MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Up-Regulation of the Long Noncoding RNA X-Inactive-Specific Transcript and the Sex Bias in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a sex-biased disease. Increased expression and activity of the long-noncoding RNA X-inactive-specific transcript (Xist), essential for X-chromosome inactivation and dosage compensation of X-linked genes, may explain the sex bias of PAH. The present studies used a murine model of plexiform PAH, the intersectin-1s (ITSN) heterozygous knockout (KOITSN+/-) mouse transduced with an ITSN fragment (EHITSN) possessing endothelial cell proliferative activity, in conjunction with molecular, cell biology, biochemical, morphologic, and functional approaches. The data demonstrate significant sex-centered differences with regard to EHITSN-induced alterations in pulmonary artery remodeling, lung hemodynamics, and p38/ETS domain containing protein/c-Fos signaling, altogether leading to a more severe female lung PAH phenotype. Moreover, the long-noncoding RNA-Xist is up-regulated in the lungs of female EHITSN-KOITSN+/- mice compared with that in female wild-type mice, leading to sex-specific modulation of the X-linked gene ETS domain containing protein and its target, two molecular events also characteristic to female human PAH lung. More importantly, cyclin A1 expression in the S and G2/M phases of the cell cycle of synchronized pulmonary artery endothelial cells of female PAH patients is greater versus controls, suggesting functional hyperproliferation. Thus, Xist up-regulation leading to female pulmonary artery endothelial cell sexual dimorphic behavior may provide a better understanding of the origin of sex bias in PAH. Notably, the EHITSN-KOITSN+/- mouse is a unique experimental animal model of PAH that recapitulates most of the sexually dimorphic characteristics of human disease.

A highly powerful nonspecific strategy to reduce COVID-19 deaths.

The coronavirus disease 2019 (COVID-19) pandemic caused by the coronavirus severe acute respiratory syndrome coronavirus 2 remains risky worldwide. We elucidate here that good IDM (isolation, disinfection, and maintenance of health) is powerful to reduce COVID-19 deaths based on the striking differences in COVID-19 case fatality rates among various scenarios. IDM means keeping COVID-19 cases away from each other and from other people, disinfecting their living environments, and maintaining their health through good nutrition, rest, and treatment of symptoms and pre-existing diseases (not through specific antiviral therapy). Good IDM could reduce COVID-19 deaths by more than 85% in 2020 and more than 99% in 2022. This is consistent with the fact that good IDM can minimize co-infections and maintain body functions and the fact that COVID-19 has become less pathogenic (this fact was supported with three novel data in this report). Although IDM has been frequently implemented worldwide to some degree, IDM has not been highlighted sufficiently. Good IDM is relative, nonspecific, flexible, and feasible in many countries, and can reduce deaths of some other relatively mild infectious diseases. IDM, vaccines, and antivirals aid each other to reduce COVID-19 deaths. The IDM concept and strategy can aid people to improve their health behavior and fight against COVID-19 and future pandemics worldwide.

Extraction methods, physiological activities and high value applications of tea residue and its active components: a review.

Tea is a traditional plant beverage originating from China as one of the most popular beverages worldwide, which has been an important companion in modern society. Nevertheless, as the waste after tea processing, tea residues from agriculture, industry and kitchen waste are discarded in large quantities, resulting in waste of resources and environmental pollution. In recent years, the comprehensive utilization of tea residue resources has attracted people's attention. The bioactive components remaining in tea residues demonstrate a variety of health benefits and can be recycled using advanced extraction processes. Furthermore, researchers have been devoted to converting tea residues into derivatives such as biosorbents, agricultural compost, and animal feeds through thermochemical techniques and biotechnology. This review summarized the chemical composition and physiological activities of bioactive components from tea residue. The extraction methods of bioactive components in tea residue were elucidated and the main high-value applications of tea residues were proposed. On this basis, the utilization of tea residues can be developed from a single way to a multi-channel or cascade way to improve its economic efficiency. Novel applications of tea residues in different fields, including food development, environmental remediation, energy production and composite materials, are of far-reaching significance. This review aims to provide new insights into developing the utilization of tea residue using a comprehensive strategy and exploring the mechanism of active components from tea residue on human health and their potential applications in different areas.HighlightsThe composition and function of tea residue active components were introduced.The extraction methods of active components from tea residue were proposed.The main high-value applications of tea residues were summarized.The current limitations and future directions of tea residue utilization were concluded.

