Endothelial Cell Injury Research Articles

LncRNA MIR4697HG Alleviates Endothelial Cell Injury and Atherosclerosis Progression in Mice via the FUS/ANXA5 Axis.

Atherosclerosis (AS) manifests with arterial intimal injury, lipid deposition and chronic inflammation, which is a key pathogenic cause of cardio-cerebrovascular disorders. LncRNA MIR4697HG was downregulated in human advanced atherosclerotic plaques. This study probed the precise biological functions and downstream regulatory mechanisms of MIR4697HG during AS progression. MIR4697HG levels in atherosclerotic plaque tissues and normal arterial intima were measured by RT-qPCR. An injury model of human umbilical vein endothelial cells (HUVECs) was induced through treating with oxidative low-density lipoprotein (ox-LDL). MIR4697HG overexpression plasmids (pcDNA-MIR4697HG) was transfected into ox-LDL-treated HUVECs, and then cell viability, apoptosis, reactive oxygen species (ROS) level, oxidative stress marker protein malondialdehyde (MDA) level and superoxide dismutase (SOD) activity, and adhesion molecule intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels in HUVECs were determined. Moreover, the binding between MIR4697HG and fused in sarcoma (FUS) was checked with RNA pull-down assay. The interaction between FUS and annexin A5 (ANXA5) was gauged with Co-immunoprecipitation. Then MIR4697HG/FUS/ANXA5 axis mediated HUVEC functions were accessed with rescue experiments. Additionally, an AS model was established via feeding a high-fat diet for ApoE-/- mice, and lentivirus MIR4697HG overexpression vector (Lv-MIR4697HG) was injected into AS mice followed by detection of atherosclerotic plaque area in mice. MIR4697HG was downregulated in atherosclerotic plaque tissues and HUVECs stimulated by ox-LDL. MIR4697HG overexpression attenuated ox-LDL-induced HUVEC viability inhibition, apoptosis, oxidative stress and adhesion molecule release. Moreover, MIR4697HG bound with FUS and facilitated FUS expression in HUVECs. FUS knockdown abrogated the functions of lncRNA MIR4697HG overexpression in ox-LDL induced HUVEC injury. Besides, FUS could bind with ANXA5. FUS overexpression inhibited ox-LDL induced HUVEC injury, while ANXA5 knockdown reversed these effects. Additionally, Lv-MIR4697HG reduced atherosclerotic plaque area in ApoE-/- mice. LncRNA MIR4697HG mitigated ox-LDL-induced apoptosis, oxidative stress and adhesion molecule release in HUVECs and alleviated AS progression in mice through the FUS/ANXA5 axis.

Knockdown of ADAMDEC1 ameliorates ox-LDL-induced endothelial cell injury and atherosclerosis progression.

This study was designed to investigate the role of a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC-1) in atherosclerosis (AS). The Gene Expression Omnibus (GEO) database was utilized to identify differentially expressed genes (DEGs) between carotid atheroma plaque and carotid tissue adjacent atheroma plaque obtained from AS patients. Gene functional enrichment analysis was conducted on DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). QRT-PCR was employed to quantify mRNAs expression. AS animal model was established using ApoE-/- mice; serum triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were detected. Aortic sinus atherosclerotic lesions were observed using H&E staining and Oil Red O staining. ADAMDEC-1 was silenced using small interfering RNAs (siRNAs) in human vascular smooth muscle cells (HVSMCs). Cell proliferation, migration, and cell cycle progression were detected by cell count kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EDU), wound scratch healing assay, transwell assay, and flow cytometry, respectively. Western blot was used to evaluate various protein expression levels. Our results showed that ADAMDEC-1 was highly expressed in the serum of AS patients, consistent with the in silico results. The elevated TG, LDL-C, and HDL-C levels along with H&E and Oil Red O staining confirmed the successful establishment of the AS mouse model. ADAMDEC-1 expression was also elevated in AS mice. ADAMDEC-1 knockdown in HVSMCs suppressed cell proliferation, inhibited the expression of proliferating cell nuclear antigen (PCNA), and reduced the levels of matrix metalloproteinases (MMP2 and MMP9) proteins. Protein-protein interaction (PPI) analysis indicated that ADAMDEC-1 was associated with CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50. The expression levels of CXCL9, CCR5, TNF-α, TNFR1, and NF-κB-p50 increased, while ADAMDEC-1 knockdown attenuated the expression of these proteins. Our study findings substantiate that ADAMDEC-1 may represent a novel target for AS.

Methylation and transcriptomic expression profiles of HUVEC in the oxygen and glucose deprivation model and its clinical implications in AMI patients.

The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)-clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood.

