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Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3′-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell–specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

Rationale Pulmonary arterial hypertension (PAH) is a vascular disease characterized by chronic increases in pulmonary vascular resistance (PVR), pulmonary arterial pressure (PAP), and right ventricular (RV) hypertrophy. Increased activation of receptor tyrosine kinase (RTK) -mediated signaling pathways leads to increased proliferation and migration of pulmonary smooth vascular muscle cells (PASMCs) which promote vascular remodeling processes. We identified the catalytic subunit p110alpha of phosphatidylinositol-3-kinase as a key enzyme for these processes and showed that both genetic ablation of p110alpha in SMCs and pharmacological inhibition can prevent experimental PH. Here, the effects of the orally bioavailable p110alpha selective PI3K inhibitor BYL719 on the RTK-mediated proliferation and chemotaxis of PASMCs, as well as the effects in the hypoxia-induced mouse and in the Sugen / hypoxia (SuHx) -induced rat model of PH were investigated. Methods Human and murine PASMCs were pretreated with different concentrations of BYL719 and stimulated with a mixture of growth factors (PDGF [30ng/ml], EGF [0,5ng/ml], bFGF [2ng/ml], insulin [0,5ng/ml], and FBS [5%]). Proliferation was investigated using a BrdU incorporation ELISA assay (Roche). Chemotaxis was quantified using modified Boyden chambers. Male BL/6 mice were subjected to hypoxia (10% O2) for 21 days. Treatment with BYL719 (or vehicle) was carried out via daily gavage of 50mg/kg bodyweight. In addition, a therapeutic approach was investigated using male Sprague Dawley rats in the SuHx model, which were treated with BYL719 (20 mg / kg body weight) or vehicle for 2 weeks after a three-week hypoxia phase. The RV pressure (RVSP) was measured using a Millar® or liquid-filled catheter. The RV hypertrophy is shown as the quotient of the weights of the RV to the LV + septum (RV / (LV + S)). Results Growth factor-induced proliferation and chemotaxis of the PASMCs were significantly and concentration-dependently inhibited by BYL719. The exposure to hypoxia led to an increase of the RVSP (24.5±0.95 to 35.2±1.28 mmHg) and the development of right ventricular hypertrophy (RV / LV + S 0.24±0.01 to 0.37±0.073), which was significantly reduced in the BYL719 treated group (RVSP 31.4±0.53 mmHg; RV / LV + S 0.31±0.01) (p Conclusion These results show that inhibition of p110alpha using the BYL719 reduced growth factor-mediated pathological processes in PASMCs in vitro, as well as hypoxia-induced (mouse) and already established SuHx-induced PH (rat). Thus, the inhibition of p110a using BYL719 represents a promising approach for the treatment of PAH. Funding Acknowledgement Type of funding sources: None.

