distress syndrome (ARDS) work and the experiences from COVID-19 to define conceptual barriers that have so far hindered therapeutic breakthroughs in this deadly disease, and to open up new avenues of thinking and thus, ultimately of therapy.

Pulmonary arterial hypertension (PAH) is a chronic, incurable condition characterized by pulmonary vascular remodeling, perivascular inflammation, and right heart failure. Regulatory T cells (Tregs) stave off autoimmunity, and there is increasing evidence for their compromised activity in the inflammatory milieu of PAH. Abnormal Treg function is strongly correlated with a predisposition to PAH in animals and patients. Athymic Treg-depleted rats treated with SU5416, an agent causing pulmonary vascular injury, develop PAH, which is prevented by infusing missing CD4+CD25highFOXP3+ Tregs. Abnormal Treg activity may also explain why PAH disproportionately affects women more than men. This mini review focuses on the role of Tregs in PAH with a special view to sexual dimorphism and the future promise of Treg therapy.

Background The p53 signaling pathway may be important for the pathogenesis of emphysematous changes in the lungs of smokers. Polymorphism of p53 at codon 72 is known to affect apoptotic effector proteins, and the polymorphism of mouse double minute 2 homolog ( MDM2 ) single nucleotide polymorphism (SNP)309 is known to increase MDM2 expression. The aim of this study was to assess polymorphisms of the p53 and MDM2 genes in smokers and confirm the role of SNPs in these genes in the pathogenesis of pulmonary emphysema. Methods This study included 365 patients with a smoking history, and the polymorphisms of p53 and MDM2 genes were identified. The degree of pulmonary emphysema was determined by means of CT scanning. SNPs, MDM2 mRNA, and p53 protein levels were assessed in human lung tissues from smokers. Plasmids encoding p53 and MDM2 SNPs were used to transfect human lung fibroblasts (HLFs) with or without cigarette smoke extract (CSE), and the effects on cell proliferation and MDM2 promoter activity were measured. Results The polymorphisms of the p53 and MDM2 genes were associated with emphysematous changes in the lung and were also associated with p53 protein and MDM2 mRNA expression in the lung tissue samples. Transfection with a p53 gene-coding plasmid regulated HLF proliferation, and the analysis of P2 promoter activity in MDM2 SNP309-coding HLFs showed the promoter activity was altered by CSE. Conclusions Our data demonstrated that p53 and MDM2 gene polymorphisms are associated with apoptotic signaling and smoking-related emphysematous changes in lungs from smokers.

Pulmonary arterial hypertension is a fatal disease associated with pulmonary vascular remodeling and right ventricular hypertrophy. Pre-clinical animal models that reproduce the human pulmonary arterial hypertension process and pharmacological response to available therapies are critical for future drug development. The most prevalent animal model reproducing many aspects of angioobliterative forms of pulmonary arterial hypertension is the rat Sugen/hypoxia model in which Sugen, a vascular endothelial growth factor receptor antagonist, primarily causes initiation of endothelial injury and later in the presence of hypoxia promotes proliferation of apoptosis-resistant endothelial cells. We previously demonstrated that exposure of human pulmonary microvascular endothelium to morphine and HIV-proteins results in initial apoptosis followed by increased proliferation. Here, we demonstrate that the double-hit of morphine and Sugen 5416 (Sugen–morphine) in rats leads to the development of pulmonary arterial hypertension with significant medial hypertrophy of pre-acinar pulmonary arteries along with neo-intimal thickening of intra-acinar vessels. In addition, the pulmonary smooth muscle and endothelial cells isolated from Sugen–morphine rats showed hyperproliferation and apoptotic resistance, respectively, in response to serum starvation. Our findings support that the dual hit model of Sugen 5416 and morphine provides another experimental strategy to induce significant pulmonary vascular remodeling and development of severe pulmonary arterial hypertension pathology in rats without exposure to hypoxia.

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.

International audience; In order to intervene appropriately and develop disease-modifying therapeutics for pulmonary arterial hypertension, it is crucial to understand the mechanisms of disease pathogenesis and progression. We herein discuss four topics of disease mechanisms that are currently highly debated, yet still unsolved, in the field of pulmonary arterial hypertension. Is pulmonary arterial hypertension a cancer-like disease? Does the adventitia play an important role in the initiation of pulmonary vascular remodeling? Is pulmonary arterial hypertension a systemic disease? Does capillary loss drive right ventricular failure? While pulmonary arterial hypertension does not replicate all features of cancer, anti-proliferative cancer therapeutics might still be beneficial in pulmonary arterial hypertension if monitored for safety and tolerability. It was recognized that the adventitia as a cell-rich compartment is important in the disease pathogenesis of pulmonary arterial hypertension and should be a therapeutic target, albeit the data are inconclusive as to whether the adventitia is involved in the initiation of neointima formation. There was agreement that systemic diseases can lead to pulmonary arterial hypertension and that pulmonary arterial hypertension can have systemic effects related to the advanced lung pathology, yet there was less agreement on whether idiopathic pulmonary arterial hypertension is a systemic disease per se. Despite acknowledging the limitations of exactly assessing vascular density in the right ventricle, it was recognized that the failing right ventricle may show inadequate vascular adaptation resulting in inadequate delivery of oxygen and other metabolites. Although the debate was not meant to result in a definite resolution of the specific arguments, it sparked ideas about how we might resolve the discrepancies by improving our disease modeling (rodent models, large-animal studies, studies of human cells, tissues, and organs) as well as standardization of the models. Novel experimental approaches, such as lineage tracing and better three-dimensional imaging of experimental as well as human lung and heart tissues, might unravel how different cells contribute to the disease pathology.

