Eva Berghausen

Role of Src Tyrosine Kinases in Experimental Pulmonary Hypertension

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 &mgr;mol/L), nilotinib (0.3 &mgr;mol/L), and imatinib (1 &mgr;mol/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.

Imatinib mesylate for the treatment of pulmonary arterial hypertension.

Introduction: Despite recent advances, pulmonary arterial hypertension (PAH) remains a devastating disease which harbors a poor prognosis. Novel therapeutic approaches directly targeting pulmonary vascular remodeling are warranted. Areas covered: This review delineates the current limitations in the management of PAH and focuses on a novel, anti-proliferative therapeutic concept. It will help readers understand the mechanisms of receptor tyrosine kinase signaling, with a special focus on platelet-derived growth factor (PDGF) receptors and their role in the pathobiology of PAH. Furthermore, it provides a comprehensive summary regarding the rationale, efficacy and safety of the tyrosine kinase inhibitor imatinib mesylate, which potently inhibits the PDGF receptor, as an additional treatment option in PAH. Expert opinion: PDGF is a potent mitogen for pulmonary vascular smooth muscle cells and represents an important mediator of pulmonary vascular remodeling. Imatinib mesylate, a compound that inhibits the Bcr-Abl kinase and was developed for the treatment of chronic myeloid leukemia, also targets PDGF receptors. Both experimental and clinical data indicate that it reverses the vascular remodeling process even when it is fully established. Results from Phase II and III clinical trials suggest potent and prolonged efficacy in patients with severe PAH (i.e., pulmonary vascular resistance > 800 dynes*s*cm-5). Future studies should evaluate the long-term clinical efficacy and safety of imatinib, including patients with less impaired hemodynamics. Based on the current knowledge, this compound is likely to become an additional treatment option for patients with PAH and has the potential to at least partially correct the pathology of the disease.

Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

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.

Genetic Ablation of PDGF-Dependent Signaling Pathways Abolishes Vascular Remodeling and Experimental Pulmonary Hypertension

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 &bgr; PDGF receptor (&bgr;PDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLC&ggr; at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of &bgr;PDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated &bgr;PDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLC&ggr; (&bgr;PDGFRF3/F3). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in &bgr;PDGFRF3/F3 pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in &bgr;PDGFRF3/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 &bgr;PDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLC&ggr; activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.

Targeting of Platelet-Derived Growth Factor Signaling in Pulmonary Arterial Hypertension

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.

Class IA Phosphatidylinositol 3-Kinase Isoform p110α Mediates Vascular Remodeling

Objective—Neointima formation after vascular injury remains a significant problem in clinical cardiology, and current preventive strategies are suboptimal. Phosphatidylinositol 3′-kinase is a central downstream mediator of growth factor signaling, but the role of phosphatidylinositol 3′-kinase isoforms in vascular remodeling remains elusive. We sought to systematically characterize the precise role of catalytic class IA phosphatidylinositol 3′-kinase isoforms (p110&agr;, p110&bgr;, p110&dgr;), which signal downstream of receptor tyrosine kinases, for vascular remodeling in vivo. Approach and Results—Western blot analyses revealed that all 3 isoforms are abundantly expressed in smooth muscle cells. To analyze their significance for receptor tyrosine kinases–dependent cellular responses, we used targeted gene knockdown and isoform-specific small molecule inhibitors of p110&agr; (PIK-75), p110&bgr; (TGX-221), and p110&dgr; (IC-87114), respectively. We identified p110&agr; to be crucial for receptor tyrosine kinases signaling, thus affecting proliferation, migration, and survival of rat, murine, and human smooth muscle cells, whereas p110&bgr; and p110&dgr; activities were dispensable. Surprisingly, p110&dgr; exerted noncatalytic functions in smooth muscle cell proliferation, but had no effect on migration. Based on these results, we generated a mouse model of smooth muscle cell–specific p110&agr; deficiency (sm-p110&agr;−/−). Targeted deletion of p110&agr; in sm-p110&agr;−/− mice blunted growth factor–induced cellular responses and abolished neointima formation after balloon injury of the carotid artery in mice. In contrast, p110&dgr; deficiency did not affect vascular remodeling in vivo. Conclusions—Receptor tyrosine kinases–induced phosphatidylinositol 3′-kinase signaling via the p110&agr; isoform plays a central role for vascular remodeling in vivo. Thus, p110&agr; represents a selective target for the prevention of neointima formation after vascular injury, whereas p110&bgr; and p110&dgr; expression and activity do not play a significant role.

Myeloperoxidase aggravates pulmonary arterial hypertension by activation of vascular Rho-kinase

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.

Pathobiology, pathology and genetics of pulmonary hypertension: Update from the Cologne Consensus Conference 2018

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.

The p110alpha subunit of PI 3-kinase is crucially involved in neointima formation by mediating smooth muscle cell proliferation, migration and survival

The p110alpha subunit of PI 3-kinase is crucially involved in neointima formation by mediating smooth muscle cell proliferation, migration and survivalRadiation exposure during electrophysiology procedures : results from the EPIC global survey