S1PR1 attenuates pulmonary fibrosis by inhibiting EndMT and improving endothelial barrier function.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease of unknown etiology. Its pathological manifestations include excessive proliferation and activation of fibroblasts and deposition of extracellular matrix. Endothelial cell-mesenchymal transformation (EndMT), a novel mechanism that generates fibroblast during IPF, is responsible for fibroblast-like phenotypic changes and activation of fibroblasts into hypersecretory cells. However, the exact mechanism behind EndMT-derived fibroblasts and activation is uncertain. Here, we investigated the role of sphingosine 1-phosphate receptor 1 (S1PR1) in EndMT-driven pulmonary fibrosis. METHODS: We treated C57BL/6 mice with bleomycin (BLM) in vivo and pulmonary microvascular endothelial cells with TGF-ß1 in vitro. Western blot, flow cytometry, and immunofluorescence were used to detect the expression of S1PR1 in endothelial cells. To evaluate the effect of S1PR1 on EndMT and endothelial barrier and its role in lung fibrosis and related signaling pathways, S1PR1 agonist and antagonist were used in vitro and in vivo. RESULTS: Endothelial S1PR1 protein expression was downregulated in both in vitro and in vivo models of pulmonary fibrosis induced by TGF-ß1 and BLM, respectively. Downregulation of S1PR1 resulted in EndMT, indicated by decreased expression of endothelial markers CD31 and VE-cadherin, increased expression of mesenchymal markers α-SMA and nuclear transcription factor Snail, and disruption of the endothelial barrier. Further mechanistic studies found that stimulation of S1PR1 inhibited TGF-ß1-mediated activation of the Smad2/3 and RhoA/ROCK1 pathways. Moreover, stimulation of S1PR1 attenuated Smad2/3 and RhoA/ROCK1 pathway-mediated damage to endothelial barrier function. CONCLUSIONS: Endothelial S1PR1 provides protection against pulmonary fibrosis by inhibiting EndMT and attenuating endothelial barrier damage. Accordingly, S1PR1 may be a potential therapeutic target in progressive IPF.

Single-cell transcriptomics uncovers phenotypic alterations in the monocytes in a Chinese population with chronic cadmium exposure.

BACKGROUND: Cadmium is the most prevalent form of heavy metal contaminant globally and its exposure rises serious health concern. Chronic exposure to cadmium causes immune disturbances. However, few studies have addressed how it affects circulating immune cells, one of the most essential elements for the host defense system, at both population and molecular level. Therefore, this is the first single-cell transcriptomic analysis of the response of the human circulating immune system to plasma cadmium level. METHODS: We conducted a cross-sectional study in Hunan province, which has the highest level of cadmium land contamination in China. A total of 3283 individuals were eligible for analyzing the association between plasma cadmium levels and the monocyte counts and its subgroups. Another 780 individuals were assigned for validation. Thirty propensity-matched individuals without chronic disease from the lowest- and highest-quartile groups according to serum cadmium levels were selected for single-cell RNA sequencing (scRNA-seq) and flow cytometry analyses. Moreover, the monocyte phenotypic alterations in the heavy metal-exposed population were validated with a cecal ligation and puncture sepsis mouse model. RESULTS: From August 2016 to July 2017, we conducted a cross-sectional study to identify phenotypic alterations in peripheral immune cells in cadmium polluted areas in China. Monocyte percentages were negatively associated with plasma cadmium levels in multivariable linear regression analysis. Peripheral blood mononuclear cell scRNA-seq revealed that the CD14+ monocyte subset was dramatically reduced in the highest-quartile cadmium-exposed group. Moreover, we assessed different hallmarks of immune cell dysfunction-such as host defense capability, apoptotic signaling, cellular diversity and malignant gene expression in monocytes. Importantly, cadmium induced phenotypic alterations in the immune system were validated in the cecal ligation and puncture sepsis mouse model, in which chronic exposure to cadmium not only increased the death rate but also decreased monocyte numbers and the ability to clear bacterial infections. CONCLUSION: This transcriptomic analysis provides molecular information about how the most important hallmarks of immune cell dysfunction are affected by plasma cadmium level. The significant phenotypic alterations in monocytes serving as early indicators of increased susceptibility to infectious and malignant diseases.

The role of sphingosine 1-phosphate and its receptors in cardiovascular diseases.

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P-based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT-1303) and the non-selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.

Rh(III)-Catalyzed Dual C-H Functionalization/Cyclization Cascade by a Removable Directing Group: A Method for Synthesis of Polycyclic Fused Pyrano[de]Isochromenes.

