Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs.

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.

Prostaglandin E2 in the Tumor Microenvironment, a Convoluted Affair Mediated by EP Receptors 2 and 4.

The involvement of the prostaglandin E2 (PGE2) system in cancer progression has long been recognized. PGE2 functions as an autocrine and paracrine signaling molecule with pleiotropic effects in the human body. High levels of intratumoral PGE2 and overexpression of the key metabolic enzymes of PGE2 have been observed and suggested to contribute to tumor progression. This has been claimed for different types of solid tumors, including, but not limited to, lung, breast, and colon cancer. PGE2 has direct effects on tumor cells and angiogenesis that are known to promote tumor development. However, one of the main mechanisms behind PGE2 driving cancerogenesis is currently thought to be anchored in suppressed antitumor immunity, thus providing possible therapeutic targets to be used in cancer immunotherapies. EP2 and EP4, two receptors for PGE2, are emerging as being the most relevant for this purpose. This review aims to summarize the known roles of PGE2 in the immune system and its functions within the tumor microenvironment. SIGNIFICANCE STATEMENT: Prostaglandin E2 (PGE2) has long been known to be a signaling molecule in cancer. Its presence in tumors has been repeatedly associated with disease progression. Elucidation of its effects on immunological components of the tumor microenvironment has highlighted the potential of PGE2 receptor antagonists in cancer treatment, particularly in combination with immune checkpoint inhibitor therapeutics. Adjuvant treatment could increase the response rates and the efficacy of immune-based therapies.

Lung Fibrosis Is Linked to Increased Endothelial Cell Activation and Dysfunctional Vascular Barrier Integrity.

Pulmonary fibrosis (PF) can be a fatal disease characterized by progressive lung scarring. It is still poorly understood how the pulmonary endothelium is involved in the disease pathogenesis. Differences of the pulmonary vasculature between patients and donors were analyzed using transmission electron microscopy, immunohistochemistry, and single-cell RNA sequencing. Vascular barrier resistance, endothelial-immune cell adhesion, and sensitivity to an inflammatory milieu were studied in vitro. Integrity and activation markers were measured by ELISA in human plasma. Transmission electron microscopy demonstrated abnormally swollen endothelial cells (ECs) in fibrotic lungs compared with donors. A more intense CD31 and von Willebrand Factor (vWF) and patchy vascular endothelial (VE)-Cadherin staining in fibrotic lungs supported the presence of a dysregulated endothelium. Integrity markers CD31, VE-Cadherin, Thrombomodulin, and VEGFR-2 (vascular endothelial growth factor receptor-2) and activation marker vWF gene expression was increased in different endothelial subpopulations (e.g., arterial, venous, general capillary, aerocytes) in PF. This was associated with a heightened sensitivity of fibrotic ECs to TNF-α or IFN-γ and elevated immune cell adhesion. The barrier strength was overall reduced in ECs from fibrotic lungs. vWF and IL-8 were increased in the plasma of patients, whereas VE-Cadherin, Thrombomodulin, and VEGFR-2 were decreased. VE-Cadherin staining was also patchy in biopsy tissue and was decreased in plasma samples of patients with PF 6 months after the initial diagnosis. Our data demonstrate highly abnormal ECs in PF. The vascular compartment is characterized by hyperactivation and increased immune cell adhesion, as well as dysfunctional endothelial barrier function. Reestablishing EC homeostasis and function might represent a new therapeutic option for fibrotic lung diseases.

Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction.

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal noncollageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and proinflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to ß1-integrin subunit clustering and Rho/ROCK activation. Blockage of the ß1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. Although pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin-induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension.NEW & NOTEWORTHY This study is the first to show that pentastatin, the matrikine of the basement membrane (BM) collagen IVα5 polypeptide, triggers rapid pulmonary arterial endothelial cell barrier disruption, activation, and apoptosis in vitro and ex vivo. Mechanistically, pentastatin partially acts through binding to the ß1-integrin subunit and the Rho/ROCK pathway. These findings are the first to link pentastatin to pulmonary endothelial dysfunction and, thus, suggest a major role for BM-matrikines in pulmonary vascular diseases such as pulmonary hypertension.

