Endothelial Cell Inflammation Research Articles

LncRNA TGFB2-OT1 Promotes Progression and Angiogenesis in Hepatocellular Carcinoma by Dephosphorylating β-Catenin.

Hepatocellular carcinoma (HCC) was the sixth most prevalent cancer worldwide. Long non-coding RNA TGFB2-OT1 has been proven to mediate inflammation and autophagy in vascular endothelial cells. However, its function in HCC is still unknown. We analyzed the relationship between TGFB2-OT1 expression and the clinicopathological features of 202 HCC patients. RT-qPCR was used to analyze the TGFB2-OT1 expression in HCC cell lines and tissues. In vitro and in vivo assays were conducted to verify the effect of TGFB2-OT1 on the phenotype of HCC. RNA pull-down assays were applied to reveal the proteins binding to the TGFB2-OT1. Western-blot assays were conducted to analyze the protein expression in HCC cell lines. TGFB2-OT1 was found to be highly expressed in HCC samples and hepatoma cells. TGFB2-OT1 expression was significantly associated with age (P = 0.001), cirrhosis (P = 0.003), tumor size (P < 0.001), tumor encapsulation (P = 0.029), tumor protruding from the liver surface (P = 0.040), and alpha fetoprotein (AFP, P < 0.001) levels. TGFB2-OT1 promoted proliferation, migration, invasion, and angiogenesis in HCC cells, both in vitro and in vivo. TGFB2-OT1 binds to β-catenin and competitively impaired the binding of β-catenin to GSK3β, thus suppressing the phosphorylation of β-catenin at Ser33, Ser37, and Thr41. TGFB2-OT1 is overexpressed in HCC and predicts the poor prognosis of HCC patients. TGFB2-OT1 impedes the phosphorylation of β-catenin and acts as an alternative activator of the Wnt/β-catenin pathway to promote the progression and angiogenesis of HCC.

Low shear stress induces inflammatory response via CX3CR1/NF-κB signal pathway in human umbilical vein endothelial cells

Low shear stress (LSS) has been reported to induce atherosclerosis. However, the molecular mechanisms underlying inflammation induced by LSS are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in low shear stress-induced inflammation in human umbilical vein endothelial cells (HUVECs) through protein profiling and cell function experiment. In this study, Western blotting analyses revealed a significant increase in the expression of CX3CR1, nucleusP65, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6), while the expression of cytosolic P65 and IκB has significantly decreased in HUVECs treated with low shear stress. CX3CR1 Sh-RNA was use to reveal its effect on LSS-induced inflammation. Further, specific NF-κB P65 inhibitors pyrrolidinedithiocarbamate (PDTC) were used to reveal the downstream NF-κB P65 exclusively involved in LSS-induced inflammation in HUVECs, this effect can be abrogated by CX3CR1 sh-RNA and NF-κB inhibitors. Monocyte adhesion assay and scratch test revealed low shear stress to promotes adhesion of monocytes and migration of cells, this effect can be abrogated by CX3CR1 sh-RNA and NF-κB inhibitors. LSS was involved in the expression of adhesion molecules and chemokines, which are important for the initiation of endothelial inflammation-related atherosclerosis. Therefore, the cell signaling pathways activated by LSS in endothelial cells may represent therapeutic targets of atherosclerosis.

Rod‐Shaped Polymeric Nanoparticles Intervene Neutrophils for Efficient Ischemic Stroke Therapy

AbstractNeutrophils are associated with pro‐inflammation and contribute to pathophysiology of ischemic stroke. Prevention of neutrophils from infiltrating into ischemic area can be a potential approach for stroke therapy. In this study, a rod‐shaped polylactic acid polyglycolic acid (PLGA) nanoparticles encapsulated with piceatannol is developed to target and intervene the interaction of neutrophils with inflamed endothelial cells. In contrast to macrophages, neutrophils exhibit enhanced endocytosis of elongated particles. The nanoparticles with aspect ratio (AR) of 5 display higher internalization by neutrophils compared to other nanoparticles. Thus, AR5 nanoparticles are screened out to load with piceatannol (Pic@AR5) for investigating the therapeutic effect against middle cerebral artery occlusion model. It is demonstrated that Pic@AR5 can block the adherence of neutrophils to endothelial cells, inhibiting the infiltration of neutrophils into blood‐brain barrier (BBB). Besides, the inflammatory cytokine of ischemic brain can also be reduced even if a small part of Pic@AR5‐carried neutrophils enter BBB. The nanoparticles release piceatannol within the ischemic region, which inhibit microglial Syk signal and result in alleviation of neuroinflammation. This strategy provides new insights into ischemic stroke therapy.