Computer simulation of two phase power-law nanofluid of blood flow through a curved overlapping stenosed artery with induced magnetic field: entropy generation optimization

PurposeThe purpose of this study is to determine the impact of two-phase power law nanofluid on a curved arterial blood flow under the presence of ovelapped stenosis. Over the past couple of decades, the percentage of deaths associated with blood vessel diseases has risen sharply to nearly one third of all fatalities. For vascular disease to be stopped in its tracks, it is essential to understand the vascular geometry and blood flow within the artery. In recent scenarios, because of higher thermal properties and the ability to move across stenosis and tumor cells, nanoparticles are becoming a more common and effective approach in treating cardiovascular diseases and cancer cells.Design/methodology/approachThe present mathematical study investigates the blood flow behavior in the overlapped stenosed curved artery with cylinder shape catheter. The induced magnetic field and entropy generation for blood flow in the presence of a heat source, magnetic field and nanoparticle (Fe3O4) have been analyzed numerically. Blood is considered in artery as two-phases: core and plasma region. Power-law fluid has been considered for core region fluid, whereas Newtonian fluid is considered in the plasma region. Strongly implicit Stone’s method has been considered to solve the system of nonlinear partial differential equations (PDE’s) with 10–6 tolerance error.FindingsThe influence of various parameters has been discussed graphically. This study concludes that arterial curvature increases the probability of atherosclerosis deposition, while using an external heating source flow temperature and entropy production. In addition, if the thermal treatment procedure is carried out inside a magnetic field, it will aid in controlling blood flow velocity.Originality/valueThe findings of this computational analysis hold great significance for clinical researchers and biologists, as they offer the ability to anticipate the occurrence of endothelial cell injury and plaque accumulation in curved arteries with specific wall shear stress patterns. Consequently, these insights may contribute to the potential alleviation of the severity of these illnesses. Furthermore, the application of nanoparticles and external heat sources in the discipline of blood circulation has potential in the medically healing of illness conditions such as stenosis, cancer cells and muscular discomfort through the usage of beneficial effects.

Ascorbate protects human kidney organoids from damage induced by cell-free hemoglobin.

Sepsis-associated acute kidney injury is associated with high morbidity and mortality in critically ill patients. Cell-free hemoglobin (CFH) is released into the circulation of patients with severe sepsis and the levels of CFH are independently associated with mortality. CFH treatment increased cytotoxicity in the human tubular epithelial cell line HK-2. To better model the intact kidney, we cultured human kidney organoids derived from induced pluripotent stem cells. We treated human kidney organoids grown using both three-dimensional and transwell protocols with CFH for 48 h. We found evidence for increased tubular toxicity, oxidative stress, mitochondrial fragmentation, endothelial cell injury and injury-associated transcripts compared to those of the untreated control group. To evaluate the protective effect of clinically available small molecules, we co-treated CFH-injured organoids with ascorbate (vitamin C) or acetaminophen for 48 h. We found significantly decreased toxicity, preservation of endothelial cells and reduced mitochondrial fragmentation in the group receiving ascorbate following CFH treatment. This study provides direct evidence that ascorbate or ascorbic acid protects human kidney cells from CFH-induced damage such as that in sepsis-associated acute kidney injury.

GPR124 induces NLRP3 inflammasome-mediated pyroptosis in endothelial cells during ischemic injury

ObjectiveG protein-coupled receptor 124 (GPR124) regulates central nervous system angiogenesis and blood-brain barrier (BBB) integrity, and its deficiency aggravates BBB breakdown and hemorrhagic transformation in ischemic mice. However, excessive GPR124 expression promotes inflammation in atherosclerotic mice. In this study, we aimed to elucidate the role of GPR124 in hypoxia/ischemia-induced cerebrovascular endothelial cell injury. MethodsbEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), and time-dependent changes in GPR124 mRNA and protein expression were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The effects of GPR124 overexpression or knockdown on the expression of pyroptosis-related genes were assessed at the mRNA and protein levels. Tadehaginoside (TA) was screened as a potential small molecule targeting GPR124, and its effects on pyroptosis-related signaling pathways were investigated. Finally, the therapeutic efficacy of TA was evaluated using a rat model of transient middle cerebral artery occlusion/reperfusion (tMCAO/R). ResultsDuring OGD, the expression of GPR124 initially increased and then decreased over time, with the highest levels observed 1 h after OGD. The overexpression of GPR124 enhanced the OGD-induced expression of NLRP3, Caspase-1, and Gasdermin D (GSDMD) in bEnd.3 cells, whereas GPR124 knockdown reduced pyroptosis. Additionally, TA exhibited a high targeting ability to GPR124, significantly inhibiting its function and expression and suppressing the expression of pyroptosis-related proteins during OGD. Furthermore, TA treatment significantly reduced the cerebral infarct volume and pyroptotic signaling in tMCAO/R rats. ConclusionsOur findings suggest that GPR124 mediates pyroptotic signaling in endothelial cells during the early stages of hypoxia/ischemia, thereby exacerbating ischemic injury.

Progress in Research on the Mechanisms and Interventions of Phlebitis from the Perspective of Vascular Endothelial Cell and Signaling Pathway.