Supplemental fibrinogen restores thrombus formation in cardiopulmonary bypass-induced platelet dysfunction ex vivo.

BACKGROUND: Major cardiac surgery related blood loss is associated with increased postoperative morbidity and mortality. Platelet dysfunction is believed to contribute to post-cardiopulmonary bypass (CPB)-induced microvascular bleeding. We hypothesised that moderately hypothermic CPB induces platelet dysfunction and that supplemental fibrinogen can restore in vitro thrombus formation. METHODS: Blood from 18 patients, undergoing first-time elective isolated aortic valve surgery was drawn before CPB, 30 min after initiation of CPB, and after CPB and protamine administration, respectively. Platelet aggregation was quantified by optical aggregometry, platelet activation by flow-cytometric detection of platelet surface expression of P-selectin, annexin V, and activated glycoprotein IIb/IIIa, thrombus formation under flow and effect of supplemental fibrinogen (4 mg ml-1) on in vitro thrombogenesis. RESULTS: Post-CPB adenosine-diphosphate and TRAP-6-induced aggregation decreased by 40% and 10% of pre-CPB levels, respectively (P<0.0001). Although CPB did not change glycoprotein IIb/IIIa receptor expression, it increased the percentage of unstimulated P-selectin (1.2% vs 7%, P<0.01) positive cells and annexin V mean fluorescence intensity (15.5 vs 17.2, P<0.05), but decreased percentage of stimulated P-selectin (52% vs 26%, P<0.01) positive cells and annexin V mean fluorescence intensity (508 vs 325, P<0.05). Thrombus area decreased from 6820 before CPB to 5230 after CPB (P<0.05, arbitrary units [a.u.]). Supplemental fibrinogen increased thrombus formation to 20 324 and 11 367 a.u. before CPB and after CPB, respectively (P<0.001), thereby restoring post-CPB thrombus area to levels comparable with or higher than pre-CPB baseline. CONCLUSIONS: Single valve surgery using moderately hypothermic CPB induces partial platelet dysfunction. Thrombus formation was restored in an experimental study design by ex vivo supplementation of fibrinogen.

Impairment of the NKT-STAT1-CXCL9 Axis Contributes to Vessel Fibrosis in Pulmonary Hypertension Caused by Lung Fibrosis.

Rationale: Pulmonary hypertension (PH) is a common, severe comorbidity in interstitial lung diseases such as pulmonary fibrosis (PF), and it has limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. Objectives: To identify a novel functional link between natural killer T (NKT) cell activation and vascular fibrosis in PF-PH. Methods: Multicolor flow cytometry, secretome, and immunohistological analyses were complemented by pharmacological NKT cell activation in vivo, in vitro, and ex vivo. Measurements and Main Results: In pulmonary vessels of patients with PF-PH, increased collagen deposition was linked to a local NKT cell deficiency and decreased IL-15 concentrations. In a mouse model of PH caused by lung fibrosis, pharmacological NKT cell activation using a synthetic α-galactosylceramide analog (KRN7000) restored local NKT cell numbers and ameliorated vascular remodeling and right ventricular systolic pressure. Supplementation with activated NKT cells reduced collagen deposition in isolated human pulmonary arterial smooth muscle cells (hPASMCs) and in ex vivo precision-cut lung slices of patients with end-stage PF-PH. Coculture with activated NKT cells induced STAT1 signaling in hPASMCs. Secretome analysis of peripheral blood mononuclear cells identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMCs via the chemokine receptor CXCR3. Conclusions: Our results indicate that the absence of NKT cells impairs the STAT1-CXCL9-CXCR3 axis in PF-PH and that restoration of this axis by NKT cell activation may unravel a novel therapeutic strategy to target vascular fibrosis in interstitial lung disease.

International Union of Basic and Clinical Pharmacology. CIX. Differences and Similarities between Human and Rodent Prostaglandin E2 Receptors (EP1-4) and Prostacyclin Receptor (IP): Specific Roles in Pathophysiologic Conditions.