Overexpression of FoxM1 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on acute respiratory distress syndrome

BackgroundInjury of alveolar epithelial cells and capillary endothelial cells is crucial in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) are a promising cell source for ALI/ARDS treatment. Overexpression of Fork head box protein M1 (FoxM1) facilitates MSC differentiation into alveolar type II (AT II) cells in vitro. Moreover, FoxM1 has been shown to repair the endothelial barrier. Therefore, this study explored whether overexpression of FoxM1 promotes the therapeutic effect of bone marrow-derived MSCs (BMSCs) on ARDS by differentiation of BMSCs into AT II cells or a paracrine mechanism.MethodsA septic ALI model was established in mice by intraperitoneal administration of lipopolysaccharide. The protective effect of BMSCs-FoxM1 on ALI was explored by detecting pathological variations in the lung, total protein concentration in bronchoalveolar lavage fluid (BALF), wet/dry (W/D) lung weight ratio, oxidative stress levels, cytokine levels, and retention of BMSCs in the lung. In addition, we assessed whether FoxM1 overexpression promoted the therapeutic effect of BMSCs on ALI/ARDS by differentiating into AT II cells using SPC−/− mice. Furthermore, the protective effect of BMSCs-FoxM1 on lipopolysaccharide-induced endothelial cell (EC) injury was explored by detecting EC proliferation, apoptosis, scratch wounds, tube formation, permeability, and oxidative stress, and analyzing whether the Wnt/β-catenin pathway contributes to the regulatory mechanism in vitro using a pathway inhibitor.ResultsCompared with BMSCs-Vector, treatment with BMSCs-FoxM1 significantly decreased the W/D lung weight ratio, total BALF protein level, lung injury score, oxidative stress, and cytokine levels. With the detected track of BMSCs-FoxM1, we observed a low residency rate and short duration of residency in the lung. Notably, SPC was not expressed in SPC−/− mice injected with BMSCs-FoxM1. Furthermore, BMSCs-FoxM1 enhanced EC proliferation, migration, and tube formation; inhibited EC apoptosis and inflammation; and maintained vascular integrity through activation of the Wnt/β-catenin pathway, which was partially reversed by XAV-939.ConclusionOverexpression of FoxM1 enhanced the therapeutic effect of BMSCs on ARDS, possibly through a paracrine mechanism rather than by promoting BMSC differentiation into AT II cells in vivo, and prevented LPS-induced EC barrier disruption partially through activating the Wnt/β-catenin signaling pathway in vitro.

Neutrophil-inspired photothermo-responsive drug delivery system for targeted treatment of bacterial infection and endotoxins neutralization