Phlebitis is a common complication of intravenous administration and greatly affects clinical outcomes, patient satisfaction, and health-care expenditure. Numerous studies have revealed venous injuries only through visual and histopathological examination. Although sporadic studies have explored the cellular and molecular biological mechanisms of phlebitis and the outcomes of pharmacological interventions, an updated review over the last decade is not available. Progress in research on the mechanisms and interventions of phlebitis was summarized from the perspective of endothelial cells and signaling pathways by retrieving the PubMed, Web of Science Core Collection, MEDLINE, Embase, and CNKI. Phlebitis involves multiple signaling pathways (eg, nuclear factor kappa B, Wnt/β-catenin, focal adhesion kinase/protein kinase B, Toll-like receptor, protein kinase C beta/NADPH oxidase, PI3K/AKT/TNF, and JAK2/STAT3), upregulation of E-selectin, GBP5/NLRP3 inflammasome axis, cell apoptosis, intracellular ROS generation, SOD reduction, stimulation of angiogenesis, and induction of autophagy-associated cell death. Preventive and curative interventions included α-solanine, baicalein, escin, intermedin, Y15, micro-ribonucleic acid-223, sotrastaurin, cimetidine, aescin, resveratrol, α-chaconine, Chahuang ointment, QingLuoTongMai, Mailuo Shutong, and N-acetylcysteine. Laboratory models included vascular endothelial cells, real-time cell-monitoring analysis, network pharmacology analysis and experimental verification in vivo, animal models of phlebitis (rat, rabbit, and mouse), rabbit models with peripherally inserted central catheters (PICC) catheterization, models of PICC/central venous catheter indwelling with combined drugs in human umbilical vein endothelial cells, and compatibility with endothelial cells. Factors affecting vascular endothelial cell injury include difference in the same class of drugs, concentration and exposure time of precipitant, and infusion strategy. Phlebitis is accompanied by endothelial dysfunction and may involve multiple molecular and cellular mechanisms. These findings improve our understanding of the molecular targets of interventions and help identify effective candidates for the prophylaxis and treatment of phlebitis. Vascular health and risk management should be considered when initiating intravenous administration.

A Phase II Study to Evaluate the Safety and Efficacy of Defibrotide and Changes in Plasma Biomarkers in Sickle Cell Disease-Related Acute Chest Syndrome (IND 127812)

Background: Acute Chest Syndrome (ACS) is the greatest contributor to the morbidity and mortality of Sickle Cell Disease (SCD) patients with a rate of 12.8 cases per 100 patient-years. The mortality rates of ACS are 4 times higher in adults than in children (Vichinsky et al, Blood, 1997). The root cause of ACS in SCD is related to endothelial dysfunction (Paul et al, European Journal of Hematology 2011). While existing therapies target infection, inflammation, and alveolar hypoxia, they do not address endothelial dysfunction, a major contributor to SCD associated ACS. Defibrotide is a polydisperse mixture of predominantly single stranded oligonucleotides derived from porcine intestinal mucosa. Several pre-clinical studies indicate that defibrotide primarily protects endothelium, particularly in small vessels, and reduces endothelial cell injury (Falanga et al, Leukemia, 2003) (Cairo/Cooke et al, British Journal of Haematology 2020). In a randomized phase III study, a decrease in incidence of SOS/VOD was observed in pediatric hematopoietic stem cell transplantation patients who received prophylactic defibrotide (Corbacioglu et al, Lancet, 2012). We hypothesized that Defibrotide would be safe and well tolerated in children and adolescents with SCD-associated ACS. Primary Aims: To determine the safety and efficacy of defibrotide in the treatment of patients with SCD-associated ACS. Design/Methods: Patients with SCD- (Homozygous Hemoglobin S disease, Hemoglobin SC Disease or Hemoglobin Sβ 0/+ Thalassemia) associated ACS 2 to 40 years of age were enrolled. Defibrotide, generously supplied by Jazz Pharmaceuticals, was administered at 6.25 mg/kg IV Q6H (total daily dose 25 mg/kg/day) within 24 hours of consent and continued for 7 days or until the patient was discharged from hospital (IND 127812) (NCT 03805581). All patients were treated with current standard of care, including antibiotics, analgesics, oxygen, intravenous fluids, and packed red blood cells (PRBC)/exchange transfusion. Complete blood counts and coagulation studies were performed at baseline, day 7 and day 30, while chest radiograph (CXR), CT chest angiogram (CTA), pulmonary function test, and transthoracic echocardiogram were performed at baseline and 30 days after defibrotide was first given. Blood plasma was collected from patients at baseline, day 7, day 30, and age matched SCD patients without ACS and analyzed for biomarkers of inflammation through ELISA and Luminex multiplex assays and significance was calculated using two-tailed T tests. Results: We have enrolled 20 patients (median age 9.5 years) with a gender ratio of M/F 8/12. Patients had hemoglobin SS (n=14), hemoglobin Sβ 0/+ (n=2), or hemoglobin SC (n=4). The median doses of defibrotide per patient was 18.5 (range 6-28) and the average hospital admission length was 6.1 days (range 3-13 days). There were no serious adverse events possibly, probably, or directly related to defibrotide, as judged by investigators. Two patients had a grade I/II epistaxis that did not disrupt defibrotide's course. Pulmonary infiltrate on CXR and/or CTA was the most common finding at diagnosis, detected in 18/20 study participants. Thirteen patients had fever as a presenting symptom, with an average fever duration of 2.8 days (range 1-6 days). Nine patients required O 2 supplementation, with an average duration of 4.1 days (range 2-7 days). Thirteen patients were treated with blood transfusions with a median of 1.9 units (PRBC unit was defined as 15ml/kg) transfused per patient (range, 1-5 units) and 1 patient underwent an exchange transfusion (Table 1). Soluble Intercellular Adhesion Molecule-1 (sICAM-1) was significantly elevated at the onset of ACS (p &amp;lt; 0.01) compared to control SCD plasma and was reduced by Day 30 after defibrotide treatment. Other markers of inflammation, including Angiopoetin-2 (Ang2), Interleukin-6 (IL-6), and Phospholipase A2-IIA (PLA2G2A) were elevated at baseline in patient plasma samples but were significantly decreased following defibrotide treatment (p &amp;lt; 0.05, 0.01, 0.01, respectively) (Figure 1). Conclusion: Our data suggests that defibrotide is safe and well tolerated in SCD patients with ACS. sICAM-1, Ang2, IL-6, and PLA2G2A may serve as important biomarkers in SCD associated ACS. A randomized phase II/III multi-center clinical trial will need to be conducted to further investigate efficacy of defibrotide in patients with SCD-associated ACS.