Prostaglandins are derived from arachidonic acid metabolism through cyclooxygenase activities. Among prostaglandins (PGs), prostacyclin (PGI2) and PGE2 are strongly involved in the regulation of homeostasis and main physiologic functions. In addition, the synthesis of these two prostaglandins is significantly increased during inflammation. PGI2 and PGE2 exert their biologic actions by binding to their respective receptors, namely prostacyclin receptor (IP) and prostaglandin E2 receptor (EP) 1-4, which belong to the family of G-protein-coupled receptors. IP and EP1-4 receptors are widely distributed in the body and thus play various physiologic and pathophysiologic roles. In this review, we discuss the recent advances in studies using pharmacological approaches, genetically modified animals, and genome-wide association studies regarding the roles of IP and EP1-4 receptors in the immune, cardiovascular, nervous, gastrointestinal, respiratory, genitourinary, and musculoskeletal systems. In particular, we highlight similarities and differences between human and rodents in terms of the specific roles of IP and EP1-4 receptors and their downstream signaling pathways, functions, and activities for each biologic system. We also highlight the potential novel therapeutic benefit of targeting IP and EP1-4 receptors in several diseases based on the scientific advances, animal models, and human studies. SIGNIFICANCE STATEMENT: In this review, we present an update of the pathophysiologic role of the prostacyclin receptor, prostaglandin E2 receptor (EP) 1, EP2, EP3, and EP4 receptors when activated by the two main prostaglandins, namely prostacyclin and prostaglandin E2, produced during inflammatory conditions in human and rodents. In addition, this comparison of the published results in each tissue and/or pathology should facilitate the choice of the most appropriate model for the future studies.

Pirfenidone exacerbates Th2-driven vasculopathy in a mouse model of systemic sclerosis-associated interstitial lung disease.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterised by severe vasculopathy and fibrosis of various organs including the lung. Targeted treatment options for SSc-associated interstitial lung disease (SSc-ILD) are scarce. We assessed the effects of pirfenidone in a mouse model of SSc-ILD. METHODS: Pulmonary function, inflammation and collagen deposition in response to pirfenidone were assessed in Fra-2-overexpressing transgenic (Fra-2 TG) and bleomycin-treated mice. In Fra-2 TG mice, lung transcriptome was analysed after pirfenidone treatment. In vitro, pirfenidone effects on human eosinophil and endothelial cell function were analysed using flow cytometry-based assays and electric cell-substrate impedance measurements, respectively. RESULTS: Pirfenidone treatment attenuated pulmonary remodelling in the bleomycin model, but aggravated pulmonary inflammation, fibrosis and vascular remodelling in Fra-2 TG mice. Pirfenidone increased interleukin (IL)-4 levels and eosinophil numbers in lung tissue of Fra-2 TG mice without directly affecting eosinophil activation and migration in vitro. A pronounced immune response with high levels of cytokines/chemokines and disturbed endothelial integrity with low vascular endothelial (VE)-cadherin levels was observed in pirfenidone-treated Fra-2 TG mice. In contrast, eosinophil and VE-cadherin levels were unchanged in bleomycin-treated mice and not influenced by pirfenidone. In vitro, pirfenidone exacerbated the IL-4 induced reduction of endothelial barrier resistance, leading to higher leukocyte transmigration. CONCLUSION: This study shows that antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high T-helper type 2 inflammation in a model of SSc-ILD. Careful ILD patient phenotyping may be required to exploit benefits of pirfenidone while avoiding therapy failure and additional lung damage in some patients.

miR-1183 Is a Key Marker of Remodeling upon Stretch and Tachycardia in Human Myocardium.

Many cardiac insults causing atrial remodeling are linked to either stretch or tachycardia, but a comparative characterization of their effects on early remodeling events in human myocardium is lacking. Here, we applied isometric stretch or sustained tachycardia at 2.5 Hz in human atrial trabeculae for 6 h followed by microarray gene expression profiling. Among largely independent expression patterns, we found a small common fraction with the microRNA miR-1183 as the highest up-regulated transcript (up to 4-fold). Both, acute stretch and tachycardia induced down-regulation of the predicted miR-1183 target genes ADAM20 and PLA2G7. Furthermore, miR-1183 was also significantly up-regulated in chronically remodeled atrial samples from patients with persistent atrial fibrillation (3-fold up-regulation versus sinus rhythm samples), and in ventricular myocardium from dilative cardiomyopathy hearts (2-fold up-regulation) as compared to non-failing controls. In sum, although stretch and tachycardia show distinct transcriptomic signatures in human atrial myocardium, both cardiac insults consistently regulate the expression of miR-1183 and its downstream targets in acute and chronic remodeling. Thus, elevated expression of miR-1183 might serve as a tissue biomarker for atrial remodeling and might be of potential functional significance in cardiac disease.