Background In smooth muscle cells (SMCs), the PI 3-kinase isoform p110α mediates receptor tyrosine kinase dependent proliferation, chemotaxis and cell survival. Since mice, harbouring a smooth muscle specific p110α deficiency (SM-p110α−/−), display reduced vascular wall thickness, we hypothesized that SM-p110α−/− mice might be prone to aortic aneurysm (AA) formation. The pathogenesis of AA is characterized by increased dedifferentiation of SMCs, extracellular matrix (ECM) degeneration and inflammation in the aortic wall. Herein, we investigated how p110α-dependent signal transduction in SMCs affects these processes. Methods and results We examined AA formation in SM-p110α−/− mice and wild-type littermates using the “porcine pancreatic elastase” (PPE) AA model. PPE was infused into the infrarenal aorta to induce AA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. The aortic diameter in SM-p110α−/− mice (0.46±0.12 mm) was significantly increased compared to wild-type animals (0.18±0.03 mm, p Conclusion These data show that deficiency of p110α in SMCs promotes the formation and progression of AA. Causative are impaired SMC plasticity and ECM homeostasis as well as inflammatory processes in the vascular wall. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Introduction and purpose Atherosclerosis is a chronic inflammatory disease of arteries, critically involving leukocytes like macrophages as well as T and B lymphocytes. Macrophages are major drivers of disease through the ingestion of lipoproteins, foam cell formation, and secretion of inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B cells can shape the course of disease by promoting or mitigating inflammatory responses. Leukocytes highly express the phosphoinositide 3-kinase isoform delta (PI3Kd), exerting a key role in the regulation of immune responses including activation, proliferation, differentiation, and effector functions. Therefore, PI3Kd represents a promising target for the modulation of inflammatory diseases. Consequently, we aimed to analyse the role of PI3Kd in leukocytes during atherogenesis. Methods and results To investigate the role of PI3Kd in atherosclerosis, bone marrow from PI3Kd−/− or PI3Kd+/+ mice was transplanted into LDLR−/− mice. After a 6-weeks-challenge by high fat diet, PI3Kd−/− recipient LDLR−/− mice displayed profoundly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T-cell differentiation, and proatherogenic CD4+ T-helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in LDLR−/− mice. Whereas plaque content and functions of macrophages including foam cell formation, efferocytosis, and cytokine secretion remained unaffected, haematopoietic PI3Kd ablation strongly reduced mature B cells and serum immunoglobulins in LDLR−/− mice. Importantly, PI3Kd deficiency severely impaired numbers and immunosuppressive functions of regulatory CD4+ T cells (Tregs) in spleen, para-aortic lymph nodes, and plaques of LDLR−/− mice. Consequently, adoptive transfer of PI3Kd+/+ Tregs fully constrained the plaque burden in PI3Kd−/− transplanted LDLR−/− mice without affecting B-cell numbers and serum immunoglobulins, whereas adoptively transferred PI3Kd−/− Tregs were unable to relieve atherosclerosis progression. Conclusions Here, we demonstrate that PI3Kd plays a crucial role in Tregs, Th1 cells, and B cells during atherogenesis. Lack of PI3Kd signalling specifically in atheroprotective Treg responses outplays its impact on proatherogenic Th1 and B-cell responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Marga and Walter Boll Foundation

Introduction Pulmonary hypertension (PH) is a pulmonary vascular disease that is associated with unacceptably high morbidity and mortality. PH is characterized by chronically increased pulmonary arterial pressure, increased pulmonary vascular resistance and right ventricular (RV) dysfunction and hypertrophy. Underlying mechanisms include increased proliferation and reduced apoptosis of both vascular smooth muscle cells (SMC) and endothelial cells (EC), as well as dysregulated immune responses. We have previously shown that class IA phosphatidylinositol-3-kinase (PI3K) isoforms, activated via receptor tyrosine kinases, are critically involved in the pathogenesis of PH. However, recent findings suggest that the class IB isoform PI3Kγ, which is activated downstream of G protein coupled receptors, is also important. It has been shown that PI3Kγ is involved in numerous processes that promote both vascular remodelling and maladaptive cardiac hypertrophy, including leukocyte recruitment, expression of proinflammatory chemokines and cytokines, as well as SMC and EC proliferation and survival. Therefore, the aim of our study was to investigate the role of PI3Kγ in the pathogenesis of PH. Methods The impact of PI3Kγ on the pathogenesis of PH was analysed in vivo using mice expressing a catalytically inactive form of PI3Kγ (PI3KγKD/KD) in the hypoxia-induced mouse model of PH. Mice were kept at 10%O2 (HOX) for 21 days or left under normoxic conditions (NOX). Subsequently, systolic right ventricular pressure (RVSP) was measured with a pressure catheter. RV hypertrophy was expressed as the ratio of RV weight to left ventricular + septum weight. Migration and proliferation of human pulmonary arterial SMC (hPASMC) as well as EC (hMVEC) were analysed using a PI3Kγ isoform-specific inhibitor (AS605240 [0.1; 0.3; 1μM]). Chemotaxis was determined by means of a modified Boyden chamber, and proliferation was quantified by a Bromodeoxyuridine (BrdU) incorporation assay. Results Whereas PI3Kγ inactivation had no effect on NOX animals, hypoxia led to increased RVSP and RV hypertrophy in WT animals (34.67±2.02 mmHg; 0.38±0.087) which were unexpectedly further increased in PI3KγKD/KD mice (37.67±1.3 mmHg, p=0.0104 vs. HOX WT; 0.47±0.06, p=0.0155 vs. HOX WT). Heart rate and systemic blood pressure remained unchanged. Inhibition of PI3Kγ by means of AS605240 did not affect proliferation of hPASMC and hMVEC, induced by multiple stimuli (FCS [10%], PDGF-BB [30ng/ml], or CXCL12 [100ng/ml], VEGF [50ng/ml]), respectively. However, FCS-induced migration of these cells was significantly reduced by AS605240 [0.3μM] (p Conclusion Contrary to our expectations, the results show that kinase inactivation of PI3Kγ was not able to attenuate the pathogenesis of PH, but surprisingly led to a significant increase without critically changing cellular responses of SMC and EC. Therefore, our results indicate an unexpected protective effect of PI3Kγ on PH. Funding Acknowledgement Type of funding source: None

