Endothelial Cell Injury Research Articles

#1071 The effect and mechanism of SGLT2 inhibitor on improving inflammatory injury of glomerular endothelial cells in diabetes

Abstract Background and Aims ATP-binding cassette transporter A1 (ABCA1), which reduces intracellular cholesterol accumulation, is highly expressed in the kidney. Studies have shown that sodium-glucose co-transporter 2 inhibitors (SGLT2) inhibitors promote ABCA1 expression in multiple tissues and organs. However, the effect of SGLT2 inhibitors on ABCA1 expression in diabetic kidney disease (DKD) is unclear. In this study, the effect of empagliflozin, an SGLT2 inhibitor, on ABCA1 in glomerular endothelial cells(GECs) of DKD and the possible mechanisms linking ABCA1 to GECs inflammatory injury were investigated. Method Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. db/db mice were used as animal models of type 2 diabetes and the effects of SGLT2 inhibitors on ABCA1 were analyzed by treatment with empagliflozin. The effects of ABCA1 overexpression on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 overexpression (db/db + ABCA1 mice), and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. Results The expression of ABCA1 in the glomeruli of DKD patients and db/db mice was significantly decreased. Enhancing ABCA1 expression in the kidney tissue of diabetic mice by adenovirus transduction improved renal cholesterol deposition, inhibited the overactivation of C3a/C5a, and improved renal inflammatory injury. In addition, in cultured HRGECs under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposit of cellular cholesterol, contributed to overactivation of C3a/C5a, and induced inflammatory injury. Overexpression of ABCA1 enhancing cholesterol efflux or inhibition of C3a/C5a activation in vitro significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. Moreover, SGLT2 inhibitor empagliflozin up-regulated the expression of ABCA1 and down-regulated the levels of cholesterol, C3a/C5a and inflammatory cytokines in diabetic mice and HRGECs. Conclusion SGLT2 inhibitor (empagliflozin) might play a renal protective role by up-regulating ABCA1-mediated cholesterol outflow, inhibiting the activation of C3a/C5a, and then ameliorating the inflammatory injury of GECs.

Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis

BackgroundShiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2–5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. MethodsVWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. ResultsStx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. ConclusionsIn the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

Inflammation and coronary microvascular disease: relationship, mechanism and treatment.

Coronary microvascular disease (CMVD) is common in patients with cardiovascular risk factors and is linked to an elevated risk of adverse cardiovascular events. Although modern medicine has made significant strides in researching CMVD, we still lack a comprehensive understanding of its pathophysiological mechanisms due to its complex and somewhat cryptic etiology. This greatly impedes the clinical diagnosis and treatment of CMVD. The primary pathological mechanisms of CMVD are structural abnormalities and/or dysfunction of coronary microvascular endothelial cells. The development of CMVD may also involve a variety of inflammatory factors through the endothelial cell injury pathway. This paper first reviews the correlation between the inflammatory response and CMVD, then summarizes the possible mechanisms of inflammatory response in CMVD, and finally categorizes the drugs used to treat CMVD based on their effect on the inflammatory response. We hope that this paper draws attention to CMVD and provides novel ideas for potential therapeutic strategies based on the inflammatory response.

PCSK9 induces endothelial cell autophagy by regulating the PI3K/ATK pathway in atherosclerotic coronary heart disease.

Atherosclerosis is a chronic inflammatory disease of the arteries, and its pathogenesis is related to endothelial dysfunction. It has been found that the protein convertase subtilin/kexin9 type (PCSK9) plays an important role in AS, but its specific mechanism is still unclear. In this study, we first cultured human umbilical vein endothelial cells (HUVECs) with 50 or 100μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 hours to establish a coronary atherosclerosis cell model. The results showed that ox-LDL induced HUVEC injury and autophagy and upregulated PCSK9 protein expression in HUVECs in a concentration-dependent manner. Silencing PCSK9 expression with siRNA inhibited ox-LDL-induced HUVEC endothelial dysfunction, inhibited the release of inflammatory factors, promoted HUVEC proliferation and inhibited apoptosis. In addition, ox-LDL increased the expression of LC3B-I and LC3B-II and decreased the expression of p62. However, these processes are reversed by sh-PCSK9. In addition, sh-PCSK9 can inhibit PI3K, AKT and mTOR phosphorylation and promote autophagy. Taken together, our research shows that silencing PCSK9 inhibits the PI3K/ATK/mTOR pathway to activate ox-LDL-induced autophagy in vascular endothelial cells, alleviating endothelial cell injury and inflammation.

