Matthias Hecker

Dysregulated Bone Morphogenetic Protein Signaling in Monocrotaline-Induced Pulmonary Arterial Hypertension

Background—Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. Methods and Results—Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38MAPK phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. Conclusion—BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.

Continuous cytomegalovirus seroconversion in a large group of healthy blood donors.

Background and Objectives  Transmission of cytomegalovirus (CMV) to seronegative, immunocompromised recipients can cause serious and fatal complications. Although the seroprevalence of CMV is high, the risk of primary CMV infection among healthy blood donors has not yet been analysed in a large population.

A novel polymorphism in the 5′ promoter region of the human interleukin-4 receptor α-chain gene is associated with decreased soluble interleukin-4 receptor protein levels

Abstract. Interleukin (IL)-4 exerts its biological effects through binding to the IL-4 receptor (IL4R) complex, plays a central role in stimulating B-cell differentiation, and is crucial for the development of T helper 2 cells. Recently, a soluble form of the human IL4R α chain (sIL4Rα), which is produced by alternate mRNA splicing of exon 8, was discovered. sIL4R is thought to play an important role in either enhancing or inhibiting IL-4 signalling. We analyzed the 5′ promoter region of the human IL4R α-chain gene (IL4RA) of healthy volunteers by DNA sequencing and found three novel single-nucleotide polymorphisms (SNPs; T–890C, T–1914C, C–3223T) and one novel short tandem repeat [(CAAAA)5–7–3600]. The two common promoter region SNPs T–1914C and C–3223T as well as six known coding SNPs in the IL4RA gene were genotyped in healthy blood donors by PCR with sequence-specific primers; total sIL4R levels were measured by ELISA. Results revealed a highly significant association of the –3223T variant with lowered sIL4R levels (two-tailed t-test, P=0.0002). Results remained highly significant after Bonferroni adjustment for multiple comparisons (P=0.0017). Moreover, the C–3223T variant was found to be in strong linkage disequilibrium with the extracellular I50V variant (P<0.001), which was recently described to be associated with atopic asthma in a Japanese population. Since this novel IL4RA promoter region SNP is common (allele frequency 29.8%), we conclude that it may be of importance for the genetic regulation of the IL-4 signalling pathway.

SNAI transcription factors mediate epithelial–mesenchymal transition in lung fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of transforming growth factor β1 (TGFβ1)-induced epithelial–mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF. Methods: Using quantitative reverse transcription-PCR (RT-PCR), immunofluorescence, immunohistochemistry, western blotting, as well as gain- and loss-of-function studies and functional assays, the role of SNAI1 and SNAI2 in TGFβ1-induced EMT in ATII cells in vitro was assessed; and the expression of SNAI transcription factors was analysed in experimental and human IPF in vivo. Results: TGFβ1 treatment increased the expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT, and small interfering RNA (siRNA)-mediated SNAI depletion attenuated TGFβ1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human IPF and localised to hyperplastic ATII cells in vivo. Conclusions: The results demonstrate that TGFβ1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and human IPF in vivo suggests that SNAI-mediated EMT may contribute to the fibroblast pool in idiopathic pulmonary fibrosis.

Acute Lung Injury Is Reduced in fat-1 Mice Endogenously Synthesizing n-3 Fatty Acids