An interesting Rh(III)-catalyzed dual C-H functionalization/cyclization cascade of azomethine imine with diazophosphonate by a removable directing group for the synthesis of highly fused pyrano[de]isochromene has been achieved. The transformation shows that the desired pyrano[de]isochromenes with two oxygen atoms on its core scaffold could be constructed with good to excellent yields (up to 86%) via a facile one-pot, multiple-step cascade reaction, along with broad generality and versatility.

Vildagliptin improves high glucose-induced endothelial mitochondrial dysfunction via inhibiting mitochondrial fission.

The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti-diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti-diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin-related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl-CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1-mediated mitochondrial fission in an AMPK-dependent manner.

Assessment of the Dermatology Life Quality Index (DLQI) in a homogeneous population under lifetime arsenic exposure.

PURPOSE: The psychometric property of the Dermatology Life Quality Index (DLQI) is underappreciated in public health settings. Our study aimed to assess the reliability, validity, and measurement invariance of DLQI in a homogeneous population with arsenic-related skin lesions and symptoms. METHODS: A cross-sectional study was conducted in communities under lifetime arsenic exposure. The DLQI was measured through a face-to-face interview. Skin examinations were performed by certificated dermatologists. The intensity of itching was measured by a numerical rating scale. Reliability, structural validity, and measurement invariance were determined using classical and modern test theories, including confirmatory factor analysis and item response models. RESULTS: 465 participants with arsenic-related skin lesions and symptoms completed the DLQI assessment. The Cronbach's alpha was 0.79, and the split-half reliability was 0.77. A two-factor model exhibited the best model fit among models evaluated, but local dependencies among items were identified. The model showed good root mean square error of approximation (0.031) and acceptable Tucker-Lewis index (0.92). Multi-group confirmatory factor analysis showed no measurement invariance across subgroups of age, gender, ethnicity, and intensity of itching. CONCLUSIONS: The DLQI had acceptable psychometric properties, but measurement invariance was not observed across different groups of participants.

Seasonal Effect on Association between Atmospheric Pollutants and Hospital Emergency Room Visit for Stroke.

BACKGROUND: The relationship between air pollution and stroke is conflicting. This study was conducted to document the relationship between daily changes in atmospheric pollutants and hospital emergency room visits (ERVs) for stroke. METHODS: Data of daily hospital ERVs for stroke and atmospheric pollutants in Changsha city between 2008 and 2009 were collected. Using a time-stratified bidirectional case-crossover design, we analyzed the association between atmospheric pollutants and stroke incidence in 4 seasons. RESULTS: In the single-pollutant model, we found changes in sulfur dioxide (SO2), nitrogen dioxide (NO2), and particulate matters (PM10) were significantly associated with cerebral hemorrhage and cerebral infarction (P < .05) in lags of 0-2 days in autumn. A 10-µg/m3 increase in SO2 in autumn was significantly associated with ERVs for both cerebral hemorrhage (odds ratio [OR], 1.166; 95% confidence interval [CI], 1.012-1.343) and cerebral infarction (OR, 1.214; 95% CI, 1.018-1.448). NO2 in autumn was significantly associated with ERVs for cerebral hemorrhage and infarction with OR = 1.162 (95% CI, 1.005-1.344) and OR = 1.137 (95% CI, 1.011-1.279), respectively. PM10 in autumn was significantly associated with ERVs for cerebral hemorrhage and infarction with OR = 1.147 (95% CI, 1.045-1.259) and OR = 1.091 (95% CI, 1.019-1.168), respectively. Results of the multipollutant model showed that in autumn after PM10 and NO2 adjustment, only a 10-µg/m3 increase in SO2 was significantly associated with ERVs for cerebral infarction (OR, 1.158; 95% CI, 1.006-1.333; P < .05). SO2, NO2, and PM10 were not associated with ERVs for cerebral hemorrhage (P > .05). CONCLUSIONS: This study demonstrates that the change in atmospheric SO2 levels in Changsha is significantly associated with the stroke incidence in autumn.