Protective Effects of Polysaccharide of Atractylodes Macrocephala Koidz against Porcine Aortic Valve Endothelial Cells Damage Induced by di (2-ethylhexyl) Phthalate.

The activation, injury, and dysfunction of endothelial cells are considered to be the initial key events in the development of atherosclerosis. Di (2-ethylhexyl) phthalate (DEHP), a prevalent organic pollutant, can cause damage to multiple organs. Polysaccharide of Atractylodes macrocephala Koidz (PAMK) is a bioactive compound extracted from A. macrocephala Koidz with various biological activities. This study investigates the protective effects of PAMK on porcine aortic valve endothelial cells (PAVEC) damaged by DEHP. PAVECs treated with DEHP alone or with PAMK showed reduced cell apoptosis and death in PAMK-pretreated cells. PAMK up-regulated Bcl-2 expression and down-regulated Bax protein, suppressing apoptosis. Flow cytometry analysis demonstrated that PAMK protected PAVECs from DEHP-induced damage. These findings suggest that PAMK inhibits cell apoptosis and protects against DEHP damage in endothelial cells.

Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

BackgroundLocal brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by...

Homeobox B4 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on endotoxin-associated acute lung injury in rats

BackgroundAlveolar capillary endothelial cell (EC) injury has a pivotal role in driving acute respiratory distress syndrome (ARDS) progression and maintaining endothelial homeostasis. A previous ex vivo study revealed that overexpression of homeobox B4 (HOXB4) in bone marrow mesenchymal stem cells (BMSCs) enhanced protection against lipopolysaccharide (LPS)-induced EC injury by activating the Wnt/β-catenin pathway. This in vivo study was performed to verify whether BMSCs overexpressing HOXB4 exert similar protective effects on LPS-induced acute lung injury (ALI) in an animal model. MethodsThe ALI rat model was established by intraperitoneal injection of LPS. Wildtype BMSCs or BMSCs overexpressing HOXB4 were then injected via the tail vein. The lung characteristics of rats were visualized by computed tomography. Lung histopathological characteristics and collagen deposition were assessed by hematoxylin-eosin and Masson's staining, respectively, which were combined with the lung wet/dry ratio and proinflammatory factor levels in bronchoalveolar lavage fluid to further evaluate therapeutic effects. Expression of β-catenin and VE-cadherin was assessed by western blotting and immunofluorescence. ResultsCompared with wildtype BMSCs, overexpression of HOXB4 optimized the therapeutic effects of BMSCs, which manifested as improvements in lung exudation and histopathological features, reduced lung collagen deposition, amelioration of lung permeability, attenuation of lung inflammation, and enhanced expression of β-catenin and VE-cadherin proteins. ConclusionsHOXB4-overexpressing BMSCs optimized the protective effect against LPS-induced ALI by partially activating Wnt/β-catenin signaling.

Abstract 2066: Relationship Between Stat3 And Endoplasmic Reticulum Stress Induces Lipopolysaccharide-induced Endothelial To Mesenchymal Transition In Vascular Endothelial Cell