BackgroundP. aeruginosa, a highly virulent Gram-negative bacterium, can cause severe nosocomial infections, and it has developed resistance against most antibiotics. New therapeutic strategies are urgently needed to treat such bacterial infection and reduce its toxicity caused by endotoxin (lipopolysaccharide, LPS). Neutrophils have been proven to be able to target inflammation site and neutrophil membrane receptors such as Toll-like receptor-4 (TLR4) and CD14, and exhibit specific affinity to LPS. However, antibacterial delivery system based on the unique properties of neutrophils has not been reported.MethodsA neutrophil-inspired antibacterial delivery system for targeted photothermal treatment, stimuli-responsive antibiotic release and endotoxin neutralization is reported in this study. Specifically, the photothermal reagent indocyanine green (ICG) and antibiotic rifampicin (RIF) are co-loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP-ICG/RIF), followed by coating with neutrophil membrane to obtain antibacterial delivery system (NM-NP-ICG/RIF). The inflammation targeting properties, synergistic antibacterial activity of photothermal therapy and antibiotic treatment, and endotoxin neutralization have been studied in vitro. A P. aeruginosa-induced murine skin abscess infection model has been used to evaluate the therapeutic efficacy of the NM-NP-ICG/RIF.ResultsOnce irradiated by near-infrared lasers, the heat generated by NP-ICG/RIF triggers the release of RIF and ICG, resulting in a synergistic chemo-photothermal antibacterial effect against P. aeruginosa (~ 99.99% killing efficiency in 5 min). After coating with neutrophil-like cell membrane vesicles (NMVs), the nanoparticles (NM-NP-ICG/RIF) specifically bind to inflammatory vascular endothelial cells in infectious site, endowing the nanoparticles with an infection microenvironment targeting function to enhance retention time. Importantly, it is discovered for the first time that NMVs-coated nanoparticles are able to neutralize endotoxins. The P. aeruginosa murine skin abscess infection model further demonstrates the in vivo therapeutic efficacy of NM-NP-ICG/RIF.ConclusionThe neutrophil-inspired antibacterial delivery system (NM-NP-ICG/RIF) is capable of targeting infection microenvironment, neutralizing endotoxin, and eradicating bacteria through a synergistic effect of photothermal therapy and antibiotic treatment. This drug delivery system made from FDA-approved compounds provides a promising approach to fighting against hard-to-treat bacterial infections.

Ox-LDL induced profound changes of small non-coding RNA in rat endothelial cells.

Atherosclerosis (AS) is a common cardiovascular disease with a high incidence rate and mortality. Endothelial cell injury and dysfunction are early markers of AS. Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of AS. Ox-LDL promotes endothelial cell apoptosis and induces inflammation and oxidative stress in endothelial cells. Small non-coding RNAs (sncRNAs) mainly include Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), microRNAs (miRNAs) and repeat-associated RNAs. Studies have shown that small non-coding RNAs play an increasingly important role in diseases. We used ox-LDL to treat rat endothelial cells to simulate endothelial cell injury. The expression changes of sncRNA were analyzed by small RNA high-throughput sequencing, and the expression changes of piRNA, snoRNA, snRNA, miRNA and repeat-associated RNA were verified by quantitative polymerase chain reaction (qPCR). Small RNA sequencing showed that 42 piRNAs were upregulated and 38 piRNAs were downregulated in endothelial cells treated with ox-LDL. PiRNA DQ614630 promoted the apoptosis of endothelial cells. The snoRNA analysis results showed that 80 snoRNAs were upregulated and 68 snoRNAs were downregulated in endothelial cells with ox-LDL treatment, and snoRNA ENSRNOT00000079032.1 inhibited the apoptosis of endothelial cells. For snRNA, we found that 20 snRNAs were upregulated and 26 snRNAs were downregulated in endothelial cells with ox-LDL treatment, and snRNA ENSRNOT00000081005.1 increased the apoptosis of endothelial cells. Analysis of miRNAs indicated that 106 miRNAs were upregulated and 91 miRNAs were downregulated in endothelial cells with ox-LDL treatment, and miRNA rno-novel-136-mature promoted the apoptosis of endothelial cells. The repeat RNA analysis results showed that 4 repeat RNAs were upregulated and 6 repeat RNAs were downregulated in endothelial cells treated with ox-LDL. This study first reported the expression changes of sncRNAs in endothelial cells with ox-LDL treatment, which provided new markers for the diagnosis and treatment of endothelial cell injury.

Gambogic acid affects high glucose-induced apoptosis and inflammation of retinal endothelial cells through the NOX4/NLRP3 pathway.

