Biventricular diastolic dysfunction, thrombocytopenia, and red blood cell macrocytosis in experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension is a fatal disease, where death is associated with right heart failure and reduced cardiorespiratory reserve. The Sugen 5416, hypoxia and normoxia Fischer rat model mimics human pulmonary arterial hypertension, although the cause(s) of death remains incompletely understood. Here, we hypothesized that these animals develop biventricular diastolic dysfunction that contributes to tissue hypoperfusion coincident with severe pulmonary arterial hypertension. We performed comprehensive echocardiographic and hematologic assessments. Serial echocardiogram at 3-5 weeks was performed followed by blood sampling via aortic or cardiac puncture. Echocardiogram revealed pulmonary arterial hypertension in pulmonary artery Doppler waves, including notched wave envelopes, and decreased pulmonary artery acceleration time/pulmonary artery ejection time ratio and right ventricular outflow tract velocity time integral. Impaired right ventricular systolic function, assessed by decreased tricuspid annular plane systolic excursion and tricuspid tissue Doppler systolic positive wave velocity, was observed in pulmonary arterial hypertension. Tricuspid and mitral pulsed wave and tissue Doppler findings suggested biventricular diastolic dysfunction, with dynamic changes in early and late diastolic filling waves, their fusion patterns, and a decrease in e' velocity. Heart rate and ejection fraction did not change, but cardiac output, stroke volume, and end-diastolic volume were decreased, and inferior vena cava respiratory variation was decreased. Blood electrolyte values were suggestive of intravascular volume expansion early in the disease followed by volume contraction and tissue hypoperfusion in the latter stages of disease. Complete blood count showed thrombocytopenia and non-anemic macrocytosis with reticulocytosis and an increase in red blood cell distribution width. Thus, pulmonary, cardiac, and hematological findings in Fischer animals with pulmonary arterial hypertension are characteristic of humans and provide an insightful experimental platform to resolve mechanisms of disease progression.

The changing face of pulmonary hypertension diagnosis: a historical perspective on the influence of diagnostics and biomarkers.

Pulmonary hypertension is a complex, multifactorial disease that results in right heart failure and premature death. Since the initial reports of pulmonary hypertension in the late 1800s, the diagnosis of pulmonary hypertension has evolved with respect to its definition, screening tools, and diagnostic techniques. This historical perspective traces the earliest roots of pulmonary hypertension detection and diagnosis through to the current recommendations for classification. We highlight the diagnostic tools used in the past and present, and end with a focus on the future directions of early detection. Early detection of pulmonary hypertension and pulmonary arterial hypertension and the proper determination of etiology are vital for the early therapeutic intervention that can prolong life expectancy and improve quality of life. The search for a non-invasive screening tool for the identification and classification of pulmonary hypertension is ongoing, and we discuss the role of animal models of the disease in this search.

Sphingosine-1-phosphate is involved in the occlusive arteriopathy of pulmonary arterial hypertension.

Despite several advances in the pathobiology of pulmonary arterial hypertension (PAH), its pathogenesis is not completely understood. Current therapy improves symptoms but has disappointing effects on survival. Sphingosine-1-phosphate (S1P) is a lysophospholipid synthesized by sphingosine kinase 1 (SphK1) and SphK2. Considering the regulatory roles of S1P in several tissues leading to vasoconstriction, inflammation, proliferation, and fibrosis, we investigated whether S1P plays a role in the pathogenesis of PAH. To test this hypothesis, we used plasma samples and lung tissue from patients with idiopathic PAH (IPAH) and the Sugen5416/hypoxia/normoxia rat model of occlusive PAH. Our study revealed an increase in the plasma concentration of S1P in patients with IPAH and in early and late stages of PAH in rats. We observed increased expression of both SphK1 and SphK2 in the remodeled pulmonary arteries of patients with IPAH and PAH rats. Exogenous S1P stimulated the proliferation of cultured rat pulmonary arterial endothelial and smooth-muscle cells. We also found that 3 weeks of treatment of late-stage PAH rats with an SphK1 inhibitor reduced the increased plasma levels of S1P and the occlusive pulmonary arteriopathy. Although inhibition of SphK1 improved cardiac index and the total pulmonary artery resistance index, it did not reduce right ventricular systolic pressure or right ventricular hypertrophy. Our study supports that S1P is involved in the pathogenesis of occlusive arteriopathy in PAH and provides further evidence that S1P signaling may be a novel therapeutic target.

Participant expectations in pulmonary hypertension-related research studies.