Background: Endothelial to mesenchymal transition (EndMT) is a process that involves the loss of endothelial cell (EC) phenotype and induced plaque instability during the progression of atherosclerosis. Primarily, EC injury, caused by an accelerated inflammatory signaling pathway, plays a crucial role in activating EC dysfunction by inducing endoplasmic reticulum (ER) stress and disrupting EC junctions. In the present study, we investigated the molecular mechanisms between EndMT and ER stress on STAT3-mdeiated inflammation under LPS-treated human umbilical vein EC (HUVEC). Methods: To explore the EndMT progression under chronic inflammation, LPS-treated HUVEC were used. Western blot, siRNA transfection, and immunofluorescence assay were employed to evaluate signaling pathways and target proteins. The phenotypic switching of ECs on the endothelium of the mouse aorta after challenging 10 mg/kg LPS was assessed by en face staining. Results: Deactivation of STAT3 by stattic pretreatment abolished not only LPS-mediated STAT3 phosphorylation, but also VCAM-1 and ICAM-1. In addition, STAT3 inhibition surprisingly reversed LPS-induced mesenchymal markers, including SM22α and vimentin, and the suppression of endothelial marker VE-cadherin. Mechanistically, stattic treatment suppressed LPS-induced EndMT progression by inhibiting protein expression and nuclear translocation of Smad2/3. Furthermore, LPS-induced chronic inflammation disrupted EC homeostasis by PERK or IRE-1α/CHOP-mediated ER stress, whereas stattic abolished LPS-mediated PERK and CHOP expression. Interestingly, depletion of CHOP by siRNA transfection significantly abolished LPS-mediated EndMT progression (p<0.001. Figure 1). Notably, the absence of CHOP significantly suppressed phosphorylation of STAT3 and its nuclear expression as well. Consistent to in vitro data, STAT3 inhibition by stattic injection significantly suppressed LPS-mediated STAT3 activation as well as CHOP expression in the mouse endothelium. Conclusion: Collectively, the STAT3-CHOP axis may contribute to the loss of EC function and induce mesenchymal differentiation by aggravating chronic inflammation and EndMT.

Up-regulation of Trim28 in pregnancy-induced hypertension is involved in the injury of human umbilical vein endothelial cells through the p38 signaling pathway.

The present study is to analyze the regulation and potential molecular mechanism of Trim28 on vascular endothelial injury induced by pregnancy-induced hypertension (PIH). Trim28 mRNA in placental tissues and peripheral blood from PIH patients were determined by quantitative real-time polymerase chain reaction. The serum from PIH was used to stimulate human umbilical vein endothelial cells (HUVECs). After silencing Trim28 in HUVECs, we used CCK-8 assay, Transwell assay and flow cytometry to investigate proliferation, migration and apoptosis. Western blotting was used to measure Trim28 protein level and p38 phosphorylation level. After addition of p38 inhibitor, the proliferation, migration and apoptosis of HUVECs with silenced Trim28 were studied again. Trim28 expression in placental tissues and peripheral blood from PIH patients is elevated, and serum from these patients can up-regulate the expression of Trim28 in HUVECs in vitro. Trim28 silencing significantly inhibits the proliferation and migration of HUVECs by affecting the cell cycle. Down-regulation of Trim28 expression promotes the apoptosis of HUVECs. Trim28 regulates the biological function of HUVECs by affecting the activity of the p38 signaling pathway. The present study demonstrates that Trim28 is up-regulated in peripheral blood of patients with PIH and participates in HUVECs injury through the p38 signaling pathway.

Reducing the injury of hippocampal vascular endothelial cells after stroke via targeting SIRT1 by butylphthalide

Butylphthalide (NBP) can inhibit various pathological processes of ischemic stroke. This experiment explored the mechanism of NBP and SIRT1 on damage of hippocampal vascular endothelial cells after stroke. The neurons in the hippocampus of rats were stained with HE, and morphology and density of neurons were observed. Flow cytometry, commercial kits and Western blotting detected apoptosis of endothelial cells, levels of antioxidant enzymes and apoptotic proteins, intracellular calcium level and activity of Ca2+-ATPase. The damage to rat nerve cells was alleviated by butylphthalide to varying degrees, and the lost parts of rat nerve cells were recovered with decreased Bax and cleaved caspase-3 expression after butylphthalide treatment, and increased Bcl-2 (P <0.05), as well as decreased serum malondialdehyde (MDA) content and activity of catalase (CAT) decreased and elevated Superoxide dismutase (SOD) activity (P <0.05). The concentration of calcium ion also decreased but activity of Ca2+-ATPase increased (P <0.05) and mitochondria in the model group appeared with severe vacuolation and swelling. The vacuolation and swelling of mitochondria in the treatment group were improved. Additionally, mitochondrial membrane fluidity, potential and rat hippocampal ATPase activity in butylphthalide group were also increased. Compared to normal control group, model group, SIRT1 inhibitor group and butylphthalide+SIRT1 inhibitor group had lower levels of SIRT1 and higher p-NF-kB p65/p-IkBα levels. Butylphthalide has a protective effect on hippocampal neurons in stroke rats and can alleviate the damage degree of rat nerve cells.