Heart failure (HF) is usually defined by the dominantly affected heart chamber; therefore, termed right or left HF (RHF or LHF). Pulmonologists understand RHF as a complex syndrome characterized by insufficient delivery of blood from the right ventricle associated with elevated systemic venous pressure at rest or exercise. Cardiologists specify LHF by its clinical functional class and the relation to a reduced (HFrEF), preserved (HFpEF) or mid-range ejection fraction (HFmrEF). Pediatric cardiologist, dealing also with patients with a failing single ventricle, define HF as a condition of insufficient systemic oxygen delivery (DO(2)). Certainly, pediatricians do not think of the right and left heart, or even a single ventricle as an isolated, independently acting entity. Because of the importance of cardiac interactions, the creation of a restrictive atrial communication aims at a palliative approach with the goal to diminish the congestive consequences of a dysfunctional ventricle; further to serve as a pop-off valve in order to prevent syncope and cardiovascular collapse. This review covers the background, the particular indications, the techniques and preliminary results achieved following the creation of a restrictive atrial septum defect (rASD) in different pathophysiological settings. Based on the institutional experience, percutaneous trans-catheter perforation of the atrial septum, followed by gradual balloon dilatation can be performed at any age and location worldwide. Medical institutions in low resource countries can make use of such palliating procedures in the setting of right as well as LHF independent of their pharmacological facilities.

BACKGROUND: Right ventricular (RV) adaptation to acute and chronic pulmonary hypertensive syndromes is a significant determinant of short- and long-term outcomes. Although remarkable progress has been made in the understanding of RV function and failure since the meeting of the NIH Working Group on Cellular and Molecular Mechanisms of Right Heart Failure in 2005, significant gaps remain at many levels in the understanding of cellular and molecular mechanisms of RV responses to pressure and volume overload, in the validation of diagnostic modalities, and in the development of evidence-based therapies.METHODS: A multidisciplinary working group of 20 international experts from the American Thoracic Society Assemblies on Pulmonary Circulation and Critical Care, as well as external content experts, reviewed the literature, identified important knowledge gaps, and provided recommendations.RESULTS: This document reviews the knowledge in the field of RV failure, identifies and prioritizes the most pertinent research gaps, and provides a prioritized pathway for addressing these preclinical and clinical questions. The group identified knowledge gaps and research opportunities in three major topic areas: 1) optimizing the methodology to assess RV function in acute and chronic conditions in preclinical models, human studies, and clinical trials; 2) analyzing advanced RV hemodynamic parameters at rest and in response to exercise; and 3) deciphering the underlying molecular and pathogenic mechanisms of RV function and failure in diverse pulmonary hypertension syndromes.CONCLUSIONS: This statement provides a roadmap to further advance the state of knowledge, with the ultimate goal of developing RV-targeted therapies for patients with RV failure of any etiology.

In PAH, a wide range of abnormal pulmonary vascular manifestations exist, all arising from the endothelium http://ow.ly/IWH2305bXpQ

Improved survival among HIV-1-infected individuals with the advent of antiretroviral therapy has clearly led to a greater prevalence of noninfectious complications. One of the most devastating sequelae in these individuals is the development of pulmonary arterial hypertension (PAH). Various epidemiological studies suggest worse survival of HIV-PAH patients when compared with other forms of PAH. Given that only a subset and not all HIV-infected individuals develop HIV-PAH, it is suggested that an additional second-hit of genetic or environmental trigger is needed for the development of PAH. In this context, it has been well documented that HIV patients who abuse illicit drugs such as stimulants, opioids, and the like, are more susceptible to develop PAH. In this review, we highlight the studies that support the significance of a double hit of HIV and drug abuse in the incidence of PAH and focus on the research that has been undertaken to unravel the pathobiology and vascular remodeling mechanisms underlying the deleterious synergy between HIV infection and drugs of abuse in orchestrating the development of PAH.

The SU5416 + hypoxia (SuHx) rat model is a commonly used model of severe pulmonary arterial hypertension. While it is known that exposure to hypoxia can be replaced by another type of hit (e.g., ovalbumin sensitization) it is unknown whether abnormal pulmonary blood flow (PBF), which has long been known to invoke pathological changes in the pulmonary vasculature, can replace the hypoxic exposure. Here we studied if a combination of SU5416 administration combined with pneumonectomy (PNx), to induce abnormal PBF in the contralateral lung, is sufficient to induce severe pulmonary arterial hypertension (PAH) in rats. Sprague Dawley rats were subjected to SuPNx protocol (SU5416 + combined with left pneumonectomy) or standard SuHx protocol, and comparisons between models were made at week 2 and 6 postinitiation. Both SuHx and SuPNx models displayed extensive obliterative vascular remodeling leading to an increased right ventricular systolic pressure at week 6. Similar inflammatory response in the lung vasculature of both models was observed alongside increased endothelial cell proliferation and apoptosis. This study describes the SuPNx model, which features severe PAH at 6 wk and could serve as an alternative to the SuHx model. Our study, together with previous studies on experimental models of pulmonary hypertension, shows that the typical histopathological findings of PAH, including obliterative lesions, inflammation, increased cell turnover, and ongoing apoptosis, represent a final common pathway of a disease that can evolve as a consequence of a variety of insults to the lung vasculature.