A Phase II Study to Evaluate the Safety and Efficacy of Defibrotide in Children with High Risk Kawasaki Disease (IND 127812)

Background: Kawasaki disease (KD) is the leading cause of acquired heart disease in children in the developed world. It is an acute, generalized vasculitis, particularly in the coronary arteries, that occurs predominantly in infants and young children (Newburger et al, 2016). The acute phase of KD produces diffuse vascular inflammation that is thought to be associated with activation of the IL-1 pathway. Endothelium, located in the innermost layer of the coronary arteries, serves as an interface between the vascular media and circulating inflammatory cells and inflammatory cytokines. The coronary artery endothelium is the first target of the inflammatory attack in the early stages of KD (Qiu et al, Frontiers Cardiovascular Medicine, 2022) (Liu et al, Molecular Cell Biochem, 2021). There are pathologic changes during an acute arteritis with neutrophilic infiltration leading to necrosis in all the vessel walls, although the usual target is the medium sized extra-parenchymal muscular arteries. Current treatment is directed at reducing inflammation with IVIG and aspirin. Usually, this leads to rapid clinical improvement. However, 10-20% of patients are IVIG resistant and require further methods of reducing inflammation (Newburger et al, 2004). These patients that are IVIG resistant, have recrudescent fevers and evidence of ongoing inflammation, with subsequent increased risk for aneurysm formation (Tremoulet et al, 2008). Furthermore, other particular patient groups have been identified as having a higher risk (HR) of coronary artery aneurysms. Boys have a higher incidence of developing KD, but even accounting for that, are at a higher risk for developing coronary artery aneurysms (Belay et al, 2006). Defibrotide is a polydisperse mixture of predominantly single-stranded oligonucleotides (90% single-stranded and 10% double-stranded) derived from porcine intestinal mucosa. Several in vivo studies indicate that defibrotide primarily targets endothelium, particularly in small vessels, and appears to reduce endothelial cell injury and stabilize endothelial function (Falanga et al, Leukemia, 2003) (Cairo et al, BJH, 2020). Defibrotide is currently approved for SOS/VOD, with renal or pulmonary dysfunction post HSCT and has been well studied in infants with SOS post HSCT (Corbacioglu et al, Lancet, 2012). We hypothesized that Defibrotide will be safe and well tolerated in HR KD patients who fail IVIG and may be effective in difficult to treat KD patients, by mitigating ongoing endothelial dysfunction. Primary Aims: To determine the safety and efficacy of defibrotide in infants with HR KD. Design/Methods: Patients with KD as defined by the American Heart Association, have HR features (Table 1), age birth - 11 years of age, PT/PTT WNL, platelet count ≥100K/mm 3, and without active bleeding, hypersensitivity to defibrotide or receiving systemic anti-coagulant therapy, were eligible. Defibrotide, generously supplied by Jazz Pharmaceuticals, was administered within 96 hours of the conclusion of IVIG at 6.25 mg/kg IV Q6H (total daily dose 25 mg/kg/day) and continued for 7 days or until the patient was discharged from hospital, whichever occurred first (IND 127812) (NCT04777422). Stopping rules included: ≥ 15% grade III-IV allergic reaction or anaphylaxis or hemorrhage. All patients were treated with current standard of care, including: IVIG, aspirin (max 5mg/kg/dose), antibiotics, and intravenous fluids. Complete blood counts, coagulation studies and echocardiogram were performed at baseline, day 10, day 21, and 6 weeks after defibrotide was first given for disease assessment. Results: We have preliminary enrolled 2 patients (mean age of 31 months, range 15 months to 4 years) with a gender ratio of M/F 1/1 (Table 2). The median dose of defibrotide per patient was 5.5 (range 1-11) and the average hospital admission length was 5.5 days (range 5-6 days). No patients demonstrated heart abnormalities on baseline echocardiogram. There were no serious adverse events (SAE) probably or directly related to defibrotide, as evaluated by investigators. One patient had a grade II epistaxis, but no patients had any SAEs. Conclusion: This preliminary data suggests defibrotide is safe in children with HR KD failing IVIG. Further patients will be required to determine the safety and preliminary efficacy of defibrotide in children with HR KD. Supported in part by a research grant from Jazz Pharmaceuticals.

