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Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH., Competing Interests: Declaration of competing interest The following authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Beate Christiane Schlueter, Karin Quanz, Julia Baldauf, Aleksandar Petrovic, Clemens Ruppert, Andreas Guenther, Henning Gall, Khodr Tello, Friedrich Grimminger, Hossein-Ardeschir Ghofrani, Norbert Weissmann, Ralph Theo Schermuly, Astrid Weiss. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Werner Seeger received consulting fees from United Therapeutics, Tiakis Biotech AG, Liquidia, Pieris Pharmaceuticalsm Abivax, Pfitzer, Medspray BV., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone., Methods and Results: Mice deficient for p66shc (p66shc-/-), CypD (CypD-/-), or both proteins (p66shc/CypD-/-) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD-/- mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD-/- and p66shc/CypD-/- mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling., Conclusions: We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO 2 ). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO 2 have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions., (© 2021 The Authors. Small Methods published by Wiley-VCH GmbH.)

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.

Pulmonary hypertension (PH) is characterized by pulmonary artery remodeling that can subsequently culminate in right heart failure and premature death. Emerging evidence suggests that hypoxia-inducible factor (HIF) signaling plays a fundamental and pivotal role in the pathogenesis of PH. This Review summarizes the regulation of HIF isoforms and their impact in various PH subtypes, as well as the elaborate conditional and cell-specific knockout mouse studies that brought the role of this pathway to light. We also discuss the current preclinical status of pan- and isoform-selective HIF inhibitors, and propose new research areas that may facilitate HIF isoform-specific inhibition as a novel therapeutic strategy for PH and right heart failure.

Aims: The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV)., Methods: Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs., Results: Pharmacological inhibition of EET synthesis with MS-PPOH, application of the EET antagonist 14,15-EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET-degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre-contracted with thromboxane mimetic U46619. However, in the presence of KCl-induced depolarization, 5,6-EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch-clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6-EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential., Conclusions: The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6-EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6-EET-induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6-EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV-related diseases., (© 2020 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Chronic obstructive pulmonary disease (COPD) is one of the most important causes of death worldwide, and in addition to its impact on the patient's health, it poses a major socioeconomic burden. Tobacco smoke, indoor cooking, and air pollution are major triggers of the disease. This article summarizes evidence for the concept that lung microvascular molecular alterations can be a driver of lung emphysema. If findings from preclinical models allow a transfer to the human situation, this concept can offer new approaches for curative treatment of lung emphysema.

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system ( p -values from 0.00% to 16.30%) compared to the open system ( p -value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

New Findings: What is the central question of this study? Hypoxic pulmonary vasoconstriction has never been characterized in isolated mouse pulmonary arteries of different generations in detail. What is the main finding and its importance? We found that only small intrapulmonary arteries (80-200 μm in diameter) exhibit hypoxic pulmonary vasoconstriction. The observed response was sustained, significantly potentiated by depolarization-induced preconstriction and not dependent on the endothelium or TRPC6 channels., Abstract: Hypoxic pulmonary vasoconstriction (HPV) is a physiological response of pulmonary arteries, which adapts lung perfusion to regional ventilation. The properties of HPV vary significantly between animal species. Despite extensive use of mouse models in studies of HPV, this physiological response has never been characterized in isolated mouse pulmonary arteries in detail. Using wire myography, we investigated the effect of 80 min exposure to hypoxia on the tone in mouse pulmonary arteries of different generations in the presence and absence of preconstriction. Hypoxia induced a sustained relaxation in non-preconstricted extrapulmonary arteries (500-700 μm in diameter), but not in the presence of KCl-induced preconstriction. Large intrapulmonary arteries (450-650 μm in diameter) did not exhibit a significant response to the hypoxic challenge. In contrast, in small intrapulmonary arteries (80-200 μm in diameter), hypoxia elicited a slowly developing sustained constriction, which was independent of the endothelium. The response was significantly potentiated in arteries preconstricted with KCl, but not with U46619. Hypoxic pulmonary vasoconstriction was not altered in pulmonary arteries of TRPC6-deficient mice, which suggests that this response corresponds to the sustained phase of biphasic HPV observed earlier in isolated, buffer-perfused and ventilated mouse lungs. In conclusion, we have established a protocol that allows the study of sustained HPV in isolated mouse pulmonary arteries. The data obtained might be useful for future studies of the mechanisms of HPV in mice., (© 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.)