In spite of treatment, severe angioproliferative pulmonary arterial hypertension (PAH) remains a disease characterized by great morbidity and shortened survival. New treatment strategies for patients with PAH are needed, and after drug development, preclinical studies are best conducted in animal models which present with pulmonary angio-obliterative disease and right heart failure. A rat model of severe pulmonary hypertension and right heart failure, described a decade ago, continues to be investigated and provide insight into the nature of the lung vascular lesions and mechanisms of cardiac adaptation to an altered lung circulation. This rat model is based on the combination of VEGF receptor blockade with Su5416 and chronic hypoxia; use of this pulmonary hypertension induction strategy led to developing the concept of apoptosis-dependent compensatory vascular cell growth. Although, often employed in experimental designs, chronic hypoxia is not necessary for the development of angio-obliterative pulmonary hypertension. Left pneumonectomy combined with Su5416 also results in severe pulmonary hypertension in normoxic conditions. Similarly, the immune insufficiency component of severe PAH can be modeled in athymic rats (lacking T-lymphocytes). In these rats housed under normoxic conditions, treatment with the VEGFR receptor blocker results in angioproliferative pulmonary hypertension; cardiopulmonary disease in these animals can be prevented by immune reconstitution of regulatory T-cells (Tregs). Finally, chronic hypoxia can be replaced with another stimulator of HIF-1α: Ovalbumin (Ova). Immunization of rats with Ova increases lung tissue HIF-1α protein expression, and in Su5416-treated rats causes lethal pulmonary hypertension. Finally, we postulate that these models may also be useful for “reverse translation”; that is, the mechanisms of lung vascular cell death and growth and the modifying influences of immune and bone marrow cells that have been identified in the Su5416 VEGFR inhibitor models can be informative about heretofore undescribed processes in human PAH.

In this perspective, we review published data which support the concept that many or most chronic and progressive lung diseases also involve the lung vessels and that microvascular abnormalities and endothelial cell death contribute to the pathobiology of emphysema. Lung vessel maintenance depends on Vascular Endothelial Growth Factor signaling and both are compromised in the emphysematous lung tissue. Although hypoxic pulmonary vasoconstriction has been considered as an important factor contributing to the vascular remodeling in chronic obstructive pulmonary disease (COPD) (COPD/emphysema, it is now clear that inhaled cigarette smoke can damage the lung vessels independent of the lung vascular tone. We propose that a "sick lung circulation" rather than the right heart afterload may better explain the cardiac abnormalities in COPD patients which are usually summarized with the term "cor pulmonale." The mechanisms and causes of pulmonary hypertension are likely complex and include vessel loss, in situ thrombosis, and endothelial cell dysfunction. Assessment of the functional importance of pulmonary hypertension in COPD requires hemodynamic measurements during exercise.

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.

The mechanisms involved in the development of severe angioobliterative pulmonary arterial hypertension (PAH) are multicellular and complex. Many of the features of human severe PAH, including angioobliteration, lung perivascular inflammation, and right heart failure, are reproduced in the Sugen 5416/chronic hypoxia (SuHx) rat model. Here we address, at first glance, the confusing and paradoxical aspect of the model, namely, that treatment of rats with the antiangiogenic vascular endothelial growth factor (VEGF) receptor 1 and 2 kinase inhibitor, Sugen 5416, when combined with chronic hypoxia, causes angioproliferative pulmonary vascular disease. We postulated that signaling through the unblocked VEGF receptor VEGFR3 (or flt4) could account for some of the pulmonary arteriolar lumen–occluding cell growth. We also considered that Sugen 5416–induced VEGFR1 and VEGFR2 blockade could alter the expression pattern of VEGF isoform proteins. Indeed, in the lungs of SuHx rats we found increased expression of the ligand proteins VEGF-C and VEGF-D as well as enhanced expression of the VEGFR3 protein. In contrast, in the failing right ventricle of SuHx rats there was a profound decrease in the expression of VEGF-B and VEGF-D in addition to the previously described reduction in VEGF-A expression. MAZ51, an inhibitor of VEGFR3 phosphorylation and VEGFR3 signaling, largely prevented the development of angioobliteration in the SuHx model; however, obliterated vessels did not reopen when animals with established PAH were treated with the VEGFR3 inhibitor. Part of the mechanism of vasoobliteration in the SuHx model occurs via VEGFR3. VEGFR1/VEGFR2 inhibition can be initially antiangiogenic by inducing lung vessel endothelial cell apoptosis; however, it can be subsequently angiogenic via VEGF-C and VEGF-D signaling through VEGFR3.

Animal models of diseases are of pivotal importance for the investigation of the normal organ function, the pathobiology of frequent and rare disorders as well as for preclinical proof of principle studies. It has been noticed that there are substantial gaps between our knowledge and understanding of left and right heart failure. To a large extent the concepts underlying right ventricular failure (RVF) have been borrowed from models of left heart failure or extrapolated from models of acute RVF. Today, several models of pulmonary hypertension are used by investigators and the aim of this chapter is to review current models of adaptive and maladaptive right ventricular hypertrophy in small and large animals. We discuss the pros and cons of each model depending on the particular disease aspect which the investigator attempts to reproduce, and we make a case for the development of new animal models of chronic RVF.

