Anna Zakrzewicz

Fhl-1, a New Key Protein in Pulmonary Hypertension

Background— Pulmonary hypertension (PH) is a severe disease with a poor prognosis. Different forms of PH are characterized by pronounced vascular remodeling, resulting in increased vascular resistance and subsequent right heart failure. The molecular pathways triggering the remodeling process are poorly understood. We hypothesized that underlying key factors can be identified at the onset of the disease. Thus, we screened for alterations to protein expression in lung tissue at the onset of PH in a mouse model of hypoxia-induced PH. Methods and Results— Using 2-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight analysis, we identified 36 proteins that exhibited significantly altered expression after short-term hypoxic exposure. Among these, Fhl-1, which is known to be involved in muscle development, was one of the most prominently upregulated proteins. Further analysis by immunohistochemistry, Western blot, and laser-assisted microdissection followed by quantitative polymerase chain reaction confirmed the upregulation of Fhl-1, particularly in the pulmonary vasculature. Comparable upregulation was confirmed (1) after full establishment of hypoxia-induced PH, (2) in 2 rat models of PH (monocrotaline-treated and hypoxic rats treated with the vascular endothelial growth factor receptor antagonist SU5416), and (3) in lungs from patients with idiopathic pulmonary arterial hypertension. Furthermore, we demonstrated that regulation of Fhl-1 was hypoxia-inducible transcription factor dependent. Abrogation of Fhl-1 expression in primary human pulmonary artery smooth muscle cells by small-interfering RNA suppressed, whereas Fhl-1 overexpression increased, migration and proliferation. Coimmunoprecipitation experiments identified Talin1 as a new interacting partner of Fhl-1. Conclusions— Protein screening identified Fhl-1 as a novel protein regulated in various forms of PH, including idiopathic pulmonary arterial hypertension.

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.

Pivotal Advance: Up‐regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts

During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti‐inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half‐life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT‐PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double‐labeling with ED1‐antibody, directed to a CD68‐like antigen. ACh content was measured by HPLC. [Ca2+]i was monitored by Fura‐2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up‐regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1‐positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP‐induced increase in [Ca2+]i was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up‐regulate non‐neuronal ACh synthesis and develop auto‐/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.

Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1β Release

IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro–IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro–IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

Neuropeptide Y Is Expressed by Rat Mononuclear Blood Leukocytes and Strongly Down-Regulated during Inflammation

Neuropeptide Y (NPY), a classical sympathetic comediator, regulates immunological functions including T cell activation and migration of blood leukocytes. A NPY-mediated neuroimmune cross-talk is well conceivable in sympathetically innervated tissues. In denervated, e.g., transplanted organs, however, leukocyte function is not fundamentally disturbed. Thus, we hypothesized that NPY is expressed by blood leukocytes themselves and regulated during inflammation. NPY mRNA and peptide expression were analyzed in mononuclear leukocytes isolated from the blood vessels of healthy rat kidneys, as well as from the blood vessels of isogeneic and allogeneic renal grafts transplanted in the Dark Agouti to Lewis or in the Fischer 344 to Lewis rat strain combination. Depending on the donor strain, acute allograft rejection is either fatal or reversible but both experimental models are characterized by massive accumulation of intravascular leukocytes. Leukocytes, predominantly monocytes, isolated from the blood vessels of untreated kidneys and isografts expressed high amounts of NPY mRNA and peptide, similar to expression levels in sympathetic ganglia. During acute allograft rejection, leukocytic NPY expression drastically dropped to ∼1% of control levels in both rat strain combinations. In conclusion, NPY is an abundantly produced and tightly regulated cytokine of mononuclear blood leukocytes.

Protein Arginine Methyltransferases (PRMTs): Promising Targets for the Treatment of Pulmonary Disorders

Protein arginine methylation is a novel posttranslational modification that plays a pivotal role in a variety of intracellular events, such as signal transduction, protein-protein interaction and transcriptional regulation, either by the direct regulation of protein function or by metabolic products originating from protein arginine methylation that influence nitric oxide (NO)-dependent processes. A growing body of evidence suggests that both mechanisms are implicated in cardiovascular and pulmonary diseases. This review will present and discuss recent research on PRMTs and the methylation of non-histone proteins and its consequences for the pathogenesis of various lung disorders, including lung cancer, pulmonary fibrosis, pulmonary hypertension, chronic obstructive pulmonary disease and asthma. This article will also highlight novel directions for possible future investigations to evaluate the functional contribution of arginine methylation in lung homeostasis and disease.

Phosphocholine – an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors

We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions.

The interaction of enolase-1 with caveolae-associated proteins regulates its subcellular localization.

Cell-surface-associated proteolysis plays a crucial role in embryonic development, monocyte/macrophage recruitment and tumour cell invasion. The glycolytic enzyme ENO-1 (enolase-1) is translocated from the cytoplasm to the cell surface, where it binds PLG (plasminogen) to enhance pericellular plasmin production and cell motility. In the present study, ENO-1 was found to localize to a specialized subset of lipid rafts called caveolae as demonstrated by fluorescence confocal microscopy and sucrose gradient ultracentrifugation. Co-immunoprecipitation studies revealed that ENO-1 interacts with Cav-1 (caveolin-1), but not with Cav-2, via the CSD (Cav-scaffolding domain). Moreover, an evolutionarily conserved CBM (Cav-binding motif) F296DQDDWGAW304 was identified within ENO-1. The point mutation W301A within the ENO-1 CBM was, however, not sufficient to disrupt ENO-1-Cav-1 interaction, whereas the mutations F296A and W304A markedly affected ENO-1 protein expression. Furthermore, ENO-1 was found associated with Annx2 (annexin 2), representing another caveolar protein, and this interaction was dependent on Cav-1 expression. Knockdown of Cav-1 and Annx2 markedly decreased cell surface expression of ENO-1. ENO-1 overexpression increased cell migration and invasion in a Cav-1-dependent manner. Thus the differential association of ENO-1 with caveolar proteins regulates ENO-1 subcellular localization and, consequently, ENO-1-dependent cell migration and invasion.