RATIONALE Acute lung injury (ALI) remains an important cause of mortality in intensive care units. Inflammation is controlled by cytokines and eicosanoids derived from the n-6 fatty acid (FA) arachidonic acid (AA). The n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and mediators derived from EPA and DHA possess reduced inflammatory potency. OBJECTIVES To determine whether the ability of fat-1 mice to endogenously convert n-6 to n-3 FA, and thus generate an increased ratio of n-3 to n-6 FA, impacts experimental ALI. METHODS We investigated ALI induced by intratracheal instillation of endotoxin in fat-1 and wild-type (WT) mice, assessing leukocyte numbers, protein concentration, and prostaglandin and cytokine levels in bronchoalveolar lavage fluid, as well as free FA in plasma, and lung ventilator compliance. Body temperature and motor activity of mice--markers of sickness behavior--were also recorded. MEASUREMENTS AND MAIN RESULTS In ALI, fat-1 mice exhibited significantly reduced leukocyte invasion, protein leakage, and macrophage inflammatory protein-2 and thromboxane B(2) levels in lavage fluid compared with WT mice. Free AA levels were increased in the plasma of WT mice in response to endotoxin, whereas EPA and DHA were increased in the fat-1 group. Ventilator compliance was significantly improved in fat-1 mice. Body temperature and motor activity were decreased in ALI. fat-1 Mice recovered body temperature and motor activity faster. CONCLUSIONS fat-1 Mice exhibited reduced features of ALI and sickness behavior. Increasing the availability of n-3 FA may thus be beneficial in critically ill patients with ALI.

Receptor for activated C-kinase 1, a novel interaction partner of type II bone morphogenetic protein receptor, regulates smooth muscle cell proliferation in pulmonary arterial hypertension

Background— Pulmonary arterial hypertension (PAH) is characterized by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the narrowing of pulmonary arterioles secondary to endothelial cell dysfunction and smooth muscle cell proliferation. Heterozygous mutations in BMPR2, encoding the type II bone morphogenetic protein receptor (BMPRII), were identified in PAH, suggesting that alterations to BMPRII function are involved in disease onset and/or progression. Methods and Results— We identified the receptor for activated C-kinase (RACK1) as a novel interaction partner of BMPRII by yeast 2-hybrid analyses using the kinase domain of BMPRII as a bait. Glutathione-S-transferase pull-down and coimmunoprecipitation confirmed the interaction of RACK1 with BMPRII in vitro and in vivo. RACK1–BMPRII interaction was reduced when kinase domain mutations occurring in patients with PAH were introduced to BMPRII. Immunohistochemistry of lung sections from PAH and control patients and immunofluorescence analysis of primary pulmonary arterial smooth muscle cells demonstrated colocalization of BMPRII and RACK1 in vivo. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed significant downregulation of RACK1 expression in the rat model of monocrotaline-induced PAH but not in pulmonary arterial smooth muscle cells from PAH patients. Abrogation of RACK1 expression in pulmonary arterial smooth muscle cells led to decreased Smad1 phosphorylation and increased proliferation, whereas overexpression of RACK1 led to increased Smad1 phosphorylation and decreased proliferation. Conclusions— RACK1, a novel interaction partner of BMPRII, constitutes a new negative regulator of pulmonary arterial smooth muscle cell proliferation, suggesting a potential role for RACK1 in the pathogenesis of PAH.

Dysregulation of the IL-13 receptor system: a novel pathomechanism in pulmonary arterial hypertension.

RATIONALE Idiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (paSMC) hyperplasia. Inflammation is proposed to play a role in vessel remodeling associated with IPAH. IL-13 is emerging as a regulator of tissue remodeling; however, the contribution of the IL-13 system to IPAH has not been assessed. OBJECTIVES The objective of this study was to assess the possible contribution of the IL-13 system to IPAH. METHODS Expression and localization of IL-13, and IL-13 receptors IL-4R, IL-13Rα1, and IL-13Rα2 were assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry, and flow cytometry in lung tissue, paSMC, and microdissected vascular lesions from patients with IPAH, and in lung tissue from rodents with hypoxia- or monocrotaline-induced pulmonary hypertension. A whole-genome microarray analysis was used to study IL-13-regulated genes in paSMC. MEASUREMENTS AND MAIN RESULTS Pulmonary expression of the IL-13 decoy receptor IL-13Rα2 was up-regulated relative to that of the IL-13 signaling receptors IL-4R and IL-13Rα1 in patients with IPAH and in two animal models of IPAH. IL-13, signaling via STAT3 and STAT6, suppressed proliferation of paSMC by promoting G(0)/G(1) arrest. Whole-genome microarrays revealed that IL-13 suppressed endothelin-1 production by paSMC, suggesting that IL-13 controlled paSMC growth by regulating endothelin production. Ectopic expression of the il13ra2 gene resulted in partial loss of paSMC growth control by IL-13 and blunted IL-13 suppression of endothelin-1 production by paSMC, whereas small-interfering RNA knockdown of il13ra2 gene expression had the opposite effects. CONCLUSIONS The IL-13 system is a novel regulator of paSMC growth. Dysregulation of IL-13 receptor expression in IPAH may partially underlie smooth muscle hypertrophy associated with pathological vascular remodeling in IPAH.