Right from the start of this essay I must dispense with the royal “we” to make it clear for the readers: what comes next is the product of a personal gedankenexperiment (thought experiment). Pulmonary vascular disorders, which frequently lead to pulmonary hypertension, are complex and multifactorial. One glance at the pulmonary hypertension classification list, which is revised every decade, makes this clear. According to Daniel Kahneman,1 complexity reduces the validity of a forecast, claim, or hypothesis—hence our need for algorithms and large databases, which help us in making decisions. Both diagnostic or treatment algorithms and databases are hypothesis-free. These tools, which are based on statistical information gathered from large but sometimes not homogenous cohorts, are used for forecasting and prognostication. As the clinical pulmonary hypertension community is surviving the 6-minute walk crisis in search of new end points, it has occurred to many in this community that hypothesis testing first requires hypothesis generation. According to Kahneman, this gets us into the territory of uncertainty and unpredictability—in his words, into “low-validity environments.” Depending on your degree of risk aversion, you may not be comfortable in such an environment. However, comfort comes from the testing of the hypotheses, which is the step that follows the hypothesis-generating first step. There are many ways to skin a cat and many ways to generate a hypothesis. For this particular exercise, my approach is to identify the spokes—and I will need to explain why I call a spoke a spoke, and I will need to declare to what degree the identification or recognition of a spoke may be biased. Similar to the level or validity of evidence rating, I will rate my bias the following way: little bias (A), moderate bias (B), and probable high bias (C). My hope is that the reader will enjoy this exercise as much as I did.

Right ventricular failure (RVF) is the most frequent cause of death in patients with pulmonary arterial hypertension (PAH); however, specific therapies targeted to treat RVF have not been developed. Chronic treatment with carvedilol has been shown to reduce established maladaptive right ventricle (RV) hypertrophy and to improve RV function in experimental PAH. However, the mechanisms by which carvedilol improves RVF are unknown. We have previously demonstrated by microarray analysis that RVF is characterized by a distinct gene expression profile when compared with functional, compensatory hypertrophy. We next sought to identify the effects of carvedilol on gene expression on a genome-wide basis. PAH and RVF were induced in male Sprague-Dawley rats by the combination of VEGF-receptor blockade and chronic hypoxia. After RVF was established, rats were treated with carvedilol or vehicle for 4 wk. RNA was isolated from RV tissue and hybridized for microarray analysis. An initial prediction analysis of carvedilol-treated RVs showed that the gene expression profile resembled the RVF prediction set. However, a more extensive analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes in pathways involved in cardiac hypertrophy, mitochondrial dysfunction, and protein ubiquitination. Genes encoding proteins in the cardiac hypertrophy and protein ubiquitination pathways were downregulated in the RV by carvedilol, while genes encoding proteins in the mitochondrial dysfunction pathway were upregulated by carvedilol. These gene expression changes may explain some of the mechanisms that underlie the functional improvement of the RV after carvedilol treatment.

Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs., Article, PLOS ONE.8(1):e53927(2013)

Purpose The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo. Methods The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment. Results In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin. Conclusion This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.

Background Given the relative abundance and toxic potential of acrolein in inhaled cigarette smoke, it is surprising how little is known about the pulmonary and systemic effects of acrolein. Here we test the hypothesis whether systemic administration of acrolein could cause endoplasmic reticulum (ER) stress, and lung cell apoptosis, leading to the enlargement of the alveolar air spaces in rats. Methods Acute and chronic effects of intraperitoneally administered acrolein were tested. Mean alveolar airspace area was measured by using light microscopy and imaging system software. TUNEL staining and immunohistochemistry (IHC) for active caspase 3 and Western blot analysis for active caspase 3, and caspase 12 were performed to detect apoptosis. The ER-stress related gene expression in the lungs was determined by Quantitative real-time PCR analysis. Acrolein-protein adducts in the lung tissue were detected by IHC. Results Acute administration of acrolein caused a significant elevation of activated caspase 3, upregulation of VEGF expression and induced ER stress proteins in the lung tissue. The chronic administration of acrolein in rats led to emphysematous lung tissue remodeling. TUNEL staining and IHC for cleaved caspase 3 showed a large number of apoptotic septal cells in the acrolein-treated rat lungs. Chronic acrolein administration cause the endoplasmic reticulum stress response manifested by significant upregulation of ATF4, CHOP and GADd34 expression. In smokers with COPD there was a considerable accumulation of acrolein-protein adducts in the inflammatory, airway and vascular cells. Conclusions Systemic administration of acrolein causes endoplasmic reticulum stress response, lung cell apoptosis, and chronic administration leads to the enlargement of the alveolar air spaces and emphysema in rats. The substantial accumulation of acrolein-protein adducts in the lungs of COPD patients suggest a role of acrolein in the pathogenesis of emphysema.