The Pulmonary Hypertension Association (PHA) is a patient advocacy organization seeking to find ways to prevent, improve treatment for, and cure pulmonary hypertension (PH) and to provide hope for the PH community through support, education, research, advocacy, and awareness. Many patients involved with PHA are also involved in various PH-specific research studies; however, the patient expectations and priorities for PH-specific research are currently unknown or not well examined. Our objective was to identify the current modes of study entry, priorities within research, and expectations over the course of study involvement for patient constituents of PHA. A 29-question online survey was designed by PHA and disseminated to the PHA patient constituency on its Facebook page through a post on November 29, 2012. Responses were collected on SurveyMonkey through December 10, 2012. Respondents were divided into parallel survey tracks, depending on whether the respondent indicated previous participation in research studies. These two cohorts of individuals were analyzed and, where appropriate, compared with tests of association. A total of 234 respondents were included in the final data analysis, with 95 (40.6%) reporting previous participation in research studies. These respondents reported an overall positive experience in their research studies (64.9% very good, 21.3% good, 12.8% neutral, 1.1% bad). Of the respondents with previous research study participation, 91.1% indicated that receipt of the study outcome after participation would positively influence their decision to participate in future research; despite this, only 41.17% reported receiving information of this sort after their participation. Research participation is a strong interest of PHA patient constituents; clear and consistent communication from the research team is an expectation of many participants. Despite this expectation, 58.83% of respondents indicated they did not receive communication from the research team after participation. This offers an opportunity not only to improve participants' experiences but also to increase the likelihood of future study participation.

Dehydroepiandrosterone restores right ventricular structure and function in rats with severe pulmonary arterial hypertension.

Current therapy of pulmonary arterial hypertension (PAH) is inadequate. Dehydroepiandrosterone (DHEA) effectively treats experimental pulmonary hypertension in chronically hypoxic and monocrotaline-injected rats. Contrary to these animal models, SU5416/hypoxia/normoxia-exposed rats develop a more severe form of occlusive pulmonary arteriopathy and right ventricular (RV) dysfunction that is indistinguishable from the human disorder. Thus, we tested the effects of DHEA treatment on PAH and RV structure and function in this model. Chronic (5 wk) DHEA treatment significantly, but moderately, reduced the severely elevated RV systolic pressure. In contrast, it restored the impaired cardiac index to normal levels, resulting in an improved cardiac function, as assessed by echocardiography. Moreover, DHEA treatment inhibited RV capillary rarefaction, apoptosis, fibrosis, and oxidative stress. The steroid decreased NADPH levels in the RV. As a result, the reduced reactive oxygen species production in the RV of these rats was reversed by NADPH supplementation. Mechanistically, DHEA reduced the expression and activity of Rho kinases in the RV, which was associated with the inhibition of cardiac remodeling-related transcription factors STAT3 and NFATc3. These results show that DHEA treatment slowed the progression of severe PAH in SU5416/hypoxia/normoxia-exposed rats and protected the RV against apoptosis and fibrosis, thus preserving its contractile function. The antioxidant activity of DHEA, by depleting NADPH, plays a central role in these cardioprotective effects.

Alveolar hypoxia promotes murine lung tumor growth through a VEGFR-2/EGFR-dependent mechanism.

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

Tyrosine kinase inhibitors are potent acute pulmonary vasodilators in rats.

Tyrosine kinase inhibitors are promising for the treatment of severe pulmonary hypertension. Their therapeutic effects are postulated to be due to inhibition of cell growth-related kinases and attenuation of vascular remodeling. Their potential vasodilatory activities have not been explored. Vasorelaxant effects of the tyrosine kinase inhibitors imatinib, sorafenib, and nilotinib were examined in isolated pulmonary arterial rings from normal and pulmonary hypertensive rats. Phosphorylation of myosin light chain phosphatase and myosin light chain was assessed by Western blots. Acute hemodynamic effects of imatinib were tested in the pulmonary hypertensive rats. In normal pulmonary arteries, imatinib reversed serotonin- and U46619-induced contractions in a concentration-dependent and endothelium-independent manner. Sorafenib and nilotinib relaxed U46619-induced contraction. Imatinib inhibited activation of myosin phosphatase induced by U46619 in normal pulmonary arteries. All three tyrosine kinase inhibitors concentration-dependently and completely reversed the spontaneous contraction of hypertensive pulmonary arterial rings unmasked by inhibition of nitric oxide synthase. Acute intravenous administration of imatinib reduced high right ventricular systolic pressure in pulmonary hypertensive rats, with little effect on left ventricular systolic pressure and cardiac output. We conclude that tyrosine kinase inhibitors have potent pulmonary vasodilatory activity, which could contribute to their long-term beneficial effect against pulmonary hypertension. Vascular smooth muscle relaxation mediated via activation of myosin light chain phosphatase (Ca(2+) desensitization) appears to play a role in the imatinib-induced pulmonary vasodilation.