The effects of repeat mild traumatic brain injuries on choroid plexus endothelial cells in-vitro

Chronic Traumatic Encephalitis (CTE) is a degenerative brain disorder seen in college and professional football players, and those that play other contact sports like hockey and soccer. It is characterized by a dementia-like phenotype and declination in memory, executive dysfunction, and mood and is associated with repetitive head trauma, as seen in those contact sports. Even with rule changes to protect the players, it is predicted that, on average, college football players experience 1,400-1,500 head impact exposures per year, or roughly 14 per game (Crisco et al., 2010). This is significant because existing literature provides evidence that it is the accumulation of repetitive head trauma, even mild, that eventually leads to lasting and irreparable damage seen in CTE. This study sought to characterize cytokine signaling in repeat mild traumatic brain injuries (rTBI) in choroid plexus endothelial cells (CPECs), the endothelial barrier of the blood-CSF barrier (BCSFB). We hypothesized that regulatory and pro-inflammatory cytokines and chemokines play a role in exacerbating post-rTBI symptomology even days after initial injury. Identifying key cytokines and their respective timepoints post-injury is important to allow us to better treat TBIs, while also preventing subsequent damage that might progress to CTE. To study this hypothesis, primary human CPECs were subjected to a 12% repetitive mechanical elongation strain using the FlexCell Tension system to mimic mild axonal stretching injury. The elongation mechanical strain was repeated 14 times over a 1-hour period, which is roughly the average number of impacts experienced by players during a one-hour college football game. We collected supernatant over 24-, 48-, 72-, and 96-hours post-injury and examined 36 different cytokines using RND microarrays following manufacturer’s instructions. Controls included cells subjected to no mechanical strain and cells that only received one total mechanical strain. Several notable cytokines and chemokines were altered during this period, including RANTES (CCL5), CXCL12, and macrophage migration inhibitory factor (MIF). RANTES is highly correlated with Alzheimer’s disease, a neurodegenerative disease and CTE relative, and CXCL12 and MIF are highly correlated with inflammatory processes. Many cytokine changes were more pronounced in the period between 24- and 48-hours after injury. However, there were lasting changes compared to the controls up to 96-hours later. These findings can be used to begin to understand the complexity of human CPEC cytokine signaling at the BCSFB when these cells react to the strain forces of repeat mild TBIs like those seen in college football players. This also gives us further insight into the lasting effects of rTBIs and the possible relation to CTE. With these results in mind, we plan to continue to monitor cytokines and chemokines up to two weeks post injury to determine if, and when, cytokine values return to baseline values post-injury. This project was funded by a Senior Research Grant from the Indiana Academy of Science and from a Faculty Research Development Grant from Marian University College of Osteopathic Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ZFYVE21 promotes endothelial nitric oxide signaling and vascular barrier function in the kidney during aging

ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) invivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.

Impinging Flow Mediates Vascular Endothelial Cell Injury through the PKCα/ERK/PPARγ Pathway in vitro

Introduction: This study aimed to elucidate the mechanisms underlying endothelial injury in the context of intracranial aneurysm formation and development, which are associated with vascular endothelial injury caused by hemodynamic abnormalities. Specifically, we focus on the involvement of PKCα, an intracellular signaling transmitter closely linked to vascular diseases, and its role in activating MAPK. Additionally, we investigate the protective effects of PPARγ, a vasculoprotective factor known to attenuate vascular injury by mitigating the inflammatory response in the vessel wall. Methods: The study employs a modified T-chamber to replicate fluid flow conditions at the artery bifurcation, allowing us to assess wall shear stress effects on human umbilical vein endothelial cells in vitro. Through experimental manipulations involving PKCα knockdown and Ca2+ and MAPK inhibitors, we evaluated the phosphorylation status of PKCα, NF-κB, ERK5, ERK1/2, JNK1/2/3, and P38, as well as the expression levels of PPARγ, NF-κB, and MMP2 via Western blot analysis. The cellular localization of phosphorylated NF-κB was determined using immunofluorescence. Results: Our results showed that impinging flow resulted in the activation of PKCα, followed by the phosphorylation of ERK5, ERK1/2, and JNK1/2/3, leading to a decrease in PPARγ expression, an increase in the expression of NF-κB and MMP2, and the induction of apoptotic injury. Inhibition of PKCα activation or knockdown of PKCα using shRNA leads to a suppression of ERK5, ERK1/2, JNK1/2/3, and P38 phosphorylation, an elevation in PPARγ expression, and a reduction in NF-κB and MMP2 expression, alleviated apoptotic injury. Furthermore, we observe that the regulation of PPARγ, NF-κB, and MMP2 expression is influenced by ERK5 and ERK1/2 phosphorylation, and activation of PPARγ effectively counteracts the elevated expression of NF-κB and MMP2. Conclusion: Our findings suggest that the PKCα/ERK/PPARγ pathway plays a crucial role in mediating endothelial injury under conditions of impinging flow, with potential implications for vascular diseases and intracranial aneurysm development.