Glucagon-Like Peptide-1 Receptor Agonism Improves Nephrotoxic Serum Nephritis by Inhibiting T-Cell Proliferation.

Glucagon-like peptide (GLP)-1 analogs such as liraglutide improved albuminuria in patients with type 2 diabetes in large randomized controlled trials. One of the suspected mechanisms is the anti-inflammatory potential of GLP-1 receptor (Glp1r) agonism. Thus, the anti-inflammatory action of Glp1r agonism was tested in a nondiabetic, T-cell-mediated murine model of nephrotoxic serum nephritis (NTS). The role of Glp1r in NTS was evaluated by using Glp1r-/- mice or C57BL/6 mice treated with liraglutide. In vitro, murine T cells were stimulated in the presence of liraglutide or vehicle. Glp1r-/- mice displayed increased renal infiltration of neutrophils and T cells after induction of NTS. Splenocyte proliferation and TH1 cytokine transcription were increased in spleen and lymph nodes of Glp1r-/- mice. Liraglutide treatment significantly improved the renal outcome of NTS in C57BL/6 mice by decreasing renal infiltration and proliferation of T cells, which resulted in decreased macrophage infiltration. In vitro, T cells stimulated in the presence of liraglutide showed decreased proliferation of TH1 and TH17 cells. Liraglutide blocked glycolysis in T cells and decreased their Glut1 mRNA expression. Together, Glp1r agonism protects mice from a T-cell-dependent glomerulonephritis model by inhibition of T-cell proliferation, possibly by interacting with their metabolic program. This mechanism may explain in part the renoprotective effects of Glp1r agonism in diabetic nephropathy.

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-ß and microRNA 218. OBJECTIVE: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. METHODS: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. RESULTS: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro. CONCLUSIONS: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.

Monocytes and Macrophages Serve as Potent Prostaglandin D2 Sources during Acute, Non-Allergic Pulmonary Inflammation.

Acute respiratory inflammation, most commonly resulting from bacterial or viral infection, is one of the leading causes of death and disability worldwide. The inflammatory lipid mediator prostaglandin D2 (PGD2) and its rate-limiting enzyme, hematopoietic PGD synthase (hPGDS), are well-known drivers of allergic pulmonary inflammation. Here, we sought to investigate the source and role of hPGDS-derived PGD2 in acute pulmonary inflammation. Murine bronchoalveolar monocytes/macrophages from LPS- but not OVA-induced lung inflammation released significant amounts of PGD2. Accordingly, human monocyte-derived macrophages expressed high basal levels of hPGDS and released significant levels of PGD2 after LPS/IFN-γ, but not IL-4 stimulation. Human peripheral blood monocytes secreted significantly more PGD2 than monocyte-derived macrophages. Using human precision-cut lung slices (PCLS), we observed that LPS/IFN-γ but not IL-4/IL-13 drive PGD2 production in the lung. HPGDS inhibition prevented LPS-induced PGD2 release by human monocyte-derived macrophages and PCLS. As a result of hPGDS inhibition, less TNF-α, IL-6 and IL-10 could be determined in PCLS-conditioned medium. Collectively, this dataset reflects the time-dependent release of PGD2 by human phagocytes, highlights the importance of monocytes and macrophages as PGD2 sources and suggests that hPGDS inhibition might be a potential therapeutic option for acute, non-allergic lung inflammation.

SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes.

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate-SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate-SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

The role of stretch, tachycardia and sodium-calcium exchanger in induction of early cardiac remodelling.

Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation-transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non-myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate-dependent fashion. Mechanistically, several calcium-handling proteins, including the sodium-calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX-dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX.

HDL-related biomarkers are robust predictors of survival in patients with chronic liver failure.