The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25-40 μm) and pre-acinar pulmonary arteries (inner diameter: 41-60 μm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.

New Findings: What is the central question of this study? The aim was to investigate whether complementary assessment of non-invasive ultrasound imaging together with closed chest-derived intracardiac pressure-volume catheterization is applicable to mice for an in-depth characterization of right ventricular (RV) function even upon maintained pressure overload. What is the main finding and its importance? Characterization of RV function by the complementary use of echocardiographic imaging together with pressure-volume catheterization reveals ventricular-arterial decoupling upon maintained pressure overload, where RV systolic function correlates with ventricular-arterial coupling rather than contractility, whereas diastolic function correlates well with RV diastolic pressure. This combined approach allows us to phenotype RV function and dysfunction better in genetically modified and/or pharmacologically treated mice. Assessment of right ventricular (RV) function in rodents is a challenge because of the complex RV anatomy and structure. To date, the best characterization of RV function has been achieved by accurate cardiovascular phenotyping, involving a combination of non-invasive imaging and intracardiac pressure-volume measurements. We sought to investigate the feasibility of two complementary phenotyping techniques for the evaluation of RV function in an experimental mouse model of sustained RV pressure overload. Mice underwent either sham surgery (n = 5) or pulmonary artery banding (n = 8) to induce isolated RV pressure overload. After 3 weeks, indices of RV function were assessed by echocardiography (Vevo2100) and closed chest-derived invasive pressure-volume measurements (PVR-1030). Pulmonary artery banding resulted in RV hypertrophy and dilatation accompanied by systolic and diastolic dysfunction. Invasive RV haemodynamic measurements demonstrated an increased end-systolic elastance and arterial elastance after pulmonary artery banding compared with sham operation, resulting in ventricular-arterial decoupling. Regression analysis revealed that tricuspid annular plane systolic excursion is correlated with ventricular-arterial coupling (r² = 0.77, P = 0.002) rather than with RV contractility (r² = -0.61, P = 0.07). Furthermore, the isovolumic relaxation time to ECG-derived R-R interval and the ratio of the early diastolic peak velocity measured by pulsed wave Doppler to the early diastolic peak obtained during tissue Doppler imaging correlate well with RV end-diastolic pressure (r² = 0.87, P = 0.0001 and r² = 0.82, P = 0.0009, respectively). Commonly used indices of systolic RV function are associated with RV-arterial coupling rather than contractility, whereas diastolic indices well correlate with end-diastolic pressure when there is maintained pressure overload., (© 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.)

Background: Chronic obstructive lung disease (COPD) is a common cause of death in industrialized countries often induced by exposure to tobacco smoke. A substantial number of patients with COPD also suffer from pulmonary hypertension that may be caused by hypoxia or other hypoxia-independent stimuli - inducing pulmonary vascular remodeling. The Ca(2+) binding protein, S100A4 is known to play a role in non-COPD-driven vascular remodeling of intrapulmonary arteries. Therefore, we have investigated the potential involvement of S100A4 in COPD induced vascular remodeling., Methods: Lung tissue was obtained from explanted lungs of five COPD patients and five non-transplanted donor lungs. Additionally, mice lungs of a tobacco-smoke-induced lung emphysema model (exposure for 3 and 8 month) and controls were investigated. Real-time RT-PCR analysis of S100A4 and RAGE mRNA was performed from laser-microdissected intrapulmonary arteries. S100A4 immunohistochemistry was semi-quantitatively evaluated. Mobility shift assay and siRNA knock-down were used to prove hypoxia responsive elements (HRE) and HIF binding within the S100A4 promoter., Results: Laser-microdissection in combination with real-time PCR analysis revealed higher expression of S100A4 mRNA in intrapulmonary arteries of COPD patients compared to donors. These findings were mirrored by semi-quantitative analysis of S100A4 immunostaining. Analogous to human lungs, in mice with tobacco-smoke-induced emphysema an up-regulation of S100A4 mRNA and protein was observed in intrapulmonary arteries. Putative HREs could be identified in the promoter region of the human S100A4 gene and their functionality was confirmed by mobility shift assay. Knock-down of HIF1/2 by siRNA attenuated hypoxia-dependent increase in S100A4 mRNA levels in human primary pulmonary artery smooth muscle cells. Interestingly, RAGE mRNA expression was enhanced in pulmonary arteries of tobacco-smoke exposed mice but not in pulmonary arteries of COPD patients., Conclusions: As enhanced S100A4 expression was observed in remodeled intrapulmonary arteries of COPD patients, targeting S100A4 could serve as potential therapeutic option for prevention of vascular remodeling in COPD patients.