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

NETs induce ferroptosis of endothelial cells in LPS-ALI through SDC-1/HS and downstream pathways

BackgroundExtracellular neutrophil extracellular traps (NETs) play an important role in acute lung injury (ALI), but their mechanisms are still unclear. The aim of this study is to explore the effects of NETs on endothelial glycocalyx/HGF/cMET pathway and ferroptosis in ALI and elucidate their potential mechanisms. MethodsPlasma was collected from healthy and sepsis patients to test for differences in neutrophil elastase (NE) expression of NETs components. In addition, LPS-ALI mice and endothelial cell injury models were established, and NETs were disrupted by siPAD4 (a driver gene for NETs) and sivelestat (an inhibitor of the NETs component) in the mice and by sivelestat in the endothelial cell injury models, and the effects of NETs on the SDC-1/HS/HGF/cMET pathway were studied. To verify the relationship between NETs and ferroptosis, Fer1, a ferroptosis inhibitor, was added as a positive control to observe the effect of NETs on ferroptosis indicators. ResultsThe expression level of NE was significantly higher in the plasma of sepsis patients. In ALI mice, intervention in the generation of NETs reduced pulmonary vascular permeability, protected the integrity of SDC-1/HS and promoted the downstream HGF/cMET pathway. In addition, sivelestat also improved the survival rate of mice, decreased the serious degree of ferroptosis. In the endothelial cells, the results were consistent with those of the ALI mice. ConclusionThe study indicates that inhibiting the production of NETs can protect the normal conduction of the SDC-1/HS/HGF/cMET signalling pathway and reduce the severity of ferroptosis.

Endothelial RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury

Endothelial repair is essential for restoring tissue fluid homeostasis following lung injury. R-spondin3 (RSPO3), a secreted protein mainly produced by endothelial cells (ECs), has shown its protective effect on endothelium. However, the specific mechanisms remain unknown. To explore whether and how RSPO3 regulates endothelial regeneration after inflammatory vascular injury, the role of RSPO3 in sepsis-induced pulmonary endothelial injury was investigated in EC-specific RSPO3 knockdown, inducible EC-specific RSPO3 deletion mice, EC-specific RSPO3 overexpression mice, systemic RSPO3-administration mice, in isolated mouse lung vascular endothelial cells (MLVECs), and in plasma from septic patients. Here we show that plasma RSPO3 levels are decreased in septic patients and correlated with endothelial injury markers and PaO2/FiO2 index. Both pulmonary EC-specific knockdown of RSPO3 and inducible EC-specific RSPO3 deletion inhibit pulmonary ECs proliferation and exacerbate ECs injury, whereas intra-pulmonary EC-specific RSPO3 overexpression promotes endothelial recovery and attenuates ECs injury during endotoxemia. We show that RSPO3 mediates pulmonary endothelial regeneration by a LGR4-dependent manner. Except for β-catenin, integrin-linked kinase (ILK)/Akt is also identified as a novel downstream effector of RSPO3/LGR4 signaling. These results conclude that EC-derived RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury.

Uvaol alleviates oxidative stress induced human umbilical vein endothelial cell injury by suppressing mitogen-activated protein kinase signaling pathway.