Radiation therapy (XRT) is an essential component in the management of many types of cancer 1. XRT of the thorax frequently places the heart in the radiation field, leading to cardiac injury 1–3. Improvements in early diagnosis and treatment have increased cancer survival, engendering the need to critically evaluate the long-term consequences of XRT 2–3. From a clinical standpoint, XRT-induced heart injury is heterogeneous. XRT-induced cardiomyopathy may be clinically apparent with an initial early injury or may be latent for many years before presenting with heart failure 1. From a pathologic standpoint, XRT induced cardiomyopathy is characterized by myocardial fibrosis in absence of left ventricular (LV) dilatation and associated with normal or reduced LV mass 1–3. Unfortunately, the incidence and pathophysiology of XRT-induced cardiomyopathy remains poorly described due to a lack of prospective data and a latency period that exceeds the follow-up duration of most clinical studies. Given the increasing recognition of XRT-induced heart disease and recent progress in murine cardiac imaging, we performed a systematic study of XRT-induced cardiomyopathy in the mouse. Experiments followed guidelines on the humane use and care of laboratory animals for biomedical research published by National Institutes of Health (No. 85-23, revised 1996) and received local Institutional Animal Care and Use Committee approval. Three-months old C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) were irradiated with a single 20 Gy dose of XRT (Group I, N=12). Anesthetized mice (pentobarbital 50 mg/kg) were shielded with lead plates leaving the thorax region exposed. XRT was administrated locally with a Trilogy linear accelerator equipped with state of the art guided radiation therapy (IGTR)(Varian, Medical system, Palo Alto, CA). An additional group of mice (Group II, N=6) underwent sham-irradiation. All mice were followed for 6 months (Figure, panel A). Figure The Experimental design is described in panel A: Group I (N=12) received a single thoracic radiation of 20 Gy at day 0, Groups II (N=6) sham non-irradiated mice were used as controls; an echocardiogram was performed at baseline (1 day prior to irradiation), ... Transthoracic echocardiography was performed under light anesthesia (pentobarbital 50 mg/kg) to determine cardiac dimensions and function using the Vevo770 imaging system (VisualSonics Inc. Toronto, Ontario, Canada) equipped with a 30-MHz probe 4–5. The thickness of the posterior pericardium and of the mitral valve and aortic valve leaflets was also measured in M-mode, the presence of pericardial fluid was also determined. Finally, left ventricular ejection fraction (LVEF) was measured at rest and 3 minutes after injection with the β-adrenergic receptor agonist isoproterenol (10 ng/mouse, Sigma Aldrich), to measure contractile reserve, defined as percentage change of LVEF within 3 minutes after isoproterenol injection. Tissue slides (5 μm), prepared from paraffin-embedded hearts collected at 6 months, were used for histological analysis. Collagen deposition was determined by Masson’s staining (Sigma Aldrich) and expressed as percentage fibrotic tissue in 5 random fields at 40X magnification using ImageJ software (rsbweb.nih.gov/ij/). DNA damage was detected by the TUNEL assay (Apo-Tag plus, Millipore, Billerica, MA) and expressed as percentage of the TUNEL positive cardiomyocytes on the total cardiomyocytes over 10 random fields. Statistical analysis was performed using the SPSS 15.0 package for Windows. Continuous variables were expressed as mean and standard error. The T test for paired data was used to compare variables before and after treatment. Kaplan Meier survival curves were constructed and compared among different groups. The T test for unpaired data was used to compare 2 groups. Two-tailed P values 4-fold increase risk of symptomatic heart failure and cardiac death 6. Other animal models of XRT-induced cardiomyopathy have been reported 7. In the most commonly used rat model, toxicity presents with acute pericarditis and effusion that may be lethal, but is generally not evident when doses 50% higher risk) than those treated for a right-sided breast cancer 9, and patients with XRT-induced cardiomyopathy have a reduction in peak oxygen consumptions of a degree consistent with severe heart failure 10. The mechanisms leading to the late cardiomyopathy and sudden death are not completely understood but previous investigations suggest that an acute inflammatory response occurs early after radiation promoting tissue fibrosis 11. The early decline in LV contractile reserve in our model is consistent with an early injury. From a clinical standpoint, the results of this study indicate that while the injury to the heart occurs immediately or very early after irradiation, a long latent clinical phase may occur. Consequently, many patients who have received chest irradiation in the past may have clinically latent disease and be at risk for late occurrence of heart failure or sudden death. It becomes clear that strategies are needed that are designed to prevent or limit the initial injury and/or the progression of the disease.

Here we provide background information for the pathobiological concept of a "sick lung circulation", and how it may affect cardiac function. We propose that in most chronic and progressive lung diseases, but in particular COPD/emphysema and interstitial lung diseases (ILD), there are significant pathological changes of the small pulmonary vessels. Complementing the WHO classification of pulmonary vascular diseases we provide a classification which is based on pathogenetic mechanisms such as inflammation, hypoxia and angioproliferation. We also propose that chronic lung disease - related 'cor pulmonale' is insufficiently explained by an elevation of the pulmonary arterial pressure. © 2012 Bentham Science Publishers.

Rationale: Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood. Objective: To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH. Methods and Results: Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell–lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4 + CD25 hi or CD4 + CD25 − T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3 + , IL-10- and TGF-β-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)–expressing cells, a receptor that activates endothelial cell survival pathways. Conclusions: PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

BackgroundCardiac remodelling after AMI is characterized by molecular and cellular mechanisms involving both the ischemic and non-ischemic myocardium. The extent of right ventricular (RV) dilatation and dysfunction and its relation to pulmonary hypertension (PH) following AMI are unknown. The aim of the current study was to evaluate changes in dimensions and function of the RV following acute myocardial infarction (AMI) involving the left ventricle (LV).MethodsWe assessed changes in RV dimensions and function 1 week following experimental AMI involving the LV free wall in 10 mice and assessed for LV and RV dimensions and function and for the presence and degree of PH.ResultsRV fractional area change and tricuspidal annular plane systolic excursion significantly declined by 33% (P = 0.021) and 28% (P = 0.001) respectively. Right ventricular systolic pressure measured invasively in the mouse was within the normal values and unchanged following AMI.ConclusionAMI involving the LV and sparing the RV induces a significant acute decline in RV systolic function in the absence of pulmonary hypertension in the mouse indicating that RV dysfunction developed independent of changes in RV afterload.

During heart development chamber specification is controlled and directed by a number of genes and a fetal heart gene expression pattern is revisited during heart failure. In the setting of chronic pulmonary hypertension the right ventricle undergoes hypertrophy, which is likely initially adaptive, but often followed by decompensation, dilatation and failure. Here we discuss differences between the right ventricle and the left ventricle of the heart and begin to describe the cellular and molecular changes which characterize right heart failure. A prevention and treatment of right ventricle failure becomes a treatment goal for patients with severe pulmonary hypertension it follows that we need to understand the pathobiology of right heart hypertrophy and the transition to right heart failure.