Pulmonary hypertension (PH) is characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation leading to vascular remodeling. Although, multiple factors have been associated with pathogenesis of PH the underlying mechanisms are not fully understood. Here, we hypothesize that already very short exposure to hypoxia may activate molecular cascades leading to vascular remodeling. Microarray studies from lung homogenates of mice exposed to only 3h of hypoxia revealed endothelin-1 (ET-1) and connective tissue growth factor (CTGF) as the most upregulated genes, and the mitogen-activated protein kinase (MAPK) pathway as the most differentially regulated pathway. Evaluation of these results in vitro showed that ET-1 but not CTGF stimulation of human PASMCs increased DNA synthesis and expression of proliferation markers such as Ki67 and cell cycle regulator, cyclin D1. Moreover, ET-1 treatment elevated extracellular signal-regulated kinase (Erk)-dependent c-fos expression and phosphorylation of c-fos and c-jun transcription factors. Silencing of c-fos with siRNA abrogated the ET-1-induced proliferation of PASMCs. Expression and immunohistochemical analyses revealed higher levels of total and phosphorylated c-fos and c-jun in the vessel wall of lung samples of human idiopathic pulmonary arterial hypertension patents, hypoxia-exposed mice and monocrotaline-treated rats as compared to control subjects. These findings shed the light on the involvement of c-fos/c-jun in the proliferative response of PASMCs to ET-1 indicating that already very short hypoxia exposure leads to the regulation of mediators involved in vascular remodeling underlying PH., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Pulmonary vascular remodeling, the pathological hallmark of pulmonary arterial hypertension, is attributed to proliferation, apoptosis resistance, and migration of vascular cells. A role of dysregulated matrix cross-linking and stability as a pathogenic mechanism has received little attention. We aimed to assess whether matrix cross-linking enzymes played a causal role in experimental pulmonary hypertension (PH)., Approach and Results: All 5 lysyl oxidases were detected in concentric and plexiform vascular lesions of patients with idiopathic pulmonary arterial hypertension. Lox, LoxL1, LoxL2, and LoxL4 expression was elevated in lungs of patients with idiopathic pulmonary arterial hypertension, whereas LoxL2 and LoxL3 expression was elevated in laser-capture microdissected vascular lesions. Lox expression was hypoxia-responsive in pulmonary artery smooth muscle cells and adventitial fibroblasts, whereas LoxL1 and LoxL2 expression was hypoxia-responsive in adventitial fibroblasts. Lox expression was increased in lungs from hypoxia-exposed mice and in lungs and pulmonary artery smooth muscle cells of monocrotaline-treated rats, which developed PH. Pulmonary hypertensive mice exhibited increased muscularization and perturbed matrix structures in vessel walls of small pulmonary arteries. Hypoxia exposure led to increased collagen cross-linking, by dihydroxylysinonorleucine and hydroxylysinonorleucine cross-links. Administration of the lysyl oxidase inhibitor β-aminopropionitrile attenuated the effect of hypoxia, limiting perturbations to right ventricular systolic pressure, right ventricular hypertrophy, and vessel muscularization and normalizing collagen cross-linking and vessel matrix architecture., Conclusions: Lysyl oxidases are dysregulated in clinical and experimental PH. Lysyl oxidases play a causal role in experimental PH and represent a candidate therapeutic target. Our proof-of-principle study demonstrated that modulation of lung matrix cross-linking can affect pulmonary vascular remodeling associated with PH., (© 2014 American Heart Association, Inc.)