Stratification of acute myocardial and endothelial cell injury, salvage index and final infarct size by systematic microRNA profiling in acute ST-elevation myocardial infarction.

Acute injury and subsequent remodelling responses to ST-segment elevation myocardial infarction (STEMI) are major determinants of clinical outcome. Current imaging and plasma biomarkers provide delayed readouts of myocardial injury and recovery. Here, we sought to systematically characterize all microRNAs (miRs) released during the acute phase of STEMI and relate miR release to magnetic resonance imaging (MRI) findings to predict acute and late responses to STEMI, from a single early blood sample. miRs were quantified in blood samples obtained from patients after primary PCI (PPCI) for STEMI. Cardiac MRI (cMRI) was performed to quantify myocardial edema, infarct size and salvage index. Regression models were constructed to predict these outcomes measures, which were then tested with a validation cohort. Transcoronary miR release was quantified from paired measurements of coronary artery and coronary sinus samples. A cell culture model was used to identify endothelial cell-derived miRs.A total of 72 patients undergoing PPCI for acute STEMI underwent miR analysis and cMRI. About >200 miRs were detectable in plasma after STEMI, from which 128 miRs were selected for quantification in all patients. Known myocardial miRs demonstrated a linear correlation with troponin release, and these increased across the transcoronary gradient. We identified novel miRs associated with microvascular injury and myocardial salvage. Regression models were constructed using a training cohort, then tested in a validation cohort, and predicted myocardial oedema, infarct size and salvage index. Analysis of miR release after STEMI identifies biomarkers that predict both acute and late outcomes after STEMI. A novel miR-based biomarker score enables the estimation of area at risk, late infarct size and salvage index from a single blood sample 6 hours after PPCI, providing a simple and rapid alternative to serial cMRI characterization of STEMI outcome.

Reduction of renal interstitial fibrosis by targeting Tie2 in vascular endothelial cells

BackgroundTie2, a functional angiopoietin receptor, is expressed in vascular endothelial cells and plays an important role in angiogenesis and vascular stability. This study aimed to evaluate the effects of an agonistic Tie2 signal on renal interstitial fibrosis (RIF) and elucidate the underlying mechanisms.MethodsWe established an in vivo mouse model of folic acid-induced nephropathy (FAN) and an in vitro model of lipopolysaccharide-stimulated endothelial cell injury, then an agonistic Tie2 monoclonal antibody (Tie2 mAb) was used to intervent these processes. The degree of tubulointerstitial lesions and related molecular mechanisms were determined by histological assessment, immunohistochemistry, western blotting, and qPCR.ResultsTie2 mAb attenuated RIF and reduced the level of fibroblast-specific protein 1 (FSP1). Further, it suppressed vascular cell adhesion molecule-1 (VCAM-1) and increased CD31 density in FAN. In the in vitro model, Tie2 mAb was found to decrease the expression of VCAM-1, Bax, and α-smooth muscle actin (α-SMA).ConclusionsThe present findings indicate that the agonistic Tie2 mAb exerted vascular protective effects and ameliorated RIF via inhibition of vascular inflammation, apoptosis, and fibrosis. Therefore, Tie2 may be a potential target for the treatment of this disease.ImpactThis is the first report to confirm that an agonistic Tie2 monoclonal antibody can reduce renal interstitial fibrosis in folic acid-induced nephropathy in mice.This mechanism possibly involves vascular protective effects brought about by inhibition of vascular inflammation, apoptosis and fibrosis.Our data show that Tie2 signal may be a novel, endothelium-specific target for the treatment of tubulointerstitial fibrosis.

INOS inhibitor S-methylisothiourea alleviates smoke inhalation-induced acute lung injury by suppressing inflammation and macrophage infiltration

ObjectiveWe investigated the effects of the inducible NO synthase (iNOS) inhibitor, S-methylisothiourea (SMT), in a mouse model of smoke inhalation-induced acute lung injury (ALI) and explored the underlying molecular mechanism. Methods and analysisA mouse model of smoke inhalation-induced ALI was established. RNA-sequencing (seq) analysis was conducted to identify the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed for functional annotation of DEGs. Moreover, an immunofluorescence assay using macrophage marker F4/80 was performed to assess macrophage infiltration. A hypoxia-induced HUVEC model was used to mimic smoke inhalation-induced injury in endothelial cells. Finally, a transwell assay was used to analyze the chemoattractive effects of endothelial cells on macrophages. ResultsSMT markedly alleviated the pulmonary pathological symptoms, edema, and inflammatory response in the mouse smoke inhalation-induced ALI model. RNA-seq analysis revealed that SMT may diminish lung injury by regulating the levels of genes associated with inflammatory responses, cell chemokines, and adhesion. In vivo data revealed that the protective effects of SMT against smoke inhalation-induced ALI were partly achieved by inhibiting the production of adhesion molecules and infiltration of macrophages. Furthermore, in vitro data from the hypoxia-induced HUVEC model revealed that SMT reduced macrophage chemotaxis by inhibiting the production of chemokines and adhesion molecules in endothelial cells. ConclusioniNOS inhibitor SMT protects the lungs from smoke inhalation-induced ALI by reducing the production of pro-inflammatory cytokines, adhesion molecules, and chemokines in endothelial cells, thereby inhibiting inflammation and macrophage infiltration.