Six-transmembrane protein of prostate (Stamp2) protects from diabetes and atherosclerosis in mice via anti-inflammatory mechanisms. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PAH), we investigated the role of Stamp2. Stamp2 expression was substantially reduced in the lung of humans with idiopathic PAH, as well as in experimental PAH. In Stamp2-deficient mice, hypoxia modestly aggravated pulmonary vascular remodeling and right ventricular pressure compared to WT. As endothelial cell (EC) and pulmonary arterial smooth muscle cell (PASMC) phenotypes drive remodeling in PAH, we explored the role of Stamp2. Knock-down of Stamp2 in human EC neither affected apoptosis, viability, nor release of IL-6. Moreover, Stamp2 deficiency in primary PASMC did not alter mitogenic or migratory properties. As Stamp2 deficiency augmented expression of inflammatory cytokines and numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages may drive vascular remodeling. Thus, PASMC responses were assessed following treatment with conditioned media of primary Stamp2−/− or WT macrophages. Stamp2−/− supernatants induced PASMC proliferation and migration stronger compared to WT. A cytokine array revealed CXCL12, MCP-1 and IL-6 as most relevant candidates. Experiments with neutralizing antibodies confirmed the role of these cytokines in driving Stamp2’s responses. In conclusion, Stamp2 deficiency aggravates pulmonary vascular remodeling via cross-talk between macrophages and PASMC. Despite a substantial pro-inflammatory response, the hemodynamic effect of Stamp2 deficiency is modest suggesting that additional mechanisms apart from inflammation are necessary to induce severe PAH. Electronic supplementary material The online version of this article (10.1007/s00395-020-00826-8) contains supplementary material, which is available to authorized users.

Rationale Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling in pulmonary arterioles. Increased activation of receptor tyrosine kinase (RTK)-mediated signaling pathways lead to increased proliferation and chemotaxis of pulmonary arterial smooth muscle cells (PASMCs). In previous studies, we identified phosphatidylinositol-3-kinase (PI3K) as a key enzyme for these processes. Since systemic PI3K inhibition is potentially associated with undesired side effects, we here for the first time tested the efficacy of the novel PI3K inhibitor CL27c to impede growth factor-mediated cellular effects in human PASMCs. CL27c is topically applicable and as a prodrug it is intracellularly activated and thus offers optimal efficacy in target cells while at the same time minimizing undesired effects in non-target cells. Methods Using Western blotting and immunohistochemistry, activation of the central PI3K effector AKT in human and murine lung tissue was investigated. Quantification of cell proliferation was performed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. PASMCs were incubated with CL27c [0.1; 0.3; 1; 3; 10μM] and stimulated either with PDGF-BB [30ng/ml] or with a growth factor combination (GFC) [PDGF-BB 30ng/ml, FGF 2ng/ml, EGF 0.5ng/ml, insulin 0.5μg/ml, FCS 5%]. Chemotaxis was quantified using modified Boyden chambers. The signal transduction cascade was investigated by Western blotting. Results Analysis of AKT activation revealed hyperphosphorylation in both the vascular wall and complete lung homogenates from PAH patients and hypoxia-treated mice. Examination of the mitogenic signal transduction revealed a markedly reduced phosphorylation of AKT and GSK3β in CL27c pretreated PASMCs. In addition, central cell cycle events, such as expression of cyclin D1, were reduced. This was in line with a CL27c concentration-dependent significant reduction of PDGF- and GFC-induced proliferation (control: 2.53±0.26 vs. CL27c: 0.73±0.14 and control: 5.27±0.61 vs. CL27c: 2.80±0.71 both at 10μM, p Conclusion These results indicate that CL27c reduces both proliferation and chemotaxis of PASMCs. As these processes play a key role in the development and progression of PAH, this approach is currently being investigated in preclinical models for its therapeutic potential. This “prodrug” approach could avoid non-specific effects and systemic side effects of Pan-PI3K inhibition, especially since the substance is suitable for topical application (inhalation). Thus, CL27c represents a promising treatment option.

