A Novel Interaction Between Aquaporin 1 and Caspase-3 in Pulmonary Arterial Smooth Muscle Cells.

Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into non-muscular arteries. These pathologic changes are predominantly due to abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin-1 (AQP1). However, the mechanisms underlying increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its c-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both HEK293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics.

Unlocking the protective potential of the angiotensin type 2 receptor (AT2R) in acute lung injury and age-related pulmonary dysfunction.

Despite its known importance in the cardiovascular system, the specific role and impact of the angiotensin type 2 receptor (AT2R) in lung physiology and pathophysiology remain largely elusive. In this study, we highlight the distinct and specialized lung-specific roles of AT2R, primarily localized to an alveolar fibroblast subpopulation, in contrast to the angiotensin type 1 receptor (AT1R), which is almost exclusively expressed in lung pericytes. Evidence from our research demonstrates that the disruption of AT2R (AT2R-/y), is associated with a surge in oxidative stress and impaired lung permeability, which were further intensified by Hyperoxic Acute Lung Injury (HALI). With aging, AT2R-/y mice show an increase in oxidative stress, premature enlargement of airspaces, as well as increased mortality when exposed to hyperoxia as compared to age-matched WT mice. Our investigation into Losartan, an AT1R blocker, suggests that its primary HALI lung-protective effects are channeled through AT2R, as its protective benefits are absent in AT2R-/y mice. Importantly, a non-peptide AT2R agonist, Compound 21 (C21), successfully reverses lung oxidative stress and TGFß activation in wild-type (WT) mice exposed to HALI. These findings suggest a possible paradigm shift in the therapeutic approach for lung injury and age-associated pulmonary dysfunction, from targeting AT1R with angiotensin receptor blockers (ARBs) towards boosting the protective function of AT2R.

Dimethyl Fumarate Protects against Lipopolysaccharide- (LPS-) Induced Sepsis through Inhibition of NF-κB Pathway in Mice.

Sepsis is one of the most severe complications and causes of mortality in the clinic. It remains a great challenge with no effective treatment for clinicians worldwide. Inhibiting the release of proinflammatory cytokines during sepsis is considered as an important strategy for treating sepsis and improving survival. In the present study, we have observed the effect of dimethyl fumarate (DMF) on lipopolysaccharide- (LPS-) induced sepsis and investigated the possible mechanism. By screening a subset of the Johns Hopkins Drug Library, we identified DMF as a novel inhibitor of nitric oxide synthesis in LPS-stimulated RAW264.7 cells, suggesting that DMF could be a potential drug to treat sepsis. To further characterize the effect of DMF on LPS signaling, TNF-α, MCP-1, G-CMF, and IL-6 expression levels were determined by using cytokine array panels. In addition, an endotoxemia model with C57BL/6 mice was used to assess the in vivo efficacy of DMF on sepsis. The survival rate was assessed, and HE staining was performed to investigate histopathological damage to the organs. DMF was found to increase the survival of septic mice by 50% and attenuate organ damage, consistent with the reduction in IL-10, IL-6, and TNF-α (inflammatory cytokines) in serum. In vitro experiments revealed DMF's inhibitory effect on the phosphorylation of p65, IκB, and IKK, suggesting that the primary inhibitory effects of DMF can be attributed, at least in part, to the inhibition of phosphorylation of IκBα, IKK as well as nuclear factor-κB (NF-κB) upon LPS stimulation. The findings demonstrate that DMF dramatically inhibits NO and proinflammatory cytokine production in response to LPS and improves survival in septic mice, raising the possibility that DMF has the potential to be repurposed as a new treatment of sepsis.

Xanthine oxidoreductase gene polymorphisms are associated with high risk of sepsis and organ failure.

BACKGROUND: Sepsis and associated organ failures confer substantial morbidity and mortality. Xanthine oxidoreductase (XOR) is implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders including sepsis and sepsis-associated acute respiratory distress syndrome (ARDS). We examined whether single nucleotide polymorphisms (SNPs) in the XDH gene (encoding XOR) might influence susceptibility to and outcome in patients with sepsis. METHODS: We genotyped 28 tag SNPs in XDH gene in the CELEG cohort, including 621 European American (EA) and 353 African American (AA) sepsis patients. Serum XOR activity was measured in a subset of CELEG subjects. Additionally, we assessed the functional effects of XDH variants utilizing empirical data from different integrated software tools and datasets. RESULTS: Among AA patients, six intronic variants (rs206805, rs513311, rs185925, rs561525, rs2163059, rs13387204), in a region enriched with regulatory elements, were associated with risk of sepsis (P < 0.008-0.049). Two out of six SNPs (rs561525 and rs2163059) were associated with risk of sepsis-associated ARDS in an independent validation cohort (GEN-SEP) of 590 sepsis patients of European descent. Two common SNPs (rs1884725 and rs4952085) in tight linkage disequilibrium (LD) provided strong evidence for association with increased levels of serum creatinine (Padjusted<0.0005 and 0.0006, respectively), suggesting a role in increased risk of renal dysfunction. In contrast, among EA ARDS patients, the missense variant rs17011368 (I703V) was associated with enhanced mortality at 60-days (P < 0.038). We found higher serum XOR activity in 143 sepsis patients (54.5 ± 57.1 mU/mL) compared to 31 controls (20.9 ± 12.4 mU/mL, P = 1.96 × 10- 13). XOR activity was associated with the lead variant rs185925 among AA sepsis patients with ARDS (P < 0.005 and Padjusted<0.01). Multifaceted functions of prioritized XDH variants, as suggested by various functional annotation tools, support their potential causality in sepsis. CONCLUSIONS: Our findings suggest that XOR is a novel combined genetic and biochemical marker for risk and outcome in patients with sepsis and ARDS.