HYDROGEN PREVENTS LIPOPOLYSACCHARIDE-INDUCED PULMONARY MICROVASCULAR ENDOTHELIAL CELL INJURY BY INHIBITING STORE-OPERATED Ca 2+ ENTRY REGULATED BY STIM1/ORAI1.

Background: Sepsis is a type of life-threatening organ dysfunction that is caused by a dysregulated host response to infection. The lung is the most vulnerable target organ under septic conditions. Pulmonary microvascular endothelial cells (PMVECs) play a critical role in acute lung injury (ALI) caused by severe sepsis. The impairment of PMVECs during sepsis is a complex regulatory process involving multiple mechanisms, in which the imbalance of calcium (Ca 2+ ) homeostasis of endothelial cells is a key factor in its functional impairment. Our preliminary results indicated that hydrogen gas (H 2 ) treatment significantly alleviates lung injury in sepsis, protects PMVECs from hyperpermeability, and decreases the expression of plasma membrane stromal interaction molecule 1 (STIM1), but the underlying mechanism by which H 2 maintains Ca 2+ homeostasis in endothelial cells in septic models remains unclear. Thus, the purpose of the present study was to investigate the molecular mechanism of STIM1 and Ca 2+ release-activated Ca 2+ channel protein1 (Orai1) regulation by H 2 treatment and explore the effect of H 2 treatment on Ca 2+ homeostasis in lipopolysaccharide (LPS)-induced PMVECs and LPS-challenged mice. Methods: We observed the role of H 2 on LPS-induced ALI of mice in vivo . The lung wet/dry weight ratio, total protein in the bronchoalveolar lavage fluid, and Evans blue dye assay were used to evaluate the pulmonary endothelial barrier damage of LPS-challenged mice. The expression of STIM1 and Orai1 was also detected using epifluorescence microscopy. Moreover, we also investigated the role of H 2 -rich medium in regulating PMVECs under LPS treatment, which induced injury similar to sepsis in vitro . The expression of STIM1 and Orai1 as well as the Ca 2+ concentration in PMVECs was examined. Results:In vivo , we found that H 2 alleviated ALI of mice through decreasing lung wet/dry weight ratio, total protein in the bronchoalveolar lavage fluid and permeability of lung. In addition, H 2 also decreased the expression of STIM1 and Orai1 in pulmonary microvascular endothelium. In vitro , LPS treatment increased the expression levels of STIM1 and Orai1 in PMVECs, while H 2 reversed these changes. Furthermore, H 2 ameliorated Ca 2+ influx under sepsis-mimicking conditions. Treatment with the sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase inhibitor, thapsigargin, resulted in a significant reduction in cell viability as well as a reduction in the expression of junctional proteins, including vascular endothelial-cadherin and occludin. Treatment with the store-operated Ca 2+ entry inhibitor, YM-58483 (BTP2), increased the cell viability and expression of junctional proteins. Conclusions: The present study suggested that H 2 treatment alleviates LPS-induced PMVEC dysfunction by inhibiting store-operated Ca 2+ entry mediated by STIM1 and Orai1 in vitro and in vivo .

Inhibition of PDGFRβ alleviates endothelial cell apoptotic injury caused by DRP-1 overexpression and mitochondria fusion failure after mitophagy

Kawasaki disease (KD), described as “mucocutaneous lymph node syndrome”, affects infants and toddlers. Patients with KD suffer from an inflammatory cascade leading to vasculitis with a predilection for coronary arteries. While the symptoms and pathogenesis of KD have received more and more attention, the precise mechanisms are still debated. Researches show that endothelial dysfunction process in KD leads to arterial damage and affect clinical outcome. In this study, we constructed a Candida albicans water soluble fraction (CAWS)-induced KD murine model and penetrated investigating the mechanisms behind endothelial dysfunction. CAWS-induced mice presented remarkably elevated vascular endothelial cell growth factor (VEGF) levels. Abundant expression of VEGF was documented in all vessels that showed edema from acute KD. It has been reported that Platelet-derived growth factor (PDGF) co-expression normalizes VEGF-induced aberrant angiogenesis. Hyperexpression of PDGFRβ was induced in the thickened medial layer and vascular endothelium of KD mice. Masitinib (Mas) is an oral tyrosine kinase inhibitor of numerous targets, which can selectively target PDGFR signaling. We set out to explore whether Mas could regulate coronary pathology in KD. Mas administration significantly reduced the VEGF-induced endothelial cells migration. NOX4 was activated in vascular endothelial cells to produce more ROS. Mitochondrial dysregulated fission and mitophagy caused by DRP-1 overexpression precipitated the arterial endothelial cells injury. Here, mitophagy seemed to work as the driving force of DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis. In summary, how mitophagy is regulated by DRP-1 under pathologic status is critical and complex, which may contribute to the development of specific therapeutic interventions in cardiovascular diseases patients, for example Masatinib, the inhibitor of PDGFRβ.Facts and questionsKawasaki disease causing systemic vasculitis, affects infants and toddlers.Coronary artery injury remains the major causes of morbidity and mortality.DRP-1 overexpression induces DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis.PDGFRβ was high-expressed in the thickened medial layer of CAWS-induced KD mice.Inhibition of PDGFRβ signaling alleviates arterial endothelial cells injury.