Introduction and purpose Atherosclerosis is a chronic inflammatory disease of arteries and represents the main underlying cause of death worldwide. Macrophages are major drivers of atherosclerosis by ingestion of lipoproteins, foam cell formation, and secretion of pro-inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B lymphocytes can shape the course of disease by promoting or mitigating inflammatory responses. Leukocytes highly express the phosphoinositide 3-kinase isoform delta (PI3Kd), exerting a key role in the regulation of immune responses including the activation, proliferation, differentiation, and effector function of lymphocytes. Since macrophages and lymphocytes are all major effectors of atherosclerosis, we aimed to understand the role of PI3Kd in these leukocytes during atherogenesis. Methods and results To investigate the role of haematopoietic PI3Kd in atherosclerosis, bone marrow from PI3Kd−/− or PI3Kd+/+ mice was transplanted into LDLR−/− mice. After 6 weeks of feeding on an atherogenic diet, PI3Kd−/− recipient LDLR−/− mice displayed significantly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T cells, and proatherogenic CD4+ T helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in LDLR−/− mice. Moreover, haematopoietic PI3Kd deficiency augmented macrophage accumulation in atherosclerotic plaques of LDLR−/− mice, whereas major macrophage functions including foam cell formation, efferocytosis, and cytokine secretion were unaffected by PI3Kd inactivation in these phagocytes. However, haematopoietic PI3Kd deficiency led to depletion of atheroprotective B-1 cells and reduction of proatherogenic B-2 cells in LDLR−/− mice. Moreover, haematopoietic PI3Kd deficiency caused a significant reduction of regulatory CD4+ T cells (Tregs) in plaques, para-aortic lymph nodes, and spleen of LDLR−/− mice. Furthermore, PI3Kd−/− Tregs exhibited reduced secretion of anti-inflammatory cytokines IL-10 and TGF-b as well as impaired suppression of CD4+ T-cell proliferation. Consequently, adoptive transfer of PI3Kd+/+ Tregs fully constrains the atherosclerotic burden in PI3Kd−/− transplanted LDLR−/− mice without affecting B cell numbers. Conclusions We demonstrate that PI3Kd plays a crucial role in B lymphocytes, Th1 cells, and Tregs during atherogenesis. Lack of PI3Kd signalling in atheroprotective Treg responses outplays its impact on proatherogenic Th1 responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis progression. Acknowledgement/Funding Center for Molecular Medicine Cologne (CMMC) and the Marga and Walter Boll-Stiftung

Background The six-transmembrane protein of prostate (Stamp2) is a potent anti-inflammatory player in adipocytes and also in macrophages. Stamp2's actions in these cells protects from diet-induced diabetes and from atherosclerosis mice. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PH), we sought to investigate the role of Stamp2 in PH. Methods and results Morphometric analyses of small pulmonary arteries after 3 weeks of chronic hypoxia (10% O2) showed aggravated pulmonary vascular remodeling in Stamp2−/− mice as compared to WT, demonstrated by a significantly reduced number of non-muscularized vessels and higher extent of fully-muscularized vessels. Consequently, right ventricular systolic pressure (RVSP, Millar catheter via right jugular vein) was significantly higher in Stamp2−/− mice (33.4±0.7 mmHg vs. 30.3±1.4, p Importantly, Stamp2 expression (qPCR, western blot analyses) was significantly lower in the lung of humans with idiopathic PAH (IPAH), as well as in experimental PH in rats (monocrotalin, sugen/hypoxia) and in mice (hypoxia). Conclusions Stamp2 deficiency aggravates hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in mice. On the cellular level, actions of Stamp2 in macrophages drive vascular remodelling processes in smooth muscle cells via secreted factors such as CXCL12. The finding of decreased expression of Stamp2 in human and various experimental forms of PH points towards a general protective role of Stamp2.