Plasma metabolomics in the perioperative period of defect repair in patients with pulmonary arterial hypertension associated with congenital heart disease.

The quality of life and survival rates of patients with pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) have been greatly improved by defect-repair surgery and personalized treatments. However, those who survive surgery may remain at risk of persistent PAH, the prognosis may be considerably worse than those unoperated. Dynamic monitoring of clinical measures during the perioperative period of shunt correction is therefore indispensable and of great value. In this study, we explored the plasma-metabolite profiling in 13 patients with CHD-PAH during the perioperative period of defect repair. Plasma was harvested at four time points: prior to cardiopulmonary bypass (CPB) after anesthesia (Pre), immediately after CPB (T0), 24 h (T24), and 48 h (T48) after defect repair. Untargeted metabolomics strategy based on UPLC Q-TOF MS was used to detect the metabolites. A total of 193 distinguishing metabolites were determined at different time points, enriched in pathways such as oxidation of branched-chain fatty acids. We found that 17 metabolite alterations were significantly correlated with the reduction in mean pulmonary arterial pressure (MPAP) at T48 versus Pre. Gradients in diastolic pulmonary arterial pressure (DPAP), bicarbonate in radial artery (aHCO3), bicarbonate in superior vena cava (svcHCO3), and the partial pressure of dissolved CO2 gas in radial artery (aPCO2) were positively correlated with MPAP gradient. Notably, these clinical-measure gradients were correlated with alterations in shunt-correction-associated metabolites. In total, 12 out of 17 identified metabolites in response to defect repair were increased at both T24 and T48 (all P < 0.05, except propionylcarnitine with P < 0.05 at T24). In contrast, galactinol dihydrate, guanosine monophosphate, and hydroxyphenylacetylglycine tended to decline at T24 and T48 (only galactinol dihydrate with P < 0.05 at T48). In conclusion, 17 metabolites that respond to shunt correction could be used as suitable noninvasive markers, and clinical measures, including DPAP, aHCO3, svcHCO3, and aPCO2, would be of great value in disease monitoring and evaluating future therapeutic interventions.

Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

Preparation, Physicochemical and Hypoglycemic Properties of Natural Selenium-Enriched Coarse Tea Glycoproteins.

Various functional components in tea have been well developed, but less research has been explored on glycoproteins in tea. In this paper, three types of glycoprotein fractions, namely tea selenium-binding glycoprotein1-1 (TSBGP1-1), TSBGP2-1, and TSBGP3-1, respectively, were extracted and purified from selenium-enriched coarse green tea. Chemical analysis revealed that three fractions were glycoproteins, but their selenium content, molecular weight, and monosaccharide composition were significantly different. Fourier transforms infrared (FT-IR) analysis indicated that three fractions contained characteristic absorption peaks of glycoproteins but differed in secondary structural composition. Thermogravimetric (TG) analysis showed that the thermal stability of the three fractions was dramatically distinct. The in vitro hypoglycemic activity showed that TSBGPs significantly activated the insulin receptor substrate 2 (IRS2)/protein kinase B (Akt) pathway in LO2 cells, then enhanced glucose metabolism and inhibited gluconeogenesis, and finally ameliorated insulin resistance (IR) and glucose metabolism disorders. Furthermore, Pearson correlation analysis reveals that the hypoglycemic activity was significantly correlated with Se, protein, monosaccharide composition (especially glucose), molecular weight, and secondary structure. Our results show that Se-enriched tea glycoprotein is a desirable candidate for developing anti-diabetic food, and TSBGP-2 and TSBGP-3 had a better regulation effect. Our results can provide a research reference for the extraction, physicochemical property, and function of selenium-enriched plant glycoproteins.

Spermine promotes pulmonary vascular remodelling and its synthase is a therapeutic target for pulmonary arterial hypertension.