BACKGROUND & AIMS: High-density lipoprotein cholesterol (HDL-C) levels are reduced in patients with chronic liver disease and inversely correlate with disease severity. During acute conditions such as sepsis, HDL-C levels decrease rapidly and HDL particles undergo profound changes in their composition and function. We aimed to determine whether indices of HDL quantity and quality associate with progression and survival in patients with advanced liver disease. METHODS: HDL-related biomarkers were studied in 508 patients with compensated or decompensated cirrhosis (including acute-on-chronic liver failure [ACLF]) and 40 age- and gender-matched controls. Specifically, we studied levels of HDL-C, its subclasses HDL2-C and HDL3-C, and apolipoprotein A1 (apoA-I), as well as HDL cholesterol efflux capacity as a metric of HDL functionality. RESULTS: Baseline levels of HDL-C and apoA-I were significantly lower in patients with stable cirrhosis compared to controls and were further decreased in patients with acute decompensation (AD) and ACLF. In stable cirrhosis (n = 228), both HDL-C and apoA-I predicted the development of liver-related complications independently of model for end-stage liver disease (MELD) score. In patients with AD, with or without ACLF (n = 280), both HDL-C and apoA-I were MELD-independent predictors of 90-day mortality. On ROC analysis, both HDL-C and apoA-I had high diagnostic accuracy for 90-day mortality in patients with AD (AUROCs of 0.79 and 0.80, respectively, similar to that of MELD 0.81). On Kaplan-Meier analysis, HDL-C <17 mg/dl and apoA-I <50 mg/dl indicated poor short-term survival. The prognostic accuracy of HDL-C was validated in a large external validation cohort of 985 patients with portal hypertension due to advanced chronic liver disease (AUROCs HDL-C: 0.81 vs. MELD: 0.77). CONCLUSION: HDL-related biomarkers are robust predictors of disease progression and survival in chronic liver failure. LAY SUMMARY: People who suffer from cirrhosis (scarring of the liver) have low levels of cholesterol carried by high-density lipoproteins (HDL-C). These alterations are connected to inflammation, which is a problem in severe liver disease. Herein, we show that reduced levels of HDL-C and apolipoprotein A-I (apoA-I, the main protein carried by HDL) are closely linked to the severity of liver failure, its complications and survival. Both HDL-C and apoA-I can be easily measured in clinical laboratories and are as good as currently used prognostic scores calculated from several laboratory values by complex formulas.

Apolipoprotein A-IV acts as an endogenous anti-inflammatory protein and is reduced in treatment-naïve allergic patients and allergen-challenged mice.

BACKGROUND: Recent studies pointed to a crucial role for apolipoproteins in the pathogenesis of inflammatory diseases. However, the role of apolipoprotein-IV (ApoA-IV) in allergic inflammation has not been addressed thoroughly thus far. OBJECTIVE: Here, we explored the anti-inflammatory effects and underlying signaling pathways of ApoA-IV on eosinophil effector function in vitro and in vivo. METHODS: Migratory responsiveness, Ca2+ -flux and apoptosis of human peripheral blood eosinophils were assessed in vitro. Allergen-driven airway inflammation was assessed in a mouse model of acute house dust mite-induced asthma. ApoA-IV serum levels were determined by ELISA. RESULTS: Recombinant ApoA-IV potently inhibited eosinophil responsiveness in vitro as measured by Ca2+ -flux, shape change, integrin (CD11b) expression, and chemotaxis. The underlying molecular mechanism involved the activation of Rev-ErbA-α and induced a PI3K/PDK1/PKA-dependent signaling cascade. Systemic application of ApoA-IV prevented airway hyperresponsiveness (AHR) and airway eosinophilia in mice following allergen challenge. ApoA-IV levels were decreased in serum from allergic patients compared to healthy controls. CONCLUSION: Our data suggest that ApoA-IV is an endogenous anti-inflammatory protein that potently suppresses effector cell functions in eosinophils. Thus, exogenously applied ApoA-IV may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophil-driven disorders.

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival.

BACKGROUND: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. OBJECTIVE: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. METHODS: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied in vitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry. RESULTS: For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. CONCLUSION: These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.