Background The pathobiology of aortic aneurysms is characterized by vascular inflammation, extracellular matrix degeneration, and particularly by loss and dedifferentiation of vascular smooth muscle cells (SMCs). In SMCs, the PI 3-kinase isoform p110α mediates receptor tyrosine kinase dependent proliferation, chemotaxis, and survival. Smooth muscle specific p110α deficient mice (SM-p110α−/− mice) display reduced medial wall thickness, substantially reduced neointima formation and media hypertrophy after balloon injury of the carotid artery. Objective We hypothesized that loss of p110α signaling impairs vascular integrity and promotes development and progression of abdominal aortic aneurysms (AAA). We aimed to elucidate the impact of p110α deficiency on vascular integrity, SMC phenotypic modulation, vascular inflammation, and AAA formation. Methods and results Ultra-structural characterization of aortic wall morphology in abdominal aortas from SM-p110α−/− mice by transmission electron microscopy (TEM) revealed disarranged structure of tunica media as indicated by disorganized elastic fibers, detached SMCs, and elastic fiber breaks. Western blots showed reduced elastin and fibrillin expression in SMCs from p110α−/− mice. Media thickness was significantly reduced in abdominal aortas from SM-p110α−/− mice compared to wild type (WT) controls (29.0±3.1 vs. 42.5±4.1 μm). Lack of p110α decreased expression of differentiation markers SM-α-actin and SM-MHC. p110α deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine. These data indicate loss of differentiation and impaired contractility of p110α−/− SMCs. We subjected SM-p110α−/− mice and WT littermate controls to the porcine pancreatic elastase (PPE) model of AAA. PPE was infused into the infrarenal aorta, respectively, to induce AAA formation. Ultrasonic examination of abdominal aortas demonstrated an enlarged aortic diameter in PPE challenged mice. AAA formation was significantly (p Conclusion These data indicate that p110α signaling critically contributes to vascular integrity via maintaining SMC plasticity, elastic fiber homeostasis, and anti-inflammatory processes. Consequently, lack of proper p110α signaling promotes progression of AAA formation.

Independent of the underlying cause, pulmonary hypertension (PH) remains a devastating condition that is characterized by limited survival. Cumulating evidence indicates that in addition to a dysbalance of mediators regulating vascular tone and growth factors promoting vascular remodeling, failure to resolve inflammation and altered immune processes play a pivotal role in the development and progression of PH. Here, we highlight the role of key inflammatory pathways in the pathobiology of vascular remodeling and PH, and discuss potential therapeutic interventions that may halt disease progression or even reverse pulmonary vascular remodeling. Perivascular inflammation is present in all forms of PH, and inflammatory pathways involve numerous mediators and cell types including macrophages, neutrophils, T cells, dendritic cells, and mast cells. Dysfunctional bone morphogenic protein receptor 2 (BMPR2) signaling and dysregulated immunity enable the accumulation of macrophages and other inflammatory cells in obliterative vascular lesions. Regulatory T cells (Tregs) were shown to be of particular relevance in the control of inflammatory responses. Key cytokines/chemokines include interleukin-6, functioning via classic or trans-signaling, macrophage migratory inhibitory factor (MIF), but also other mediators such as neutrophil-derived myeloperoxidase. The expanding knowledge on this topic has resulted in multiple opportunities for sophisticated therapeutic interventions.