Background The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known. Methods Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed. Results The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vβ16.1 in the lung tissue. Conclusions Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.

Severe pulmonary hypertension is a lethal group of disorders which preferentially afflicts women. It appears that in recent years the patient profile has shifted towards older, obese, and postmenopausal women, suggesting that endocrine factors may be important. Several studies have revealed an increased prevalence of thyroid disease in these patients, but no studies have evaluated for a coexistence of endocrine factors. In particular, no studies have attempted to evaluate for concurrent thyroid disease, obesity and long-term estrogen exposure in patients. 88 patients attending the Pulmonary Hypertension Association 8th International meeting completed a questionnaire and were interviewed. Information was collected regarding reproductive history, height, weight, and previous diagnosis of thyroid disease. 46% met criteria for obesity. 41% reported a diagnosis of thyroid disease. 81% of women reported prior use of hormone therapy. 70% reported greater than 10 years of exogenous hormone use. 74% of female patients reported two or more of potentially disease modifying endocrine factors (obesity, thyroid disease or estrogen therapy). The coexistent high prevalence in our cohort of exogenous estrogen exposure, thyroid disease and obesity suggests that an interaction of multiple endocrine factors might contribute to the pathogenesis of pulmonary hypertension and may represent epigenetic modifiers in genetically-susceptible individuals.

Background Hypoxia induces the proliferation of lung fibroblasts in vivo and in vitro. However, the subcellular interactions between hypoxia and expression of tumor suppressor p53 and cyclin-dependent kinase inhibitors p21 and p27 remain unclear. Methods Normal human lung fibroblasts (NHLF) were cultured in a hypoxic chamber or exposed to desferroxamine (DFX). DNA synthesis was measured using bromodeoxyuridine incorporation, and expression of p53, p21 and p27 was measured using real-time RT-PCR and Western blot analysis. Results DNA synthesis was increased by moderate hypoxia (2% oxygen) but was decreased by severe hypoxia (0.1% oxygen) and DFX. Moderate hypoxia decreased p21 synthesis without affecting p53 synthesis, whereas severe hypoxia and DFX increased synthesis of both p21 and p53. p27 protein expression was decreased by severe hypoxia and DFX. Gene silencing of p21 and p27 promoted DNA synthesis at ambient oxygen concentrations. p21 and p53 gene silencing lessened the decrease in DNA synthesis due to severe hypoxia or DFX exposure. p21 gene silencing prevented increased DNA synthesis in moderate hypoxia. p27 protein expression was significantly increased by p53 gene silencing, and was decreased by wild-type p53 gene transfection. Conclusion These results indicate that in NHLF, severe hypoxia leads to cell cycle arrest via the p53-p21 pathway, but that moderate hypoxia enhances cell proliferation via the p21 pathway in a p53-independent manner. In addition, our results suggest that p27 may be involved in compensating for p53 in cultured NHLF proliferation.

Increasing evidence demonstrates that interleukin (IL)-32 is a pro-inflammatory cytokine, inducing IL-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and chemokines via nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase (MAPK), and activating protein (AP)-1 activation. Here we report that IL-32 is expressed and is also functional in human vascular endothelial cells (EC) of various origins. Compared with primary blood monocytes, high levels of IL-32 are constitutively produced in human umbilical vein EC (HUVEC), aortic macrovascular EC, and cardiac as well as pulmonary microvascular EC. At concentrations as low as 0.1 ng/ml, IL-1beta stimulated IL-32 up to 15-fold over constitutive levels, whereas 10 ng/ml of TNFalpha or 100 ng/ml of lipopolysaccharide (LPS) were required to induce similar quantities of IL-32. IL-1beta-induced IL-32 was reduced by inhibition of the IkappaB kinase-beta/NF-kappaB and ERK pathways. In addition to IL-1beta, pro-coagulant concentrations of thrombin or fresh platelets increased IL-32 protein up to 6-fold. IL-1beta and thrombin induced an isoform-switch in steady-state mRNA levels from IL-32alpha/gamma to beta/epsilon. Adult EC responded in a similar fashion. To prove functionality, we silenced endogenous IL-32 with siRNA, decreasing intracellular IL-32 protein levels by 86%. The knockdown of IL-32 resulted in reduction of constitutive as well as IL-1beta-induced intercellular adhesion molecule-1 (ICAM-1) (of 55% and 54%, respectively), IL-1alpha (of 62% and 43%), IL-6 (of 53% and 43%), and IL-8 (of 46% and 42%). In contrast, the anti-inflammatory/anti-coagulant CD141/thrombomodulin increased markedly when IL-32 was silenced. This study introduces IL-32 as a critical regulator of endothelial function, expanding the properties of this cytokine relevant to coagulation, endothelial inflammation, and atherosclerosis.

Emphysema is one manifestation of a group of chronic, obstructive, and frequently progressive destructive lung diseases. Cigarette smoking and air pollution are the main causes of emphysema in humans, and cigarette smoking causes emphysema in rodents. This review examines the concept of a homeostatically active lung structure maintenance program that, when attacked by proteases and oxidants, leads to the loss of alveolar septal cells and airspace enlargement. Inflammatory and noninflammatory mechanisms of disease pathogenesis, as well as the role of the innate and adaptive immune systems, are being explored in genetically altered animals and in exposure models of this disease. These recent scientific advances support a model whereby alveolar destruction resulting from a coalescence of mechanical forces, such as hyperinflation, and more recently recognized cellular and molecular events, including apoptosis, cellular senescence, and failed lung tissue repair, produces the clinically recognized syndrome of emphysema.