The transforming growth factor-β/Smad2,3 signalling axis is impaired in experimental pulmonary hypertension

Mutations in genes encoding members of the transforming growth factor (TGF)-β superfamily have been identified in idiopathic forms of pulmonary arterial hypertension (PAH). The current study examined whether perturbations to the TGF-β/Smad2,3 signalling axis occurred in a monocrotaline (MCT) rodent model of experimental PAH. Expression of the TGF-β signalling machinery was assessed in the lungs and kidneys of MCT-treated rodents with severe PAH by semi-quantitative reverse-transcription (RT)-PCR, real-time RT-PCR and immunoblotting. TGF-β signalling was assessed in the lungs and in pulmonary artery smooth muscle cells (PASMC) from MCT-treated rodents by Smad2 phosphorylation, expression of the connective tissue growth factor gene, activation of the serpine promoter in a luciferase reporter system and by the induction of apoptosis. The expression of type1 TGF-β receptor (TGFBR) activin-A receptor-like kinase1, TGFBR-2, TGFBR-3 (endoglin), Smad3 and Smad4; as well as TGF-β signalling and TGF-β-induced apoptosis, were dramatically reduced in the lungs and PASMC, but not the kidneys, of MCT-treated rodents that developed severe PAH. The current data indicate that the transforming growth factor-β/Smad2,3 signalling axis is functionally impaired in monocrotaline-treated rodents with severe pulmonary arterial hypertension, underscoring the potential importance of transforming growth factor-β/Smad2,3 signalling in the onset or development of pulmonary arterial hypertension.

Mitochondrial Hyperpolarization in Pulmonary Vascular Remodeling. Mitochondrial Uncoupling Protein Deficiency as Disease Model

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Peroxisome proliferator-activated receptor-α reduces inflammation and vascular leakage in a murine model of acute lung injury

Acute lung injury (ALI) still represents a major cause of morbidity and mortality in intensive care units. Beneficial effects have been described after activation of the peroxisome proliferator-activated receptor (PPAR)-α by fibrates such as WY 14,643 (WY) in inflammatory models. In the present study, the impact of WY was investigated in a model of endotoxin (lipopolysaccharide; LPS)-induced ALI in mice. Intratracheal LPS challenge dose-dependently resulted in leukocyte invasion, protein leakage and release of tumour necrosis factor-α as well as macrophage inflammatory protein-2, prostaglandin E2 and thromboxane B2 into the alveolar space after 8 and 24 h. Lung ventilator compliance was reduced at both time-points. In isolated perfused mouse lungs, platelet-activating factor (PAF) induced an acute increase in pulmonary artery pressure (Ppa) and in capillary filtration coefficient (Kfc). WY significantly improved all features of ALI in vivo and blunted the increase in Kfc in isolated perfused mice lungs. In mice with genetic deletion of PPAR-α, all characteristics of ALI, Ppa, and Kfc were not significantly different from wild-type mice but WY failed to improve ALI and PAF-induced increase in Kfc. Activation of peroxisome proliferator-activated receptor-α by WY 14,643 reduced acute lung injury and vascular leakage. Fibrates may possess beneficial effects in acute pulmonary diseases beyond their lipid-lowering capability.