Background: Pulmonary hypertension (PH) is a life-threatening disease characterized by vascular remodeling and increased pulmonary vascular resistance. Chronic alveolar hypoxia in animals is often used to decipher pathways being regulated in PH. Here, we aimed to investigate whether chronic hypoxia-induced PH in mice can be reversed by reoxygenation and whether possible regression can be used to identify pathways activated during the reversal and development of PH by genome-wide screening., Methods and Results: Mice exposed to chronic hypoxia (21 days, 10% O2) were reoxygenated for up to 42 days. Full reversal of PH during reoxygenation was evident by normalized right ventricular pressure, right heart hypertrophy, and muscularization of small pulmonary vessels. Microarray analysis from these mice revealed s-adenosylmethionine decarboxylase 1 (AMD-1) as one of the most downregulated genes. In situ hybridization localized AMD-1 in pulmonary vessels. AMD-1 silencing decreased the proliferation of pulmonary arterial smooth muscle cells and diminished phospholipase Cγ1 phosphorylation. Compared with the respective controls, AMD-1 depletion by heterozygous in vivo knockout or pharmacological inhibition attenuated PH during chronic hypoxia. A detailed molecular approach including promoter analysis showed that AMD-1 could be regulated by early growth response 1, transcription factor, as a consequence of epidermal growth factor stimulation. Key findings from the animal model were confirmed in human idiopathic pulmonary arterial hypertension., Conclusions: Our study indicates that genome-wide screening in mice from a PH model in which full reversal of PH occurs can be useful to identify potential key candidates for the reversal and development of PH. Targeting AMD-1 may represent a promising strategy for PH therapy.

Aims: Chronic hypoxia induces pulmonary hypertension (PH) that is concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this. Recent findings suggest that ROS production by NADPH oxidase 4 (Nox4) is important in this remodeling. We investigated whether ROS production by Nox is also important in an inflammatory model of monocrotaline (MCT)-induced PH. We examined ROS production, their possible sources, and their impact on the function of pulmonary arterial smooth muscle cells (PASMC) isolated from MCT-treated and healthy rats., Results: MCT-PASMC showed increased intracellular superoxide production, migration, and proliferation compared with healthy controls due to increased Nox1 expression. A comparison of PASMC from MCT- and nontreated rats revealed an up-regulation of Sod2, Nrf2, cyclin D1, and matrix metalloproteinase-9 (MMP-9) as well as an increased phosphorylation of cofilin and extracellular signal-regulated kinases (Erk). Expression of Sod2, Nrf2, and cyclin D1 and phosphorylation of cofilin and Erk were Nox1 dependent., Innovation: The role of ROS in PH is not fully understood. Mitochondria and Nox have been suggested as sources of altered ROS generation in PH, yet it remains unclear whether increased or decreased ROS contributes to the development of PH. Our studies provide evidence that for different triggers of PH, different Nox isoforms regulate proliferation and migration of PASMC., Conclusion: In contrast to hypoxia-induced PH, Nox1 but not Nox4 is responsible for pathophysiological proliferation and migration of PASMC in an inflammatory model of MCT-induced PH via increased superoxide production. Thus, different Nox isoforms may be targeted in different forms of PH.