Rationale: Impaired endothelial cell–dependent vasodilation, inflammation, apoptosis, and proliferation are manifestations of endothelial dysfunction in chronic obstructive pulmonary disease (COPD). Prostacyclin (PGI2) is a major product of the cyclooxygenase pathway with potent vasodilatory and antimitogenic properties and may be relevant to endothelial dysfunction in COPD. Objectives: To determine if PGI2 expression is altered in smoking-related lung disease and if it may be protective in COPD-associated endothelial dysfunction. Methods: We evaluated, by immunohistochemistry, Western blotting, and polymerase chain reaction, human emphysema tissue compared with normal tissue for expression of prostacyclin synthase (PGI2S). We examined the effects of cigarette smoke extract (CSE) and aldehyde components on eicosanoid expression in primary human pulmonary microvascular endothelial cells. Finally, we used a murine model of lung-specific PGI2S overexpression and in vitro studies to determine if PGI2 expression has protective effects on cigarette smoke–induced endothelial apoptosis. Measurements and Main Results: Human emphysema lung tissue exhibited lower PGI2S expression within the pulmonary endothelium than in normal lung. In vitro studies demonstrated that CSE, and in particular the α,β unsaturated aldehyde acrolein, suppressed PGI2S gene expression, whereas CSE significantly induced the upstream mediators COX-2 and cytosolic phospholipase A2 in human pulmonary microvascular endothelial cells. Mice with lung-specific PGI2S overexpression exhibited less endothelial apoptosis after chronic smoke exposure. In vitro, iloprost exhibited protective effects on CSE-induced apoptosis. Conclusions: PGI2 has protective effects in the pulmonary vasculature after acute and chronic cigarette smoke exposure. An imbalance in eicosanoid expression may be important to COPD-associated endothelial dysfunction.

Rationale: Within the lungs of patients with severe emphysema, inflammation continues despite smoking cessation and is characterized by the presence of T lymphocytes (T cells). Disease severity has been associated with increasing numbers of T cells within the small airways and alveolar walls of patients with chronic obstructive pulmonary disease (COPD). At present, the role of the T cell in this persistent inflammation remains unknown. One important question is whether these T cells are accumulating in the lung in response to an antigen or are being nonspecifically recruited. The aim of this study was to determine if T cells recruited to the lungs of subjects with severe emphysema are composed of oligoclonal T-cell populations, suggesting their accumulation in response to conventional antigenic stimuli. Methods: Lung T-cell receptor (TCR) Vβ repertoire from seven patients with emphysema was evaluated at the time of tissue procurement (ex vivo) and after 2 weeks of culture with interleukin-2 (in vitro). Junctional region nucleotide sequencing of expanded TCR Vβ subsets was performed. Results: No significantly expanded TCR Vβ subsets were identified in ex vivo samples. However, T cells grew from all emphysema (n = 7) but no control (n = 4) lung samples when exposed to interleukin-2. Within the cultured CD4+ T cells, a total of six major TCR Vβ subset expansions were identified in four of the seven patients with emphysema. Importantly, these T-cell expansions contained T-cell clones that had already been expanded in vivo. Conclusion: From the lungs of subjects with severe emphysema, oligoclonal CD4+ T-cell populations were identified that preferentially proliferated in culture. Oligoclonal populations of T cells within the lungs of patients with severe emphysema suggest that response to an antigen plays an important role in the continuing inflammation characteristic of severe COPD.

There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of alpha1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke-induced emphysema.We propose that, in addition to its antielastolytic effects, alpha1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis. METHODS, MEASUREMENTS, AND MAIN RESULTS: Transduction of human alpha1-antitrypsin via replication-deficient adeno-associated virus attenuated airspace enlargement and emphysema caused by inhibition of vascular endothelial growth factor (VEGF) receptors with SU5416 in mice, a model of apoptosis-dependent emphysema lacking neutrophilic inflammation. The overexpressed human serine protease inhibitor accumulated in lung cells and suppressed caspase-3 activation and oxidative stress in lungs treated with the VEGF blocker or with VEGF receptor-1 and -2 antibodies. Similar results were obtained in SU5416-treated rats given human alpha1-antitrypsin intravenously.Our findings suggest that inhibition of structural alveolar cell apoptosis by alpha1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of alpha1-antitrypsin.

Resolution of alveolar epithelial/capillary membrane damage after acute lung injury requires coordinated and effective tissue repair to reestablish a functional alveolar epithelial/capillary membrane barrier. We hypothesized that signaling pathways important in lung alveolar bud ontogeny are activated in the recovery and remodeling phases after profound oxidant stress lung injury in a murine model. To test this, we characterized the expression of noncanonical beta-catenin pathway proteins E-cadherin, integrin-linked kinase-1, and beta-catenin in mice undergoing normoxic recovery after exposure to butylated hydroxytoluene (BHT, ionol) and concomitant sublethal (75% O2) hyperoxia. Mice developed early acute lung injury with subsequent inflammation, collagen deposition, interstitial cellular proliferation, and lung architectural distortion. Reduced E-cadherin expression after 6 d of BHT and hyperoxia was accompanied by enhanced expression and nuclear localization of beta-catenin and increased integrin-linked kinase-1 expression during subsequent normoxic recovery. This resulted in increased expression of the cotranscriptional regulators TCF-1 and -3 and cyclin D1. Proliferation of murine lung epithelial-12 cells in vitro after 8 h of treatment with BHT quinone-methide and hyperoxia and 48 h of normoxic recovery was enhanced 2.7-fold compared with vehicle-treated control mice at the same time point. BHT/hyperoxia-exposed mice treated with the pan-caspase inhibitor z-ASP had increased acute lung injury and reduced survival despite the presence of TUNEL-positive cells, suggesting enhanced lung cell necrosis. Beta-catenin expression was reduced in z-ASP-co-treated lungs after BHT/hyperoxia. The noncanonical cadherin-beta-catenin axis is associated with fibroproliferative repair after BHT/hyperoxia exposure and may regulate epithelial proliferation and lung matrix remodeling and repair in response to lung injury.