Pathological mechanisms of pulmonary arterial hypertension (PAH) remain largely unexplored. Effective treatment of PAH remains a challenge. The aim of this study was to discover the underlying mechanism of PAH through functional metabolomics and to help develop new strategies for prevention and treatment of PAH.Metabolomic profiling of plasma in patients with idiopathic PAH was evaluated through high-performance liquid chromatography mass spectrometry, with spermine identified to be the most significant and validated in another independent cohort. The roles of spermine and spermine synthase were examined in pulmonary arterial smooth muscle cells (PASMCs) and rodent models of pulmonary hypertension.Using targeted metabolomics, plasma spermine levels were found to be higher in patients with idiopathic PAH compared to healthy controls. Spermine administration promoted proliferation and migration of PASMCs and exacerbated vascular remodelling in rodent models of pulmonary hypertension. The spermine-mediated deteriorative effect can be attributed to a corresponding upregulation of its synthase in the pathological process. Inhibition of spermine synthase in vitro suppressed platelet-derived growth factor-BB-mediated proliferation of PASMCs, and in vivo attenuated monocrotaline-mediated pulmonary hypertension in rats.Plasma spermine promotes pulmonary vascular remodelling. Inhibiting spermine synthesis could be a therapeutic strategy for PAH.

Potential of live pathogen vaccines for defeating the COVID-19 pandemic: History and mechanism.

The whole world has entered a terrible crisis with a huge and increasing number of human deaths and economic losses in fighting the pandemic of COVID-19 caused by the novel coronavirus termed SARS-CoV-2. The live pathogen vaccine (LPV) strategy, which originated in ancient China for fighting smallpox, has been applied successfully by US military recruits for decades to control acute respiratory diseases caused by types 4 and 7 adenoviruses. This strategy has also been widely employed in veterinary medicine. These facts suggest a fast way out of the current pandemic crisis, namely that SARS-CoV-2 could be directly used as a live vaccine. Beyond the two traditional mechanisms to guarantee the LPV's safety (the LPV seed strain is properly selected; the LPV is inoculated bypassing the respiratory sites of pathology), three novel mechanisms to further ensure the LPV's safety are available (the virus replication is inhibited with early use of an antiviral drug; symptomatic LPV recipients are cured with convalescent plasma; the LPV is inoculated in the hot season). This LPV strategy has multiple potential advantages over other options and could reduce morbidity and mortality greatly as well as the economic loss caused by the pandemic. The safety and efficacy of this strategy should be investigated strictly using animal experiments and clinical trials, and even if the experiments and trials all support the strategy, it should be implemented with enough caution.

Potential for elimination of SAR-CoV-2 through vaccination as inspired by elimination of multiple influenza viruses through natural pandemics or mass vaccination.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed many lives worldwide. To combat the pandemic, multiple types of vaccines are under development with unprecedented rapidity. Theoretically, future vaccination against COVID-19 may fall into long-term costly guerrilla warfare between SARS-CoV-2 and humans. Elimination of SARS-CoV-2 through vaccination to avoid the potential long-term costly guerrilla warfare, if possible, is highly desired and worth intensive consideration. Human influenza pandemics emerging in 1957, 1968, and 2009 established strong global herd immunity and led to the elimination of three human influenza viruses, which circulated worldwide for years before the pandemics. Moreover, both clade 7.2 of subtype H5 highly pathogenic avian influenza virus and subtype H7N9 avian influenza virus circulated in poultry in China for years, and they have been virtually eliminated through mass vaccination in recent years. These facts suggest that the rapid establishment of global herd immunity through mass vaccination using an appropriate vaccine could eliminate SARS-CoV-2. The coming 2 years are a golden time for elimination through vaccination, which requires tremendous national and international collaboration. This review also prioritizes the efficacy of vaccines for COVID-19 and elucidates the importance of the development of more live vaccines for COVID-19.

Injury-Induced Shedding of Extracellular Vesicles Depletes Endothelial Cells of Cav-1 (Caveolin-1) and Enables TGF-ß (Transforming Growth Factor-ß)-Dependent Pulmonary Arterial Hypertension.