Deep venous thrombosis (DVT) is a potentially life-threatening disorder with high morbidity. Uvaol is a natural pentacyclic triterpene possessing multiple pharmacological activities. Nevertheless, the role of uvaol in DVT is unclarified. Human umbilical vein endothelial cells (HUVECs) were treated with hydrogen peroxide (H 2 O 2 ) to mimic DVT in vitro . CCK-8 assay and flow cytometry were utilized for measuring cell viability and apoptosis, respectively. Levels of the cell injury marker, thrombosis-associated factors, inflammatory cytokines, and oxidative stress-related markers were examined by commercial assay kits. Western blotting was used for evaluating the expression of mitogen-activated protein kinase (MAPK) signaling-associated proteins. Uvaol treatment attenuated H 2 O 2 -induced HUVEC apoptosis and injury. Uvaol reduced the expression of pro-thrombotic factors and inflammatory cytokines and attenuated oxidative stress in H 2 O 2 -stimulated HUVECs. Uvaol inhibited MAPK signaling pathway in H 2 O 2 -stimulated HUVECs. Activating MAPK signaling reversed uvaol-mediated protective effects on H 2 O 2 -treated HUVECs. Uvaol treatment alleviates H 2 O 2 -induced HUVEC injury, apoptosis, and oxidative stress by inactivating MAPK signaling.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Emodin Blocks mPTP Opening and Improves LPS-Induced HMEC-1 Cell Injury by Upregulation of ATP5A1.

Emodin has been shown to exert anti-inflammatory and cytoprotective effects. Our study aimed to identify a novel anti-inflammatory mechanism of emodin. An LPS-induced model of microvascular endothelial cell (HMEC-1) injury was constructed. Cell proliferation was examined using a CCK-8 assay. The effects of emodin on reactive oxygen species (ROS), cell migration, the mitochondrial membrane potential (MMP), and the opening of the mitochondrial permeability transition pore (mPTP) were evaluated. Actin-Tracker Green was used to examine the relationship between cell microfilament reconstruction and ATP5A1 expression. The effects of emodin on the expression of ATP5A1, NALP3, and TNF-α were determined. After treatment with emodin, ATP5A1 and inflammatory factors (TNF-α, IL-1, IL-6, IL-13 and IL-18) were examined by Western blotting. Emodin significantly increased HMEC-1 cell proliferation and migration, inhibited the production of ROS, increased the mitochondrial membrane potential, and blocked the opening of the mPTP. Moreover, emodin could increase ATP5A1 expression, ameliorate cell microfilament remodeling, and decrease the expression of inflammatory factors. In addition, when ATP5A1 was overexpressed, the regulatory effect of emodin on inflammatory factors was not significant. Our findings suggest that emodin can protect HMEC-1 cells against inflammatory injury. This process is modulated by the expression of ATP5A1.

Coal-fired PM2.5 induces endothelial cell injury and the expression of atherosclerosis-related adhesion molecules: Involvement of the p38 and JNK signaling pathways

It has been reported that PM2.5 causes endothelial cell injury and promotes atherosclerosis. However, the exact mechanism through which coal-fired PM2.5 induces endothelial cell injury, and the involvement of the p38 and JNK signaling pathways in this process remain unclear. In this study, EA.hy926 cells were exposed to coal-fired PM2.5 at concentrations of 25, 50, and 100 μg/mL, and the toxic effects were observed. The phosphorylation of the JNK and p38 signaling pathways was investigated through western blot analysis. Additionally, the expression of adhesion molecules (ICAM-1 and E-selectin) was assessed using ELISA and flow cytometry. Changes in cellular toxicity and adhesion molecules were evaluated after pretreatment with the p38 inhibitor SB203580 (10 μM) and the JNK inhibitor SP600125 (25 μM). We observed that exposure to coal-fired PM2.5 led to a decrease in cell survival rate and proliferation while promoting apoptosis. Exposure to coal-fired PM2.5 promoted the expression of ICAM-1 and E-selectin, as well as the phosphorylation of p38 and JNK in EA.hy926 cells. After using p38 and JNK inhibitors, there was an observed increase in cell survival rate and proliferation, accompanied by a decrease in apoptosis. The levels of ICAM-1 and E-selectin showed no significant changes with the addition of p38 and JNK inhibitors. Our results indicated that coal-fired PM2.5 caused cellular toxicity and increased the levels of ICAM-1 and E-selectin. The p38 and JNK signaling pathways might play a role in the reduction of cell viability, while the regulation of ICAM-1 and E-selectin might not be influenced by these pathways.