Discovery and Optimization of Novel Nonbile Acid FXR Agonists as Preclinical Candidates for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a multifactorial chronic inflammation of the intestine and has become a global public health concern. A farnesoid X receptor (FXR) was recently reported to play a key role in hepatic-intestinal circulation, intestinal metabolism, immunity, and microbial regulation, and thus, it becomes a promising therapeutic target for IBD. In this study, we identified a series of nonbile acid FXR agonists, in which 33 novel compounds were designed and synthesized by the structure-based drug design strategy from our previously identified hit compound. Compound 33 exhibited a potent FXR agonistic activity, high intestinal distribution, good anti-inflammatory activity, and the ability to repair the colon epithelium in a DSS-induced acute enteritis model. Based on the results of RNA-seq analysis, we further investigated the therapeutic potential of the combination of compound 33 with 5-ASA. Overall, the results indicated that compound 33 is a promising drug candidate for IBD treatment.

S1PR2/Wnt3a/RhoA/ROCK1/ß-catenin signaling pathway promotes diabetic nephropathy by inducting endothelial mesenchymal transition and impairing endothelial barrier function.

AIMS: Hyperglycemia and hyperlipidemia are key factors in the pathogenesis of diabetic nephropathy (DN), and renal fibrosis is the most common pathway leading to the disease. Endothelial mesenchymal transition (EndMT) is a crucial mechanism for the production of myofibroblasts, and impaired endothelial barrier function is one of the mechanisms for the generation of microalbuminuria in DN. However, the specific mechanisms behind these are not yet clear. MAIN METHODS: Protein expression was detected by immunofluorescence, immunohistochemistry and Western blot. Knocking down or pharmacological inhibition of S1PR2 were used to inhibit Wnt3a, RhoA, ROCK1, ß-catenin, and Snail signaling. Changes in cell function were analyzed by CCK-8 method, cell scratching assay, FITC-dextran permeability assay, and Evans blue staining. KEY FINDINGS: Consistent with increased gene expression of S1PR2 in DN patients and mice with kidney fibrosis disease, S1PR2 expression was significantly increased in glomerular endothelial cells of DN mice and HUVEC cells treated with glucolipids. Knocking down or pharmacological inhibition of S1PR2 significantly decreased the expression of Wnt3a, RhoA, ROCK1, and ß-catenin in endothelial cells. Furthermore, inhibition of S1PR2 in vivo reversed EndMT and endothelial barrier dysfunction in glomerular endothelial cells. Inhibition of S1PR2 and ROCK1 in vitro also reversed EndMT and endothelial barrier dysfunction in endothelial cells. SIGNIFICANCE: Our results suggest that the S1PR2/Wnt3a/RhoA/ROCK1/ß-catenin signaling pathway is involved in the pathogenesis of DN by inducing EndMT and endothelial barrier dysfunction.

Identifying myoglobin as a mediator of diabetic kidney disease: a machine learning-based cross-sectional study.

In view of the alarming increase in the burden of diabetes mellitus (DM) today, a rising number of patients with diabetic kidney disease (DKD) is forecasted. Current DKD predictive models often lack reliable biomarkers and perform poorly. In this regard, serum myoglobin (Mb) identified by machine learning (ML) may become a potential DKD indicator. We aimed to elucidate the significance of serum Mb in the pathogenesis of DKD. Electronic health record data from a total of 728 hospitalized patients with DM (286 DKD vs. 442 non-DKD) were used. We developed DKD ML models incorporating serum Mb and metabolic syndrome (MetS) components (insulin resistance and ß-cell function, glucose, lipid) while using SHapley Additive exPlanation (SHAP) to interpret features. Restricted cubic spline (RCS) models were applied to evaluate the relationship between serum Mb and DKD. Serum Mb-mediated renal function impairment induced by MetS components was verified by causal mediation effect analysis. The area under the receiver operating characteristic curve of the DKD machine learning models incorporating serum Mb and MetS components reached 0.85. Feature importance analysis and SHAP showed that serum Mb and MetS components were important features. Further RCS models of DKD showed that the odds ratio was greater than 1 when serum Mb was > 80. Serum Mb showed a significant indirect effect in renal function impairment when using MetS components such as HOMA-IR, HGI and HDL-C/TC as a reason. Moderately elevated serum Mb is associated with the risk of DKD. Serum Mb may mediate MetS component-caused renal function impairment.

AKT/PACS2 Participates in Renal Vascular Hyperpermeability by Regulating Endothelial Fatty Acid Oxidation in Diabetic Mice.