Objective- To determine whether pulmonary arterial hypertension is associated with endothelial cell (EC)-Cav-1 (caveolin-1) depletion, EC-derived extracellular vesicle cross talk with macrophages, and proliferation of Cav-1 depleted ECs via TGF-ß (transforming growth factor-ß) signaling. Approach and Results- Pulmonary vascular disease was induced in Sprague-Dawley rats by exposure to a single injection of VEGFRII (vascular endothelial growth factor receptor II) antagonist SU5416 (Su) followed by hypoxia (Hx) plus normoxia (4 weeks each-HxSu model) and in WT (wild type; Tie2.Cre-; Cav1 lox/lox) and EC- Cav1-/- (Tie2.Cre+; Cav1 fl/fl) mice (Hx: 4 weeks). We observed reduced lung Cav-1 expression in the HxSu rat model in association with increased Cav-1+ extracellular vesicle shedding into the circulation. Whereas WT mice exposed to hypoxia exhibited increased right ventricular systolic pressure and pulmonary microvascular thickening compared with the group maintained in normoxia, the remodeling was further increased in EC- Cav1-/- mice indicating EC Cav-1 expression protects against hypoxia-induced pulmonary hypertension. Depletion of EC Cav-1 was associated with reduced BMPRII (bone morphogenetic protein receptor II) expression, increased macrophage-dependent TGF-ß production, and activation of pSMAD2/3 signaling in the lung. In vitro, in the absence of Cav-1, eNOS (endothelial NO synthase) dysfunction was implicated in the mechanism of EC phenotype switching. Finally, reduced expression of EC Cav-1 in lung histological sections from human pulmonary arterial hypertension donors was associated with increased plasma concentration of Cav-1, extracellular vesicles, and TGF-ß, indicating Cav-1 may be a plasma biomarker of vascular injury and key determinant of TGF-ß-induced pulmonary vascular remodeling. Conclusions- EC Cav-1 depletion occurs, in part, via Cav-1+ extracellular vesicle shedding into the circulation, which contributes to increased TGF-ß signaling, EC proliferation, vascular remodeling, and pulmonary arterial hypertension.

Dihydroartemisinin Attenuates Pulmonary Hypertension Through Inhibition of Pulmonary Vascular Remodeling in Rats.

Pulmonary arterial hypertension (PAH) is a malignant disease characterized by pulmonary arterial remodeling because of the abnormal proliferation and migration of pulmonary arterial smooth muscle cells. Dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, is able to inhibit fibrosis, neovascularization, and tumor proliferation. In this study, we hypothesized that DHA can be beneficial in treating PAH. To test this hypothesis, a rat model of pulmonary hypertension induced with monocrotaline (MCT) was used. Compared with MCT treatment alone, treatment with 50 or 100 mg/kg DHA significantly reduced the mean pulmonary arterial pressure (30.11 ± 2.48 mm Hg vs. 21.35 ± 3.04 mm Hg and 19.18 ± 1.98 mm Hg, respectively, both P < 0.01), right ventricular transverse diameter (4.36 ± 0.41 mm vs. 3.72 ± 0.24 mm and 3.67 ± 0.27 mm, respectively, both P < 0.01), pulmonary artery medial wall thickness (57.93 ± 11.14% vs. 34.45 ± 4.39% and 25.01 ± 6.66%, respectively, both P < 0.01), and increased tricuspid annular plane systolic excursion (1.34 ± 0.17 mm vs. 1.62 ± 0.3 mm and 1.62 ± 0.16 mm, respectively, both P < 0.05). We also found that DHA inhibited platelet-derived growth factor-BB-mediated pulmonary arterial smooth muscle cells proliferation and migration in a dose-dependent manner. Moreover, DHA downregulated ß-catenin levels while upregulating the levels of axis inhibition protein 2 (Axin2) and glycogen synthase kinase 3ß (GSK-3ß). Our findings suggest that DHA, which may be a potential candidate for PAH therapy, attenuates experimental pulmonary hypertension possibly by inhibiting pulmonary vascular remodeling.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Epidemiological characteristics, clinical manifestations and laboratory findings in 850 patients with brucellosis in Heilongjiang Province, China.

BACKGROUND: Brucellosis has extensive clinical spectrum, clinicians have insufficient understanding of the disease, and the misdiagnosis rate is still high. By collecting and analyzing the clinical characteristics of patients with brucellosis in Heilongjiang Province to provide guidance and reference for clinicians to make timely diagnosis and treatment. METHODS: The demographic and epidemiological characteristics, clinical features, complications, laboratory findings were retrospectively evaluated in 850 brucellosis patients admitted in the Department of Infectious Diseases of the First Affiliated Hospital of Harbin Medical University and the Second Hospital of Daqing from 2012 to 2017. RESULTS: Of the 850 patients, the most common clinical manifestations were fever (93.3%), joint pain (69.8%), sweating (45.2%), fatigue (38.6%), and splenomegaly (34.0%). Peripheral arthritis, spondylitis and epididymal-orchitis were the common complications. Of the 398 patients who were followed up and completed treatment, 22 (5.5%) had relapse. CONCLUSIONS: Brucellosis is a multisystem disease with diverse clinical manifestations. In areas where brucellosis is endemic, the possibility of the disease should be considered in patients with unexplained fever and joints pain. In addition, the high rate of relapse is mainly due to the misdiagnosis of complications, so local CT or MRI examination is necessary for patients with joint pain and low back pain. Timely diagnosis, early detection of complications are essential to improve the prognosis and reduce relapse.