The 2015 European Guidelines on Diagnosis and Treatment of Pulmonary Hypertension (PH) are also valid for Germany. While the guidelines contain detailed recommendations regarding clinical aspects of pulmonary arterial hypertension (PAH) and other forms of PH, they contain only a relatively short paragraph on novel findings on the pathobiology, pathology, and genetics. However, these are of great importance for our understanding of this complex disease both from a clinical and scientific point of view, and they are essential for the development of novel treatment strategies. To this end, a number of current data are relevant, prompting a detailed commentary to the guidelines, and the consideration of new scientific data. In June 2016, a Consensus Conference organized by the PH working groups of the German Society of Cardiology (DGK), the German Society of Respiratory Medicine (DGP) and the German Society of Pediatric Cardiology (DGPK) was held in Cologne, Germany. This conference aimed to solve practical and controversial issues surrounding the implementation of the European Guidelines in Germany. To this end, a number of working groups was initiated, one of which was specifically dedicated to the pathobiology, pathology and genetics of PH. This article summarizes the results and recommendations of this working group.

Background Obesity is associated with cardiovascular complications, including pulmonary hypertension (PH). Reports suggest that peroxisome proliferator-activated receptor-γ (PPARγ) has direct action in preventing vascular remodelling in PH. Here we dissected the specific role of high-fat-diet (HFD)-induced obesity and vascular smooth muscle cell (VSMC)-PPARγ for remodelling of small pulmonary arteries. Methods Wild-type (WT) and VSMC-specific PPARγ-knockout (SmPparγ −/−) mice were fed a low-fat-diet (LFD, 10% kcal from fat) or HFD (60% kcal from fat) for 24 weeks. Mice were metabolically phenotyped (e.g. weight development, insulin/glucose tolerance) at the beginning, and after 12 and 24 weeks, respectively. At 24 weeks additionally pulmonary pressure, heart structure, pulmonary vascular muscularization together with gene and protein expression in heart and lung tissues were determined. Results HFD increased right ventricular systolic pressure (RVSP) to a similar extent in WT and SmPparγ −/− mice. HFD decreased glucose tolerance and insulin sensitivity in both WT and SmPparγ −/− mice. Importantly, the increase in RVSP correlated with the degree of insulin resistance. However, VSMC-PPARγ deficiency increased pulmonary vascular muscularization independently of the diet-induced rise in RVSP. This increase was associated with elevated expression of early growth response protein 1 in heart and osteopontin in lung tissue. Conclusions Here we demonstrate a correlation of insulin resistance and pulmonary pressure. Further, deficiency of PPARγ in VSMCs diet-independently leads to increased pulmonary vascular muscularization.

The European guidelines, which focus on clinical aspects of pulmonary hypertension (PH), provide only minimal information about the pathophysiological concepts of PH. Here, we review this topic in greater detail, focusing on specific aspects in the pathobiology, pathology and genetics, which include mechanisms of vascular inflammation, the role of transcription factors, ion channels/ion channel diseases, hypoxic pulmonary vasoconstriction, genetics/epigenetics, metabolic dysfunction, and the potential future role of histopathology of PH in the modern era of PH therapy. In addition to new insights in the pathobiology of this disease, this working group of the Cologne Consensus Conference also highlights novel concepts and potential new therapeutic targets to further improve the treatment options in PAH.

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo-/- than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo-/- mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.

Pulmonary arterial hypertension (PAH) is a fatal disease with limited therapeutic options. Pathophysiological changes comprise obliterative vascular remodelling of small pulmonary arteries, elevated mean pulmonary arterial systolic pressure (PASP) due to elevated resistance of pulmonary vasculature, adverse right ventricular remodelling, and heart failure. Recent findings also indicate a role of increased inflammation and insulin resistance underlying the development of PAH. We hypothesized that treatment of this condition with the peroxisome proliferator-activated receptor-γ (PPARγ) activator pioglitazone, known to regulate the expression of different genes addressing insulin resistance, inflammatory changes, and vascular remodelling, could be a beneficial approach. PAH was induced in adult rats by a single subcutaneous injection of monocrotaline (MCT). Pioglitazone was administered for 2 weeks starting 3 weeks after MCT-injection. At day 35, hemodynamics, organ weights, and -indices were measured. We performed morphological and molecular characterization of the pulmonary vasculature, including analysis of the degree of muscularization, proliferation rates, and medial wall thickness of the small pulmonary arteries. Furthermore, markers of cardiac injury, collagen content, and cardiomyocyte size were analyzed. Survival rates were monitored throughout the experimental period. Pioglitazone treatment improved survival, reduced PASP, muscularization of small pulmonary arteries, and medial wall thickness. Further, MCT-induced right ventricular hypertrophy and fibrosis were attenuated. This was accompanied with reduced cardiac expression of brain natriuretic peptide, as well as decreased cardiomyocyte size. Finally, pulmonary macrophage content and osteopontin gene expression were attenuated. Based on the beneficial impact of pioglitazone, activation of PPARγ might be a promising treatment option in PAH.