Hypothesis/introduction A polymorphic marker within the angiotensin-converting enzyme (ACE) gene has been associated with circulating and tissue ACE activity and with a variety of forms of cardiovascular disease. Since angiotensin II (Ang II) causes pulmonary vasoconstriction and vascular and myocardial remodelling, we postulated a role for the renin-angiotensin system and the ACE DD genotype in the pathophysiology of primary pulmonary hypertension (PPH) and in the right ventricular response to pressure overload in these patients. Methods and results The incidence of the ACE DD genotype was evaluated in 60 patients with severe PPH compared with two normal control populations, a group of healthy population-based controls (n=158) and subjects found suitable for cardiac organ donation (n=79). Genomic DNA extracted from peripheral leukocytes was amplified using the polymerase chain reaction to detect polymorphic markers. Haemodynamics were determined by right heart catheterisation in a subset of the PPH patients. The frequency of the ACE DD genotype was 45% in the patients with PPH, compared with 24% in the organ donors, and 28% in populationbased healthy controls (p=0.01 for chi-square test). Of the 32 PPH patients with baseline haemodynamics, 12 exhibited the ACE DD genotype and 20 were non-DD. While the mean pulmonary artery pressure and the duration of symptoms attributable to pulmonary hypertension was not different between the DD and non-DD groups, cardiac output was significantly lower (3.29±0.27 vs. 5.07±0.37 L/minute, p=0.002) and the trouvemean right atrial pressure tended to be higher (8.85±1.29 vs. 4.92±1.27 mmHg, p=0.08) in the non-DD group. The reduction in cardiac output seen in the non-DD group was not due to a difference in heart rate, but to a significant reduction in stroke volume, consistent with a decreased contractile state. In addition, non-DD patients exhibited a significantly worse functional capacity (NYHA Class 3.14±0.12 vs. 2.40±0.28, p=0.02). Conclusions 1) The ACE DD genotype is significantly increased in patients with severe PPH compared with normal controls, suggesting that certain individuals may be genetically predisposed to developing pulmonary hypertension. 2) The ACE DD genotype is associated with preserved right ventricular function in PPH patients, supporting a compensatory myocardial or inotropic role for Ang II in the pressure overloaded right ventricle.

The etiology and pathogenesis of the vascular lesions characterizing primary pulmonary hypertension (PPH), an often fatal pulmonary vascular disease, are largely unknown. Plexiform lesions composed of proliferating endothelial cells occur in between 20 and 80% of the cases of this irreversible pulmonary vascular disease. Recently, technology to assess monoclonality has allowed the distinction between cellular proliferation present in neoplasms from that in reactive nonneoplastic tissue. To determine whether the endothelial cell proliferation in plexiform lesions in PPH is monoclonal or polyclonal, we assessed the methylation pattern of the human androgen receptor gene by PCR (HUMARA) in proliferated endothelial cells in plexiform lesions from female PPH patients (n = 4) compared with secondary pulmonary hypertension (PH) patients (n = 4). In PPH, 17 of 22 lesions (77%) were monoclonal. However, in secondary PH, all 19 lesions examined were polyclonal. Smooth muscle cell hyperplasia in pulmonary vessels (n = 11) in PPH and secondary PH was polyclonal in all but one of the examined vessels. The monoclonal expansion of endothelial cells provides the first marker that allows the distinction between primary and secondary PH. Our data of a frequent monoclonal endothelial cell proliferation in PPH suggests that a somatic genetic alteration similar to that present in neoplastic processes may be responsible for the pathogenesis of PPH.

Endothelial cells constitute an essential integrator of factors that effect blood vessel remodeling induced by chronic hypoxia. We hypothesized that vascular endothelial growth factor (VEGF) may participate in the lung response to acute and to chronic hypoxia. We found that ex vivo perfusion of isolated lungs under hypoxic conditions (when compared with normoxia) caused an increase in lung tissue mRNA of VEGF and of the VEGF receptors KDR/Flk and Flt. Chronic hypobaric hypoxia also increased lung tissue mRNA levels of VEGF, KDR/Flk, and Flt and the amount of VEGF protein. In situ hybridization studies demonstrated increased VEGF and KDR/flk hybridization signals in lungs from chronically hypoxic rats. Since endotoxin treatment of rats decreased lung VEGF mRNA, we postulated that nitric oxide (NO) or an NO-related metabolite might be involved in lung VEGF gene expression. Indeed, sodium nitroprusside, a NO donor, decreased and L-NAME (N-nitro-L-arginine methyl ester), an inhibitor of NO-synthesis, increased both VEGF and VEGF receptor transcripts. We conclude that VEGF in the isolated perfused lung acts as an early gene in response to hypoxia and that lung VEGF and VEGF receptor mRNA levels are influenced by hypoxia and NO-dependent mechanisms.