Pulmonary hypertension (PH) is a fatal disease characterized by remodeling processes such as increased migration and proliferation of pulmonary arterial smooth muscle cells (PASMC), enhanced matrix deposition, and dysregulation of cytoskeletal proteins. However, the contribution of cytoskeletal proteins in PH is still not fully understood. In this study, we have used a yeast two-hybrid screen to identify novel binding partners of the cytoskeletal adaptor protein four-and-a-half LIM domains 1 (Fhl-1). This identified paxillin as a new Fhl-1 interacting partner, and consequently we assessed its contribution to vascular remodeling processes. Native protein-protein binding was confirmed by co-immunoprecipitation studies in murine and human PASMC. Both proteins co-localized in PASMC in vitro and in vivo. In lung samples from idiopathic pulmonary arterial hypertension patients, paxillin expression was increased on mRNA and protein levels. Laser-microdissection of murine intrapulmonary arteries revealed elevated paxillin expression in hypoxia-induced PH. Furthermore, hypoxia-dependent upregulation of paxillin was HIF-1α dependent. Silencing of paxillin expression led to decreased PASMC adhesion, proliferation, and increased apoptosis. Regulation of these processes occurred via Akt and Erk1/2 kinases. In addition, adhesion of PASMC to the extracellular matrix protein fibronectin was critically dependent on paxillin expression. To summarize, we identified paxillin as a new regulator protein of PASMC growth., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rationale: A hallmark of the vascular remodeling process underlying pulmonary hypertension (PH) is the aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). Accumulating evidence suggests that mast cell mediators play a role in the pathogenesis of PH., Objective: In the present study we investigated the importance of protease-activated receptor (PAR)-2 and its ligand mast cell tryptase in the development of PH., Methods and Results: Our results revealed strong increase in PAR-2 and tryptase expression in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline (MCT)-treated rats. Elevated tryptase levels were also detected in plasma samples from IPAH patients. Hypoxia and platelet-derived growth factor (PDGF)-BB upregulated PAR-2 expression in PASMC. This effect was reversed by HIF (hypoxia inducible factor)-1α depletion, PDGF-BB neutralizing antibody, or the PDGF-BB receptor antagonist Imatinib. Attenuation of PAR-2 expression was also observed in smooth muscle cells of pulmonary vessels of mice exposed to hypoxia and rats challenged with MCT in response to Imatinib treatment. Tryptase induced PASMC proliferation and migration as well as enhanced synthesis of fibronectin and matrix metalloproteinase-2 in a PAR-2- and ERK1/2-dependent manner, suggesting that PAR-2-dependent signaling contributes to vascular remodeling by various mechanisms. Furthermore, PAR-2(-/-) mice were protected against hypoxia-induced PH, and PAR-2 antagonist application reversed established PH in the hypoxia mouse model., Conclusions: Our study identified a novel role of PAR-2 in vascular remodeling in the lung. Interference with this pathway may offer novel therapeutic options for the treatment of PH.

Aims: Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-))., Methods and Results: Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice., Conclusion: SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Rationale: Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia., Objectives: To investigate whether hypoxia alters PDGF β receptor (βPDGFR) signaling in the pulmonary vasculature., Methods: The impact of chronic hypoxia on signal transduction by the βPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo., Measurements and Main Results: Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect βPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated βPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced βPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. βPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH., Conclusions: Hypoxia reduces expression and activity of βPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the βPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.

Background: Generalized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV., Methods: Ex vivo isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and in vivo anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the ex vivo model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the in vivo model., Results: In the ex vivo model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the in vivo model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable., Conclusion: Nitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung matching blood perfusion to ventilation during local alveolar hypoxia. HPV thus optimizes pulmonary gas exchange. In contrast chronic and generalized hypoxia leads to pulmonary vascular remodeling with subsequent pulmonary hypertension and right heart hypertrophy. Among other non-selective cation channels, the family of classical transient receptor potential channels (TRPC) has been shown to be expressed in pulmonary arterial smooth muscle cells. Among this family, TRPC6 is essential for the regulation of acute HPV in mice. Against this background, in this chapter we give an overview about the TRPC family and their role in HPV.

Endothelin (ET)-1 causes long-lasting vasoconstriction and vascular remodeling by interacting with specific G-protein-coupled receptors in pulmonary artery smooth muscle cells (PASMCs), and thus plays an important role in the pathophysiology of pulmonary arterial hypertension. The two-pore domain K(+) channel, TASK-1, controls the resting membrane potential in human PASMCs (hPASMCs), and renders these cells sensitive to a variety of vasoactive factors, as previously shown. ET-1 may exert its vasoconstrictive effects in part by targeting TASK-1. To clarify this, we analyzed the ET-1 signaling pathway related to TASK-1 in primary hPASMCs. We employed the whole-cell patch-clamp technique combined with TASK-1 small interfering RNA (siRNA) in hPASMC and the isolated, perfused, and ventilated mouse lung model. We found that ET-1 depolarized primary hPASMCs by phosphorylating TASK-1 at clinically relevant concentrations. The ET sensitivity of TASK-1 required ET(A) receptors, phospholipase C, phosphatidylinositol 4,5-biphosphate, diacylglycerol, and protein kinase C in primary hPASMCs. The ET-1 effect on membrane potential and TASK-1 was abrogated using TASK-1 siRNA. This is the first time that the background K(+) channel, TASK-1, has been identified in the ET-1-mediated depolarization in native hPASMC, and might represent a novel pathologic mechanism related to pulmonary arterial hypertension.