Objective— Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such as platelet-derived growth factor (PDGF) are closely involved in the pathogenesis of pulmonary arterial hypertension. We investigated the effects of 2 novel PDGF inhibitors, nilotinib/AMN107 (Abl kinases/PDGF receptor inhibitor) and dasatinib/BMS-354825 (Abl kinases/PDGF receptor/Src inhibitor), on the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and on the hemodynamics and pulmonary vascular remodeling in experimental pulmonary hypertension, and determined the expression and regulation of Src family kinases. Methods and Results— Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 μmol/L), nilotinib (0.3 μmol/L), and imatinib (1 μmol/L) potently inhibited PDGF-induced signal transducer and activator of transcription 3 and Akt phosphorylation. All 3 inhibitors decreased PDGF-induced proliferation, cell cycle gene regulation, and migration. In contrast, only dasatinib inhibited multiple growth factor–induced PASMC proliferation, and this was associated with the inhibition of Src phosphorylation. Combination of specific Src inhibitors (phosphoprotein phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor–induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per body weight) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per body weight) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. Conclusion— We suggest that dual inhibition of PDGF receptor and Src kinases potently inhibits mitogenic and motogenic responses to growth factors in PASMCs and pulmonary vascular remodeling in vivo so that dual inhibition may represent an alternative therapeutic approach for pulmonary arterial hypertension.

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.To investigate whether hypoxia alters PDGF β receptor (βPDGFR) signaling in the pulmonary vasculature.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.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.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.

Atherosclerosis and its consequences such as myocardial infarction and ischemic stroke remains the leading cause of death in western countries. Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, critically involving macrophages, dendritic cells and T lymphocytes. Leukocytes highly express the catalytic phosphoinositide 3-kinase isoform p110δ (PI3Kδ), exerting a key role in the regulation of immune responses including activation, differentiation and effector function of lymphocytes. Therefore, PI3Kδ represents a promising target for the modulation of inflammatory processes during atherogenesis. To investigate the role of leukocytic PI3Kδ during atherogenesis, lethally irradiated LDLR -/- mice were either transplanted with bone marrow from PI3Kδ -/- or PI3Kδ +/+ mice. After recovery, recipient mice were fed an atherogenic diet for 6 weeks. Hypercholesterolemic PI3Kδ -/- recipient LDLR -/- mice displayed a profound reduction of peripheral B and T cells as well as strongly impaired CD4 + T-cell activation, T-helper 1 response and regulatory T-cell numbers in paraaortic lymph nodes and spleen compared with PI3Kδ +/+ transplanted controls. Surprisingly, the profound impairment of the adaptive immune system by PI3Kδ-deficiency caused a considerable aggravation of atherosclerosis in LDLR -/- mice. Atherosclerotic lesion area at the aortic root and abdominal aorta of PI3Kδ -/- recipient LDLR -/- mice was significantly increased by 72% and 218% compared with PI3Kδ +/+ recipients, respectively (n = 10[[Unable to Display Character: –]]20; P < 0.001). Importantly, atherosclerotic lesions of PI3Kδ-deficient LDLR -/- mice were characterized by a lower fraction of CD4 + T cells and a higher proportion of MOMA-2 + monocytes/macrophages compared with controls despite unaltered circulating monocyte subsets. Thus, PI3Kδ-deficiency in mononuclear phagocytes may contribute to enhanced plaque growth. In summary, we demonstrate that hematopoietic PI3Kδ plays a crucial role in regulating innate and adaptive immune responses within the arterial wall by exerting protective functions during atherogenesis. Current studies aim to dissect PI3Kδ-dependent mechanisms that modulate inflammatory and regulatory processes in multiple stages of atherosclerosis.