Tsantan Sumtang, a traditional Tibetan medicine, protects pulmonary vascular endothelial function of hypoxia-induced pulmonary hypertension rats through AKT/eNOS signaling pathway

Ethnopharmacological relevanceTsantan Sumtang (TS), originated from the Four Tantras, is an empirical Tibetan medicine prescription, which has been widely used for treating cardiovascular diseases in the clinic in Qinghai Province of China. Our previous studies found that TS alleviated hypoxia-induced pulmonary hypertension (HPH) in rats. However, the effect and bioactive fractions of TS on hypoxia-injured pulmonary vascular endothelium are unknown. Aim of the studyTo investigate the effect, bioactive fractions and pharmacological mechanism of TS on hypoxia-injured pulmonary vascular endothelium in vivo and in vitro. Materials and methodsIn vivo studies, HPH animal model was established, and TS was administrated for four weeks. Then, hemodynamic indexes, ex vivo pulmonary artery perfusion experiment, morphological characteristics, nitric oxide (NO) production, and the protein expression of protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK)/eNOS signaling were determined. In vitro studies, 1% O2-induced pulmonary artery endothelial cells (PAECs) injury model was applied for screening bioactive fractions of TS by cell proliferation assay and NO production measurement. The associated proteins of AKT/eNOS signaling were further measured to elucidate underlying mechanism of bioactive fraction of TS via using phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002. Ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) was used to reveal the chemical profile of bioactive fraction of TS. ResultsTS showed protective effect on the integrity of distal pulmonary arterial endothelium in HPH rats. Tsantan Sumtang dilated pulmonary arterial rings in HPH rats. TS enhanced NO bioavailability in lung tissue via regulating AKT/eNOS signaling. Furthermore, in the cellular level, cell viability as well as NO content of hypoxia-injured PAECs were elevated by fraction 17 of water extract of TS (WTS), through activating the AKT/eNOS signaling. Ellagic acid could be one of compositions in fraction 17 of WTS to produce NO in hypoxia-injured PAECs. ConclusionTS restored pulmonary arterial endothelial function in HPH rats. The bioactive fraction 17 was screened, which protected hypoxia-injured PAECs via upregulating AKT/eNOS signaling.

MAGNESIUM SULFATE AMELIORATES HISTONE-INDUCED COAGULATION DYSFUNCTION AND LUNG DAMAGE IN MICE.

Introduction: Extracellular histones have been determined as significant mediators of sepsis, which can induce endothelial cell injury and promote coagulation activation, and ultimately contribute to multiorgan failure. Evidence suggests that magnesium sulfate (MgSO 4 ) exerts a potential coagulation-modulating activity; however, whether MgSO 4 ameliorates histone-induced coagulation dysfunction and organ damage remains unclear. Methods: To measure circulating histone levels, blood specimens were collected from septic patients and mice, and the relationship between circulating histone levels, coagulation parameters, and Mg 2+ levels in sepsis was investigated. Furthermore, to explore the possible protective effects of MgSO 4 , we established a histone-induced coagulation model in mice by intravenous histone injection. The survival rate of mice was assessed, and the histopathological damage of the lungs (including endothelial cell injury and coagulation status) was evaluated using various methods, including hematoxylin and eosin staining, immunohistochemistry, immunofluorescence, electron microscopy, and quantitative polymerase chain reaction. Results: The circulating histone levels in septic patients and mice were significantly associated with several coagulation parameters. In septic patients, histone levels correlated negatively with platelet counts and positively with prothrombin time and D-dimer levels. Similarly, in cecal ligation and puncture mice, histones correlated negatively with platelet counts and positively with D-dimer levels. Interestingly, we also observed a positive link between histones and Mg 2+ levels, suggesting that Mg 2+ with anticoagulant activity is involved in histone-mediated coagulation alterations in sepsis. Further animal experiments confirmed that MgSO 4 administration significantly improved survival and attenuated histone-mediated endothelial cell injury, coagulation dysfunction, and lung damage in mice. Conclusion: These results suggest that therapeutic targeting of histone-mediated endothelial cell injury, coagulation dysfunction, and lung damage, for example, with MgSO 4 , may be protective in septic individuals with elevated circulating histone levels.

Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury.

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.

Tanshinone IIA attenuates ox-LDL-induced endothelial cell injury by inhibiting NF-kapaB pathway via circ_0000231/miR-590-5p/TXNIP axis.