Formation of plexiform lesions in experimental severe pulmonary arterial hypertension.

BACKGROUND: The plexiform lesion is the hallmark of severe pulmonary arterial hypertension. However, its genesis and hemodynamic effects are largely unknown because of the limited availability of lung tissue samples from patients with pulmonary arterial hypertension and the lack of appropriate animal models. This study investigated whether rats with severe progressive pulmonary hypertension developed plexiform lesions. METHODS AND RESULTS: After a single subcutaneous injection of the vascular endothelial growth factor receptor blocker Sugen 5416, rats were exposed to hypoxia for 3 weeks. They were then returned to normoxia for an additional 10 to 11 weeks. Hemodynamic and histological examinations were performed at 13 to 14 weeks after the Sugen 5416 injection. All rats developed pulmonary hypertension (right ventricular systolic pressure approximately 100 mm Hg) and severe pulmonary arteriopathy, including concentric neointimal and complex plexiform-like lesions. There were 2 patterns of complex lesion formation: a lesion forming within the vessel lumen (stalk-like) and another that projected outside the vessel (aneurysm-like). Immunohistochemical analyses showed that these structures had cellular and molecular features closely resembling human plexiform lesions. CONCLUSIONS: Severe, sustained pulmonary hypertension in a very late stage of the Sugen 5416/hypoxia/normoxia-exposed rat is accompanied by the formation of lesions that are indistinguishable from the pulmonary arteriopathy of human pulmonary arterial hypertension. This unique model provides a new and rigorous approach for investigating the genesis, hemodynamic effects, and reversibility of plexiform and other occlusive lesions in pulmonary arterial hypertension.

Rho kinase-mediated vasoconstriction in pulmonary hypertension.

Rho kinase-mediated vasoconstriction rather than fixed arterial wall thickening is responsible for increased pulmonary vascular resistance and pulmonary hypertension in chronically hypoxic and monocrotaline-injected rats. In the absence of vascular tone, the medial and adventitial thickening in these models has only minimal impact on the cross-sectional area of the pulmonary arterial bed. In contrast, increased pulmonary vascular resistance in left-pneumonectomized plus monocrotaline-injected rats and VEGF receptor blocker-injected plus chronic hypoxia rats is attributable to both Rho kinase-mediated vasoconstriction and formation of lumen obliterating lesions in small pulmonary arteries. The upstream signals responsible for activation of RhoA/Rho kinase signaling in hypertensive pulmonary arteries and whether or not they differ in different forms of pulmonary hypertension are unclear. The RhoA/Rho kinase pathway is a convergence point of several different vasoconstrictor signals, including those mediated by G protein-coupled receptors, receptor tyrosine kinases, and integrin clustering. Both isoforms of Rho kinase can also be constitutively activated by cleavage, and cleaved Rho kinase 1 has been detected in the hypertensive lungs of left-pneumonectomized plus monocrotaline-injected rats. That such diverse stimuli can lead to activation of Rho kinase, which may cause hypercontraction of smooth muscle by promoting both actomyosin interaction and remodeling of the cytoskeleton, may explain why in various rat models of pulmonary hypertension Rho kinase inhibitors are more effective pulmonary vasodilators than conventional agents such as nitric oxide, prostacyclin, and nifedipine. We suspect the same will be true in at least some forms of human pulmonary arterial hypertension.

Future perspectives for the treatment of pulmonary arterial hypertension.

Over the past 2 decades, pulmonary arterial hypertension has evolved from a uniformly fatal condition to a chronic, manageable disease in many cases, the result of unparalleled development of new therapies and advances in early diagnosis. However, none of the currently available therapies is curative, so the search for new treatment strategies continues. With a deeper understanding of the genetics and the molecular mechanisms of pulmonary vascular disorders, we are now at the threshold of entering a new therapeutic era. Our working group addressed what can be expected in the near future. The topics span the understanding of genetic variations, novel antiproliferative treatments, the role of stem cells, the right ventricle as a therapeutic target, and strategies and challenges for the translation of novel experimental findings into clinical practice.

Neprilysin null mice develop exaggerated pulmonary vascular remodeling in response to chronic hypoxia.

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Vascular dysfunction in a murine model of severe hemolysis.