Pulmonary arterial hypertension is a life-threatening, vasculoproliferative disease of the lung, which is characterized by vasoconstriction and remodeling of small pulmonary arteries. Drugs for the treatment of PAH mainly address the increased vascular tone. Substances like prostacyclin, endothelin-receptor-antagonists and phosphodiesterase-5-inhibitors have been approved for the treatment of PAH and represent the current therapeutic options. The development of a causal treatment aiming a normalization of the vessel wall structure is the current focus of research. The key events in disease progression are represented by increased proliferation, migration and a resistance to apoptosis of pulmonary vascular cells. Therefore, new non-vasoactive drugs are investigated in relevant preclinical animal models of pulmonary arterial hypertension. Some of these substances, like tyrosine kinase inhibitors, elastase inhibitors and phosphodiesterase-1-inhibitors, could not only attenuate (anti-remodeling) but reverse (reverse-remodeling) the disease. Additionally, new vasodilators, like soluble guanylate cyclase stimulators and activators, addressing well-known and new signaling pathways are currently under investigation. Taken together, with increasing insight into the pathology of PAH, several novel drug targets and treatments have emerged which may improve the management of patients and which efficacy is currently addressed in preclinical studies and clinical trials., (Georg Thieme Verlag KG Stuttgart, New York.)

Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. HPV and EET production were greater in lungs from sEH(-/-) mice than from wild-type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15-EEZE decreased the response. 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced HPV via a Rho-dependent mechanism. Both 11,12-EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6-V5 fusion protein, the latter effect was sensitive to 14,15-EEZE. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6(+/-) mice, lungs from TRPC6(-/-) mice did not respond to either stimuli. These data demonstrate that CYP-derived EETs are involved in HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6-dependent pathway.

Shroom is a PDZ-domain protein involved in the regulation and maintenance of cytoskeletal architecture by binding to actin. Hypertrophy and altered actin organisation of pulmonary arterial smooth muscle cells (PASMC) is a hallmark of pulmonary arterial hypertension (PAH). The aim of the present study was to localise and characterise Shroom expression in the lung in experimental and idiopathic PAH (IPAH). Shroom expression and localisation in hypoxia-induced PAH in mice and IPAH in humans, in vivo, as well as in primary PASMC, in vitro, was assessed by quantitative RT-PCR, immunofluorescence, laser-assisted microdissection and immunohistochemistry. Shroom localised exclusively to PASMC (both bronchial and vascular) in mouse and human lungs. Both in vivo and in primary PASMC, in vitro, Shroom exhibited spatially similar expression with alpha-smooth muscle actin (alpha-SMA). Shroom expression was significantly reduced in the mouse model of PAH, in primary murine PASMC exposed to hypoxia, and in primary PASMC isolated from patients with IPAH. The ratio between Shroom and alpha-SMA RNA expression further confirmed Shroom downregulation in both mouse and human PASMC. In summary, Shroom localises exclusively to pulmonary smooth muscle cells. Shroom downregulation in pulmonary arterial hypertension suggests a link between Shroom expression and pulmonary arterial smooth muscle cell hypertrophy in pulmonary arterial hypertension.

Alterations of the nitric oxide receptor, soluble guanylate cyclase (sGC) may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). In the present study, the expression of sGC in explanted lung tissue of PAH patients was studied and the effects of the sGC stimulator BAY 63-2521 on enzyme activity, and haemodynamics and vascular remodelling were investigated in two independent animal models of PAH. Strong upregulation of sGC in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was demonstrated by immunohistochemistry. Upregulation of sGC was detected, similarly to humans, in the structurally remodelled smooth muscle layer in chronic hypoxic mouse lungs and lungs from monocrotaline (MCT)-injected rats. BAY 63-2521 is a novel, orally available compound that directly stimulates sGC and sensitises it to its physiological stimulator, nitric oxide. Chronic treatment of hypoxic mice and MCT-injected rats, with fully established PAH, with BAY 63-2521 (10 mg x kg(-1) x day(-1)) partially reversed the PAH, the right heart hypertrophy and the structural remodelling of the lung vasculature. Upregulation of soluble guanylate cyclase in pulmonary arterial smooth muscle cells was noted in human idiopathic pulmonary arterial hypertension lungs and lungs from animal models of pulmonary arterial hypertension. Stimulation of soluble guanylate cyclase reversed right heart hypertrophy and structural lung vascular remodelling. Soluble guanylate cyclase may thus offer a new target for therapeutic intervention in pulmonary arterial hypertension.