Diabetes is a chronic metabolic disorder that can cause many microvascular and macrovascular complications, including diabetic nephropathy. Endothelial cells exhibit phenotypic and metabolic diversity and are affected by metabolic disorders. Whether changes in endothelial cell metabolism affect vascular endothelial function in diabetic nephropathy remains unclear. In diabetic mice, increased renal microvascular permeability and fibrosis, as well as increased MAMs and PACS2 in renal endothelial cells, were observed. Mice lacking PACS2 improved vascular leakage and glomerulosclerosis under high fat diet. In vitro, PACS2 expression, VE-cadherin internalization, fibronectin production, and Smad-2 phosphorylation increased in HUVECs treated with high glucose and palmitic acid (HGHF). Pharmacological inhibition of AKT significantly reduced HGHF-induced upregulation of PACS2 and p-Smad2 expression. Blocking fatty acid ß-oxidation (FAO) ameliorated the impaired barrier function mediated by HGHF. Further studies observed that HGHF induced decreased FAO, CPT1α expression, ATP production, and NADPH/NADP+ ratio in endothelial cells. However, these changes in fatty acid metabolism were rescued by silencing PACS2. In conclusion, PACS2 participates in renal vascular hyperpermeability and glomerulosclerosis by regulating the FAO of diabetic mice. Targeting PACS2 is potential new strategy for the treatment of diabetic nephropathy.

Glucolipotoxicity induces endothelial cell dysfunction by activating autophagy and inhibiting autophagic flow.

OBJECTIVES: This study aims to determine the role and mechanism of autophagy in endothelial cell dysfunction by glucolipotoxicity. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with high glucose and high palmitic acid. The number of autophagosomes was evaluated by monodansylcadaverine (MDC) staining and transmission electron microscopy (TEM). The expression of autophagy-related proteins (LC3 and P62) was assessed by Western blotting. Capillary tube-like formation was evaluated on Matrigel. Reactive oxygen species (ROS) production was detected by DCFH-DA. Cell apoptosis was measured by Hoechst 33258 staining and flow cytometry. Phosphorylation of AMPK, mTOR, and ULK1 was also analyzed by Western blotting. RESULTS: We found that glucolipotoxicity induced autophagy initiation and hindered autophagosomes degradation. Moreover, glucolipotoxicity increased the production of intracellular ROS, decreased the ability of tubular formation, and increased cell apoptosis. However, endothelial cell dysfunction was alleviated by 3-methyladenine, an early-stage autophagy inhibitor. Additionally, glucolipotoxicity promoted the phosphorylation of AMPK and ULK1 and inhibited the phosphorylation of mTOR. CONCLUSIONS: Glucolipotoxicity initiates autophagy through the AMPK/mTOR/ULK1 signaling pathway and inhibits autophagic flow, leading to the accumulation of autophagosomes, thereby inducing apoptosis and impairing endothelial cell function.

Effects of co-exposure to multiple metals on children's behavior problems in China.

Exposure to single metals have been linked to childhood behavior problems, But little is known about the effects of metals mixtures on children. We aimed to evaluate associations of multiple metals exposures in urine with childhood behavior in China. For this population-based study, the children eligible for inclusion provided urine samples and their parents agreed to take in-person interview. A total of 831 children were remained from three cities for the final analysis. Urinary metals concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS). The childhood behavior scores was calculated by the Conners' Parent Rating Scale (CPRS). Variable selection was achieved by the least absolute shrinkage and selection operator (LASSO) regularization and stepwise regression to for all metals in the study. Linear regression models and Bayesian kernel machine regression (BKMR) were applied to estimate the associations of urinary metals concentrations with children's behavior. In BKMR models, the overall effect of mixture was significantly associated with conduct problems, learning problems and hyperactive index when urinary metals concentrations were all above the 50th percentile compared to all of them at their medians. The models also suggested marginally significant interaction effects of Se and Fe as well as Se and Sb (PSe∗Fe = 0.063; PSe∗Sb = 0.061), with a decline in estimate of Se on learning problems when Sb/Fe levels were relatively high. The concentrations of 22 metals in boys were higher than girls. In summary, multiple metals are associated with an increased risk of childhood behavioral problems in China. Potential interaction effects of Se and Fe as well as Se and Sb on childhood behavior should be taken into consideration.

S1PR2/RhoA/ROCK1 pathway promotes inflammatory bowel disease by inducing intestinal vascular endothelial barrier damage and M1 macrophage polarization.