Objective— Despite modern therapies, pulmonary arterial hypertension (PAH) harbors a high mortality. Vascular remodeling is a hallmark of the disease. Recent clinical studies revealed that antiremodeling approaches with tyrosine–kinase inhibitors such as imatinib are effective, but its applicability is limited by significant side effects. Although imatinib has multiple targets, expression analyses support a role for platelet-derived growth factor (PDGF) in the pathobiology of the disease. However, its precise role and downstream signaling events have not been established. Approach and Results— Patients with PAH exhibit enhanced expression and phosphorylation of β PDGF receptor (βPDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLCγ at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of βPDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated βPDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLCγ (βPDGFR F3/F3 ). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in βPDGFR F3/F3 pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in βPDGFR F3/F3 mice compared with wild-type littermates. These alterations led to protection from hypoxia-induced PAH and right ventricular hypertrophy. Conclusions— By means of a genetic approach, our data provide definite evidence that the activated βPDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLCγ activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.

Despite recent advances in the management of patients with pulmonary arterial hypertension (PAH), this disease remains a devastating condition with limited survival. While the current therapies primarily target the vasoconstrictor/vasodilator imbalance in the pulmonary circulation, there is currently no cure for PAH, and pulmonary vascular remodeling-representing the underlying cause of the disease-is only modestly affected. Hence, novel therapeutic approaches directly targeting the vascular remodeling process are warranted. Recent studies provided compelling evidence that peptide growth factors, which elicit their signals via receptor tyrosine kinases, are important contributors to the development and progression of PAH. In particular, platelet-derived growth factor (PDGF) is a strong mitogen for pulmonary vascular smooth muscle cells and protects these cells from apoptosis, thus representing an important mediator of pulmonary vascular remodeling. PDGF ligand and receptors are upregulated in PAH, and experimental studies have shown that inhibition of PDGF receptor signaling by pharmacological or genetic approaches prevents the development of PAH in animal models and is even able to reverse pulmonary vascular remodeling once it has been established. Consistently, results from phase II and phase III clinical trials indicate that the tyrosine kinase inhibitor imatinib mesylate, which potently inhibits the PDGF receptor, is effective in improving exercise capacity and pulmonary hemodynamics as add-on therapy in patients with severe PAH (i.e., pulmonary vascular resistance800 dynes s cm(-5)). Future studies will evaluate the long-term clinical efficacy and safety of imatinib, including patients with less impaired hemodynamics. Based on the current knowledge, targeting of PDGFR signaling is likely to become an anti-proliferative treatment option for patients with PAH and has the potential to at least partially correct the pathology of the disease.

Despite recent advances in the management of patients with pulmonary arterial hypertension (PAH), this disease remains a devastating condition with limited survival. While the current therapies primarily target the vasoconstrictor/vasodilator imbalance in the pulmonary circulation, there is currently no cure for PAH, and pulmonary vascular remodeling—representing the underlying cause of the disease—is only modestly affected. Hence, novel therapeutic approaches directly targeting the vascular remodeling process are warranted. Recent studies provided compelling evidence that peptide growth factors, which elicit their signals via receptor tyrosine kinases, are important contributors to the development and progression of PAH. In particular, platelet-derived growth factor (PDGF) is a strong mitogen for pulmonary vascular smooth muscle cells and protects these cells from apoptosis, thus representing an important mediator of pulmonary vascular remodeling. PDGF ligand and receptors are upregulated in PAH, and experimental studies have shown that inhibition of PDGF receptor signaling by pharmacological or genetic approaches prevents the development of PAH in animal models and is even able to reverse pulmonary vascular remodeling once it has been established. Consistently, results from phase II and phase III clinical trials indicate that the tyrosine kinase inhibitor imatinib mesylate, which potently inhibits the PDGF receptor, is effective in improving exercise capacity and pulmonary hemodynamics as add-on therapy in patients with severe PAH (i.e., pulmonary vascular resistance >800 dynes s cm−5). Future studies will evaluate the long-term clinical efficacy and safety of imatinib, including patients with less impaired hemodynamics. Based on the current knowledge, targeting of PDGFR signaling is likely to become an anti-proliferative treatment option for patients with PAH and has the potential to at least partially correct the pathology of the disease.