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and Is a Therapeutic Target in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. METHODS: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/- mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. RESULTS: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/- mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia-induced pulmonary hypertension in rats. CONCLUSIONS: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Micro-RNA-1 is decreased by hypoxia and contributes to the development of pulmonary vascular remodeling via regulation of sphingosine kinase 1.

Sphingosine kinase 1 (SphK1) upregulation is associated with pathologic pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), but the mechanisms controlling its expression are undefined. In this study, we sought to characterize the regulation of SphK1 expression by micro-RNAs (miRs). In silico analysis of the SphK1 3'-untranslated region identified several putative miR binding sites, with miR-1-3p (miR-1) being the most highly predicted target. Therefore we further investigated the role of miR-1 in modulating SphK1 expression and characterized its effects on the phenotype of pulmonary artery smooth muscle cells (PASMCs) and the development of experimental pulmonary hypertension in vivo. Our results demonstrate that miR-1 is downregulated by hypoxia in PASMCs and can directly inhibit SphK1 expression. Overexpression of miR-1 in human PASMCs inhibits basal and hypoxia-induced proliferation and migration. Human PASMCs isolated from PAH patients exhibit reduced miR-1 expression. We also demonstrate that miR-1 is downregulated in mouse lung tissues during experimental hypoxia-mediated pulmonary hypertension (HPH), consistent with upregulation of SphK1. Furthermore, administration of miR-1 mimics in vivo prevented the development of HPH in mice and attenuated induction of SphK1 in PASMCs. These data reveal the importance of miR-1 in regulating SphK1 expression during hypoxia in PASMCs. A pivotal role is played by miR-1 in pulmonary vascular remodeling, including PASMC proliferation and migration, and its overexpression protects from the development of HPH in vivo. These studies improve our understanding of the molecular mechanisms underlying the pathogenesis of pulmonary hypertension.

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed.

Murine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin-positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH.

Hemin Causes Lung Microvascular Endothelial Barrier Dysfunction by Necroptotic Cell Death.

Hemin, the oxidized prosthetic moiety of hemoglobin, has been implicated in the pathogenesis of acute chest syndrome in patients with sickle cell disease by virtue of its endothelial-activating properties. In this study, we examined whether hemin can cause lung microvascular endothelial barrier dysfunction. By assessing transendothelial resistance using electrical cell impedance sensing, and by directly measuring trans-monolayer fluorescein isothiocyanate-dextran flux, we found that hemin does cause endothelial barrier dysfunction in a concentration-dependent manner. Pretreatment with either a Toll-like receptor 4 inhibitor, TAK-242, or an antioxidant, N-acetylcysteine, abrogated this effect. Increased monolayer permeability was found to be associated with programmed cell death by necroptosis, as evidenced by Trypan blue staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, Western blotting for activated forms of key effectors of cell death pathways, and studies utilizing specific inhibitors of necroptosis and apoptosis. Further studies examining the role of endothelial cell necroptosis in promoting noncardiogenic pulmonary edema during acute chest syndrome are warranted and may open a new avenue of potential treatments for this devastating disease.

Loss of lung WWOX expression causes neutrophilic inflammation.

The tumor suppressor WW domain-containing oxidoreductase (WWOX) exhibits regulatory interactions with an array of transcription factors and signaling molecules that are positioned at the well-known crossroads between inflammation and cancer. WWOX is also subject to downregulation by genotoxic environmental exposures, making it of potential interest to the study of lung pathobiology. Knockdown of lung WWOX expression in mice was observed to cause neutrophil influx and was accompanied by a corresponding vascular leak and inflammatory cytokine production. In cultured human alveolar epithelial cells, loss of WWOX expression resulted in increased c-Jun- and IL-8-dependent neutrophil chemotaxis toward cell monolayers. WWOX was observed to directly interact with c-Jun in these cells, and its absence resulted in increased nuclear translocation of c-Jun. Finally, inhibition of the c-Jun-activating kinase JNK abrogated the lung neutrophil influx observed during WWOX knockdown in mice. Altogether, these observations represent a novel mechanism of pulmonary neutrophil influx that is highly relevant to the pathobiology and potential treatment of a number of different lung inflammatory conditions.