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disease, characterized by vascular smooth muscle cell hyperproliferation. The calcium/calmodulin-dependent phosphodiesterase 1 (PDE1) may play a major role in vascular smooth muscle cell proliferation., Methods and Results: We investigated the expression of PDE1 in explanted lungs from idiopathic PAH patients and animal models of PAH and undertook therapeutic intervention studies in the animal models. Strong upregulation of PDE1C in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was noted on the mRNA level by laser-assisted vessel microdissection and on the protein level by immunohistochemistry. In chronically hypoxic mouse lungs and lungs from monocrotaline-injected rats, PDE1A upregulation was detected in the structurally remodeled arterial muscular layer. Long-term infusion of the PDE1 inhibitor 8-methoxymethyl 3-isobutyl-1-methylxanthine in hypoxic mice and monocrotaline-injected rats with fully established pulmonary hypertension reversed the pulmonary artery pressure elevation, structural remodeling of the lung vasculature (nonmuscularized versus partially muscularized versus fully muscularized small pulmonary arteries), and right heart hypertrophy., Conclusions: Strong upregulation of the PDE1 family in pulmonary artery smooth muscle cells is noted in human idiopathic PAH lungs and lungs from animal models of PAH. Inhibition of PDE1 reverses structural lung vascular remodeling and right heart hypertrophy in 2 animal models. The PDE1 family may thus offer a new target for therapeutic intervention in pulmonary hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches perfusion to ventilation in order to optimize pulmonary gas exchange. Despite intensive research, the underlying mechanism has not yet been fully elucidated. Reactive oxygen species (ROS) have been proposed as key mediators of HPV. However, there is ongoing discussion as to whether ROS really contribute to HPV regulation and if so, whether an increase or a decrease in ROS occurs during alveolar hypoxia. In this overview, we summarize our data that have led us to conclude that alveolar hypoxia induces an increase in superoxide and subsequently H2O2, and thus elicits HPV. This conclusion is drawn from investigations employing various inhibitors that interfere with ROS in isolated buffer-perfused rabbit lungs challenged with 10-minute periods of alveolar hypoxia. Targeting possible sources of a hypoxia-induced increase in ROS, our data are only partially in accordance with the hypothesis that mitochondria are the hypoxia-dependent ROS generators, and suggest NADPH oxidases as an alternative source. From measurements of intracellular and exhaled H2O2, we hypothesize that total lung ROS release is reduced in alveolar hypoxia, but that in specialized cells or sub-cellular structures an increased ROS release may occur, triggering HPV.

Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human FBPA exposed to hypoxia. Primary cultures of FBPA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FBPA was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF-2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the FBPA was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies.

Background: Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling., Methods and Results: Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-beta, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media., Conclusion: Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation which tends to optimize pulmonary gas exchange. Investigations using genetically engineered mice represent a promising approach to understand the underlying mechanisms. Our goal was to characterize basic features of HPV in the isolated buffer-perfused and ventilated mouse lung system. HPV was reproducible for several hours when ventilating the lungs with 1% O2 (10 min) alternated with normoxic ventilation periods (21% O2, 15 min). HPV was well elicitable and most constant using Krebs-Henseleit buffer with the addition of hydroxyethylamylopectin as an oncotic agent. Inhibition of both lung NO and prostanoid formation amplified HPV in an over-additive fashion. HPV was higher in BALB/c mive as compared to C57BL/6 mice, and was approximately threefold enhanced under positive pressure ventilation as compared to negative pressure ventilation. A three hour hypoxic ventilation period resulted in a biphasic vasoconstrictor response with loss of posthypoxic vasodilatation. In summary, we have characterised HPV and established an experimental set-up optimized for investigation of the basic mechanisms of HPV in mice.

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co-cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum-free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia-inducible-transcription-factor subtypes (HIF-1alpha, HIF-2alpha, HIF-3alpha) and its dependent target genes, carrying the hypoxia-responsive-element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF-transcription-factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia-driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF-transcription-decoy may offer a new therapeutic approach to suppress these events.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.