Tanshinone IIA (TSIIA) exhibits inhibitory function in atherosclerosis (AS) progression, and circular RNAs (circRNAs) are pivotal regulators in AS. However, the relation between TSIIA and circ_0000231 in AS pathogenesis remains unknown. In this study, oxidized low-density lipoprotein (ox-LDL) was used to establish AS cell model. Treatment of ox-LDL inhibited cell growth but promoted apoptosis, inflammation, and oxidative stress. Then, TSIIA was shown to attenuate ox-LDL-induced endothelial injury. Furthermore, the protective effect of TSIIA against ox-LDL-induced endothelial cell injury was reversed by circ_0000231. Circ_0000231 was identified as a miR-590-5p sponge. Also, miR-590-5p downregulation restored the protection of TSIIA for endothelial cell function. Moreover, circ_0000231 was found to upregulate thioredoxin interacting protein (TXNIP) level via targeting miR-590-5p. TXNIP overexpression mitigated the regulatory function of circ_0000231 knockdown after co-treatment with ox-LDL and TSIIA. TXNIP upregulation recovered the inhibitory regulation of TSIIA in ox-LDL-induced cell damage. In addition, TSIIA inactivated NF-kapaB (NF-κB) signaling pathway via regulating miR-590-5p/TXNIP axis by downregulating circ_0000231. All these results suggested that TSIIA inhibited ox-LDL-induced AS progression in endothelial cells by affecting NF-κB pathway via circ_0000231/miR-590-5p/TXNIP.

Circulating large Extracellular Vesicles (EV-L) from STEMI patients contribute to post-injury adverse myocardial remodeling

Abstract Background While primary percutaneous coronary intervention (PCI) is the treatment of choice in patients with ST-segment elevation myocardial infarction (STEMI), this treatment does not fully prevent late adverse cardiac remodeling. Infarcted myocardial tissue releases damage-associated extracellular vesicles (EV) that mediate tissue inflammation and may regulate myocardial remodeling. Here, we investigated the role of circulating EV in STEMI patients, with a focus on endothelial cell (EC) injury and myocardial fibrosis. Methods Peripheral blood samples were obtained from STEMI patients (n=30) within 4 hrs of onset of pain before PCI and from age- and gender-matched healthy control (CTRL; n=30) with documented absence of coronary artery disease. Raw serum was characterized by atomic force microscopy (AFM) single-particle morphometry. Large EV (EV-L) were isolated by differential centrifugation and used for in vitro experiments on human aortic endothelial cells (hAEC), and human cardiac fibroblast (hCF). Results AFM analysis showed a 7.5-fold increase in EV-L in STEMI vs. CTRL serum. In vitro treatment of hAEC with EV-L from STEMI patients, but not CTRL-EV-L, induced an increase in reactive oxygen species (ROS; 1.15-Fold increase), p66SHC and oxidized low-density lipoprotein receptor 1 (LOX-1) expression (1.34 and 20-Fold increase, respectively), and cell death (1.40-Fold increase). Interestingly, ELISA analysis on EV-L showed an enrichment of LOX-1 in STEMI-EV-L compared to CTRL-EV-L (3.01-Fold increase). Moreover, STEMI-EV-L, but not CTRL-EV-L, stimulated in vitro proliferation (1.4-Fold increase) of hCF and their activation in myofibroblasts, as assessed by alpha smooth muscle actin (αSMA) expression (1.8-fold increase), resulting in increased extracellular matrix (ECM) secretion (1.5-Fold increase). Conclusions Our preliminary data showed a pathological role of circulating EV-L derived from STEMI patients. In particular, results from in vitro experiments evidence their function in inducing endothelial damage and activation of cardiac fibroblast into myofibroblast; two important features in the progression of pathological myocardial remodeling.Graphical abstract

Panax notoginseng saponins reverse steroid resistance in lupus nephritis: Involvement of the suppression of exosomal P-gp levels from lymphocytes to glomerular endothelial cells

Microangiopathy is the most basic pathological manifestation of lupus nephritis (LN), and glomerular endothelial cells (GECs) injury is an important pathological mechanism. LN patients with microangiopathy are prone to steroid resistance (SR). Our previous studies confirmed that Panax notoginseng saponins (PNS) could reverse SR by downregulating the expression of P-gp in SR lymphocytes of LN mice (SLCsL/S). However, the mechanism of how circulating lymphocytes transmit SR information to GECs and thus affect the efficacy of kidney treatment is not clear. Recent studies have found that exosomes (exos) are an important carrier for intercellular bioactive substance communication. But whether exosomes derived from SLCsL/S mediate SR in GECs and PNS interventions. To solve this problem, Exosomes isolated from SLCsL/S were characterized, and in vitro cell coculture was further conducted to investigate the effect of SLCsL/S-derived exosomes in the SR of GECs and PNS intervention. Sequencing was used to define the exosomal miRNA expression profiling of SR GECs. Moreover, the in vivo experiments were performed through the injection of exosomes extracted from SLCsL/S into the tail vein of mice.Our research results indicate that exosomes derived from SLCsL/S could transmit SR information to GECs and lead to the aggravation of inflammatory injury through conferring P-gp, which were negated by a P-gp inhibitor. Further, we identified higher levels of exosomal miR-125b-5p from SR GECs were associated with SR in LN and could serve as biomarker for the risk of developing SR. PNS could reverse the SR of GECs and alleviate inflammatory injury by suppressing exosomal P-gp levels from lymphocytes to GECs in vitro and in vivo. However, the specific molecular mechanism by which PNS regulates exosomes has not yet been elucidated, and we need to conduct more in-depth research in the future. Overall, Our findings suggest that exosomal transfer of SLCsL/S derived P-gp confer SR to GECs, and PNS can target exosome communication to reverse SR in LN, which provides new ideas and a scientific basis for improving the clinical efficacy of traditional Chinese medicine in the treatment of refractory LN.