Spectrin is the backbone of the erythroid cytoskeleton; sph/sph mice have severe hereditary spherocytosis (HS) because of a mutation in the murine erythroid alpha-spectrin gene. sph/sph mice have a high incidence of thrombosis and infarction in multiple tissues, suggesting significant vascular dysfunction. In the current study, we provide evidence for both pulmonary and systemic vascular dysfunction in sph/sph mice. We found increased levels of soluble cell adhesion molecules in sph/sph mice, suggesting activation of the vascular endothelium. We hypothesized that plasma hemoglobin released by intravascular hemolysis initiates endothelial injury through nitric oxide (NO) scavenging and oxidative damage. Likewise, electron paramagnetic resonance spectroscopy showed that plasma hemoglobin is much greater in sph/sph mice. Moreover, plasma from sph/sph mice had significantly higher oxidative potential. Finally, xanthine oxidase, a potent superoxide generator, is decreased in subpopulations of liver hepatocytes and increased on liver endothelium in sph/sph mice. These results indicate that vasoregulation is abnormal, and NO-based vasoregulatory mechanisms particularly impaired, in sph/sph mice. Together, these data indicate that sph/sph mice with severe HS have increased plasma hemoglobin and NO scavenging capacity, likely contributing to aberrant vasoregulation and initiating oxidative damage.

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone.

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33+/-5 and 16+/-1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47+/-3 and 30+/-3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.

Dehydroepiandrosterone upregulates soluble guanylate cyclase and inhibits hypoxic pulmonary hypertension.

OBJECTIVE: It has been reported that dehydroepiandrosterone is a pulmonary vasodilator and inhibits chronic hypoxia-induced pulmonary hypertension. Additionally, dehydroepiandrosterone has been shown to improve systemic vascular endothelial function. Thus, we hypothesized that chronic treatment with dehydroepiandrosterone would attenuate hypoxic pulmonary hypertension by enhancing pulmonary artery endothelial function. METHODS AND RESULTS: Rats were randomly assigned to five groups. Three groups received food containing 0, 0.3, or 1% dehydroepiandrosterone during a 3-wk-exposure to simulated high altitude (HA). The other 2 groups were kept at Denver's low altitude (LA) and received food containing 0 or 1% dehydroepiandrosterone. Dehydroepiandrosterone dose-dependently inhibited hypoxic pulmonary hypertension (mean pulmonary artery pressures after treatment with 0, 0.3, and 1% dehydroepiandrosterone=45+/-5, 33+/-2*, and 25+/-1*# mmHg, respectively. *P<0.05 vs. 0% and # vs. 0.3%). Dehydroepiandrosterone (1%, 3 wks) treatment started after rats had been exposed to 3-wk hypoxia also effectively reversed established hypoxic pulmonary hypertension. Pulmonary artery rings isolated from both LA and HA rats treated with 1% dehydroepiandrosterone showed enhanced relaxations to acetylcholine and sodium nitroprusside, but not to 8-bromo-cGMP. In the pulmonary artery tissue from dehydroepiandrosterone-treated LA and HA rats, soluble guanylate cyclase, but not endothelial nitric oxide synthase, protein levels were increased. CONCLUSION: These results indicate that the protective effect of dehydroepiandrosterone against hypoxic pulmonary hypertension may involve upregulation of pulmonary artery soluble guanylate cyclase protein expression and augmented pulmonary artery vasodilator responsiveness to nitric oxide.

Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms.

Chronic hypoxic exposure induces changes in the structure of pulmonary arteries, as well as in the biochemical and functional phenotypes of each of the vascular cell types, from the hilum of the lung to the most peripheral vessels in the alveolar wall. The magnitude and the specific profile of the changes depend on the species, sex, and the developmental stage at which the exposure to hypoxia occurred. Further, hypoxia-induced changes are site specific, such that the remodeling process in the large vessels differs from that in the smallest vessels. The cellular and molecular mechanisms vary and depend on the cellular composition of vessels at particular sites along the longitudinal axis of the pulmonary vasculature, as well as on local environmental factors. Each of the resident vascular cell types (ie, endothelial, smooth muscle, adventitial fibroblast) undergo site- and time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors, and each resident cell type plays a specific role in the overall remodeling response. In addition, hypoxic exposure induces an inflammatory response within the vessel wall, and the recruited circulating progenitor cells contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation. The possibility exists that the lung or lung vessels also contain resident progenitor cells that participate in the remodeling process. Thus the hypoxia-induced remodeling of the pulmonary circulation is a highly complex process where numerous interactive events must be taken into account as we search for newer, more effective therapeutic interventions. This review provides perspectives on each of the aforementioned areas.