MK2 nonenzymatically promotes nuclear translocation of caspase-3 and resultant apoptosis.

We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.

Tumor MK2 transcript levels are associated with improved response to chemotherapy and patient survival in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

Comparison of polynomial fitting versus single time point analysis of ECIS data for barrier assessment.

Electrical cell-substrate impedance sensing (ECIS) is an in vitro methodology for measuring the barrier integrity of a variety of cell types, including pulmonary endothelial cells. These experiments are frequently used for in vitro assessment of lung injury. The data derived from ECIS experiments consists of repeated measures of resistance across an endothelial monolayer. As such, these data reflect the dynamic changes in electrical resistance that occur over time. Currently methodologies for assessing ECIS data rely on single point assessments of barrier function, such as the maximal drop in trans-endothelial electrical resistance (TERMax ). However, this approach ignores the myriad of changes in resistance that occur before and after the TERMax data point. Herein, we utilize polynomial curve fitting on experimentally generated ECIS data, thus allowing for comparing ECIS experiments by examining the mean polynomial coefficients between groups. We show that polynomial curves accurately fit a variety of ECIS data, and that concordance between TERMax and coefficient analysis varies by type of stimulus, suggesting that TERMax differences may not always correlate with a significant difference in the overall shape of the ECIS profile. Lastly, we identify factors that impact coefficient values obtained in our analyses, including the length of time devoted to baseline measurements before addition of stimuli. Polynomial coefficient analysis is another tool that can be used for more comprehensive interrogation of ECIS data to better understand the biological underpinnings that lead to changes in barrier dysfunction in vitro.

PDE9A deficiency does not prevent chronic-hypoxic pulmonary hypertension in mice.

Inhibition of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterases (PDEs) is a cornerstone of pulmonary arterial hypertension (PAH)-specific therapy. PDE9A, expressed in the heart and lung tissue, has the highest affinity for cGMP of all known PDEs. PDE9A deficiency protects mice against chronic left ventricular (LV) pressure overload via increased natriuretic peptide (NP)-dependent cGMP signaling. Chronic-hypoxic pulmonary hypertension (CH-PH) is a model of chronic right ventricular (RV) pressure overload, and previous studies have demonstrated a protective role for NPs in the murine model. Therefore, we hypothesized that PDE9A deficiency would promote NP-dependent cGMP signaling and prevent RV remodeling in the CH-PH model, analogous to findings in the LV. We exposed wild-type and PDE9A-deficient (Pde9a-/- ) C57BL/6 mice to CH-PH for 3 weeks. We measured RV pressure, hypertrophy, and levels of lung and RV cGMP, PDE9A, PDE5A, and phosphorylation of the protein kinase G substrate VASP (vasodilatory-stimulated phosphoprotein) after CH-PH. In wild-type mice, CH-PH was associated with increased circulating ANP and lung PDE5A, but no increase in cGMP, PDE9A, or VASP phosphorylation. Downstream effectors of cGMP were not increased in Pde9a-/- mice exposed to CH-PH compared with Pde9a+/+ littermates, and CH-PH induced increases in RV pressure and hypertrophy were not attenuated in knockout mice. Taken together, these findings argue against a prominent role for PDE9A in the murine CH-PH model.

Rationale and design of the Prone Position and Respiratory Outcomes in Non-intubated COVID-19 PatiEnts: The "PRONE" Study.

While benefits of prone position in mechanically-ventilated patients have been well-described, a randomized-control trial to determine the effects of prone positioning in awake, spontaneously-breathing patients with an acute pneumonia has not been previously conducted. Prone Position and Respiratory Outcomes in Non-Intubated COVID-19 PatiEnts: the "PRONE" Study (PRONE) was conducted in non-intubated hospitalized patients with coronavirus disease 2019 (COVID-19) pneumonia as defined by respiratory rate ≥ 20/min or an oxyhemoglobin saturation (SpO2) ≤ 93% without supplemental oxygen [1]. The PRONE trial was designed to investigate the effects of prone positioning on need for escalation in respiratory support, as defined by need for transition to a higher acuity level of care, increased fraction of inspired oxygen (FiO2), or the initiation of invasive mechanical ventilation. Secondary objectives were to assess the duration of effect of prone positioning on respiratory parameters such as respiratory rate and SpO2, as well as other outcomes such as time to discharge or transition in level of care.

Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by exuberant vascular remodeling leading to elevated pulmonary arterial pressure, maladaptive right ventricular remodeling, and eventual death. The factors controlling pulmonary arterial smooth muscle cell (PASMC) and endothelial cell hyperplasia and migration, hallmark features of the vascular remodeling observed in PAH, remain poorly understood. We previously demonstrated that hypoxia upregulates the expression of aquaporin 1 (AQP1), a water channel, in PASMCs, and that this upregulation was required for hypoxia-induced migration and proliferation. However, whether the same is true in a model of severe PAH and in pulmonary microvascular endothelial cells (MVECs) is unknown. In this study, we used the SU5416 plus hypoxia (SuHx) rat model of severe pulmonary hypertension, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in PASMCs and MVECs and contributed to a hyperproliferative/hypermigratory phenotype. Rats received a single injection of SU5416 (20 mg/kg) and then were placed in 10% O2 for 3 weeks, followed by a return to normoxic conditions for an additional 2 weeks. We found that AQP1 protein levels were increased in both PASMCs and MVECs from SuHx rats, even in the absence of sustained hypoxic exposure, and that in MVECs, the increase in protein expression was associated with upregulation of AQP1 mRNA levels. Silencing of AQP1 had no significant effect on PASMCs from control animals but normalized enhanced migration and proliferation observed in cells from SuHx rats. Loss of AQP1 also reduced migration and proliferation in MVECs from SuHx rats. Finally, augmenting AQP1 levels in MVECs from control rats using forced expression was sufficient to increase migration and proliferation. These results demonstrate a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in pulmonary vascular cells from a rodent model that reflects many of the features of human PAH.

Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion.

The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2 compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis ("renal tubular alkalosis"). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.

Functional Impact of Human Genetic Variants of COL18A1/Endostatin on Pulmonary Endothelium.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

SU5416 does not attenuate early RV angiogenesis in the murine chronic hypoxia PH model.

BACKGROUND: Right ventricular (RV) angiogenesis has been associated with adaptive myocardial remodeling in pulmonary hypertension (PH), though molecular regulators are poorly defined. Endothelial cell VEGFR-2 is considered a "master regulator" of angiogenesis in other models, and the small molecule VEGF receptor tyrosine kinase inhibitor SU5416 is commonly used to generate PH in rodents. We hypothesized that SU5416, through direct effects on cardiac endothelial cell VEGFR-2, would attenuate RV angiogenesis in a murine model of PH. METHODS: C57 BL/6 mice were exposed to chronic hypoxia (CH-PH) to generate PH and stimulate RV angiogenesis. SU5416 (20 mg/kg) or vehicle were administered at the start of the CH exposure, and weekly thereafter. Angiogenesis was measured after one week of CH-PH using a combination of unbiased stereological measurements and flow cytometry-based quantification of myocardial endothelial cell proliferation. In complementary experiments, primary cardiac endothelial cells from C57 BL/6 mice were exposed to recombinant VEGF (50 ng/mL) or grown on Matrigel in the presence of SU5416 (5 µM) or vehicle. RESULT: SU5416 directly inhibited VEGF-mediated ERK phosphorylation, cell proliferation, and Kdr transcription, but not Matrigel tube formation in primary murine cardiac endothelial cells in vitro. SU5416 did not inhibit CH-PH induced RV angiogenesis, endothelial cell proliferation, or RV hypertrophy in vivo, despite significantly altering the expression profile of genes involved in angiogenesis. CONCLUSIONS: These findings demonstrate that SU5416 directly inhibited VEGF-induced proliferation of murine cardiac endothelial cells but does not attenuate CH-PH induced RV angiogenesis or myocardial remodeling in vivo.

A nonapoptotic endothelial barrier-protective role for caspase-3.

Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.

Altered expression of TIAM1 in endotoxin-challenged airway epithelial cells and rodent septic models.

BACKGROUND: In sepsis, reorganization of the actin cytoskeleton in the epithelium during inflammation will lead to a breakdown of epithelial barrier integrity, and contribute to the pathogenesis of sepsis, but the exact changes of various components regulating the actin cytoskeleton pathway remain unclear. METHODS: We used lipopolysaccharide (LPS) challenged primary epithelial cells cultured at the air-liquid interface (ALI) to mimic epithelial barrier dysfunction during sepsis. Then we detected differential expression of T-lymphoma invasion and metastasis 1 (TIAM1) gene in lung epithelial cells and septic models. RESULTS: LPS induced barrier dysfunction in human tracheobronchial epithelial cells (HTBEs) as measured by statistically significant changes in ionic and macromolecular permeability. We observed differential expression of TIAM1 gene. The protein expression of TIAM1 was decreased after LPS challenge, in human bronchial epithelial cells. Furthermore, the expression levels of both TIAM1 mRNA and protein were decreased in lungs of septic rodent models. CONCLUSIONS: Given that expression levels of TIAM1 have been associated with mortality among sepsis patients, our findings have the potential for the development of diagnostic and treatment strategies relevant for patient management.

Reactive oxygen species induced Ca2+ influx via TRPV4 and microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca2+]i) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca2]i in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca2+ entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca2]i, migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca2+ influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.