Vascular and immune dysfunctions are thought to be related to the pathogenesis of inflammatory bowel disease (IBD), but behind this, the exact mechanism of mucosal vascular endothelial barrier dysfunction and macrophage phenotypic transition is not fully understood. Here, we explored the mechanistic role of sphingosine 1-phosphate receptor 2 (S1PR2) and its downstream G protein RhoA/Rho kinase 1 (ROCK1) signaling pathway in the intestinal endothelial barrier damage and M1 macrophage polarization in IBD. We found that the expression of S1PR2 in intestinal mucosal vascular endothelial cells and macrophages of IBD patients and DSS-induced colitis mice as well as vascular endothelial cells and macrophages treated with LPS in vitro was significantly increased. Knocking down or pharmacologically inhibiting S1PR2 significantly downregulated the expression of RhoA and ROCK1 in vascular endothelial cells and macrophages. Furthermore, inhibition of S1PR2 and ROCK1 reversed the impaired vascular barrier function and M1 macrophage polarization in vivo and in vitro, while reducing ER stress in vascular endothelial cells and glycolysis in macrophages. In addition, inhibition of ER stress or glycolysis reversed LPS-induced impairment of vascular endothelial cell barrier function and M1 macrophage polarization. Collectively, our results indicate that the S1PR2/RhoA/ROCK1 signaling pathway may participate in the pathogenesis of IBD by regulating vascular endothelial barrier function and M1 macrophage polarization.

Organelle dynamics of endothelial mitochondria in diabetic angiopathy.

Diabetes, a chronic non-communicable disease, has become one of the most serious and critical public health problems with increasing incidence trends. Chronic vascular complications are the major causes of disability and death in diabetic patients with endothelial dysfunction. Diabetes is intimately associated with endothelial mitochondrial dysfunction, indicated by increased oxidative stress, decreased biogenesis, increased DNA damage, and weakened autophagy in mitochondria. All these morphological and functional changes of mitochondria play important roles in diabetic endothelial dysfunction. Herein, we reviewed the roles and mechanisms of endothelial mitochondrial dysfunction, particularly mitochondrial dynamics in the vascular complications of diabetes and summarized the potential mitochondria-targeted therapies in diabetic vascular complications.

Discovery and Optimization of Non-bile Acid FXR Agonists as Preclinical Candidates for the Treatment of Nonalcoholic Steatohepatitis.

Farnesoid X receptor (FXR) plays a key role in bile acid homeostasis, inflammation, fibrosis, and metabolism of lipid and glucose and becomes a promising therapeutic target for nonalcoholic steatohepatitis (NASH) or other FXR-dependent diseases. The phase III trial results of obeticholic acid demonstrate that the FXR agonists emerge as a promising intervention in patients with NASH and fibrosis, but this bile acid-derived FXR agonist brings severe pruritus and an elevated risk of cardiovascular disease for patients. Herein, we reported our efforts in the discovery of a series of non-bile acid FXR agonists, and 36 compounds were designed and synthesized based on the structure-based drug design and structural optimization strategies. Particularly, compound 42 is a highly potent and selective FXR agonist, along with good pharmacokinetic profiles, high liver distribution, and preferable in vivo efficacy, indicating that it is a potential candidate for the treatment of NASH or other FXR-dependent diseases.

Palmitic acid-induced autophagy increases reactive oxygen species via the Ca2+/PKCα/NOX4 pathway and impairs endothelial function in human umbilical vein endothelial cells.

It is well known that the lipotoxic mechanism of palmitic acid (PA), a main constituent of triglyceride, is dependent on reactive oxygen species (ROS). Recently, it has also been reported that PA is an autophagy inducer. However, the causal association and underlying mechanism of induced autophagy and ROS in PA toxicity remain unclear. The present study demonstrates for the first time that PA-induced autophagy enhances ROS generation via activating the calcium ion/protein kinase Cα/nicotinamide adenine dinucleotide phosphate oxidase 4 (Ca2+/PKCα/NOX4) pathway in human umbilical vein endothelial cells (HUVECs). It was revealed that PA treatment resulted in a significant increase in ROS generation and autophagic activity, leading to endothelial dysfunction as indicated by downregulated nitric oxide synthesis, decreased capillary-like structure formation and damaged cell repair capability. Furthermore, PA effectively activated the Ca2+/PKCα/NOX4 pathway, which is indicative of upregulated cytosolic Ca2+ levels, activated PKCα and increased NOX4 protein expression. 3-Methyladenine was then used to inhibit autophagy, which significantly reduced PA-induced ROS generation and blocked the Ca2+/PKCα/NOX4 pathway. The endothelial dysfunction caused by PA was ameliorated by downregulating ROS generation using a NOX4 inhibitor. In conclusion, PA-induced autophagy contributes to endothelial dysfunction by increasing oxidative stress via the Ca2+/PKCα/NOX4 pathway in HUVECs.