This study aimed to investigate the effect and mechanism of gambogic acid (GA) on the apoptosis and inflammation of human retinal endothelial cells (HRECs) under high glucose conditions. HRECs were cultured in a high glucose medium to simulate retinal endothelial cell injury induced by diabetic retinopathy. Flow cytometry was used to analyze the apoptosis level of HRECs. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Western blotting was applied to detect the intracellular apoptosis-related proteins and expression levels of NADPH oxidase 4 (NOX4), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and interleukin (IL)-1β. Enzyme linked immunosorbent assay (ELISA) was utilized to detect the expression of IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) in the cell supernatants. The messenger RNA (mRNA) levels of IL-6, IL-8, IL-10, and TNF-α were detected by reverse transcription-polymerase chain reaction (RT-qPCR). We observed that high glucose induced the apoptosis and inflammation of HRECs. In addition, the high glucose environment promoted NOX/NLRP3 pathway activation. The activity of HRECs was not significantly affected by the presence of 20 μM or less of GA, and 15 μM of GA could restore the diminished activity of HRECs induced by high glucose. The apoptosis of HRECs cultured under high glucose conditions was significantly inhibited (P<0.05), the levels of IL-6, IL-8, and TNF-α in the cell supernatant were significantly decreased (P<0.05), and the levels of IL-10 were significantly increased (P<0.05). Meanwhile, the relative mRNA expression levels of IL-6, IL-8, and TNF-α in HRECs were significantly decreased (P<0.05), while those of IL-10 were significantly increased (P<0.05). The activity of the high glucose-induced NOX4/NLRP3 pathway in HRECs was significantly inhibited after treatment with 15 μM of GA (P<0.05). Following activation of the NOX4/NLRP3 pathway in HRECs, the apoptosis level was significantly increased (P<0.05), and the inflammatory response was aggravated (P<0.05). Inhibiting the activity of the intracellular NOX4/NLRP3 pathway markedly inhibited cell apoptosis and the inflammatory response (P<0.05). GA can inhibit the apoptosis and inflammation of HRECs under high glucose conditions by inhibiting the activity of the NOX4/NLRP3 pathway. This has a significant inhibitory effect on diabetic retinopathy, which is worthy of further study.

Abstract 111: Cerebrovascular Effects Of Pre/post-losartan Treatment In Humanized ACE2 Knock-in Mice After SARS-CoV-2 Spike Protein Injection

SARS-CoV-2 has killed millions of people globally and has left many with devastating health problems, including neurological deficits. SARS-CoV-2 invades endothelial cells via binding its Spike-protein with angiotensin-converting enzyme 2 (ACE2) receptors. Losartan, an angiotensin receptor blocker, upregulates ACE2 expression. We hypothesize that pre-treatment with losartan could increase the cerebrovascular dysfunction associated with SARS-CoV-2 spike-protein, while post-SARS-CoV-2 losartan would provide potential protective effects. Methods: Recombinant SARS-CoV-2 spike-protein was injected intravenously in transgenic hACE2 mice, humanized ACE2 receptors knock-in mice. Treatment with losartan was started either two weeks before or right after spike-protein injection. Cognitive function was assessed using Y-Maze. Vascular density and cerebral blood flow were evaluated in the hACE2 mice with/out spike protein injection. Western blot and RT-PCR analysis were used to measure protein/gene expression. Results: Spike-protein injection decreased cerebral vascular density and cerebral blood flow in hACE2 brains compared to control (*P&lt;0.05), which was associated with increased cognitive dysfunction in h ACE2 mice (*P&lt;0.05). Pre-losartan treatment exacerbated spike protein-induced cognitive dysfunction, while post-losartan treatment helped restore cognitive function (*P&lt;0.05). Spike-protein decreased ACE2 expression and increased endothelial cell inflammation and apoptosis, as shown by the increased expression of TNF- α ; and caspase-3 (*P&lt;0.05), which were prevented by post-losartan treatment. Conclusion: Spike-protein decreased vascular density, cerebral blood flow, and cognitive function via increasing brain endothelial cell apoptosis. Pre-losartan treatment exacerbated the detrimental effects of spike-protein, while post-losartan treatment restored ACE2 expression and decreased SARS-CoV-2 spike-protein damaging effects.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Intravenous iron supplementation in heart failure patients induces temporary endothelial dysfunction with release of endothelial microvesicles.

Intravenous iron supplementation is an established therapy for patients with heart failure (HF) and concomitant iron deficiency reducing the risk of HF hospitalization. However, concerns persist regarding potential adverse vascular effects, since iron may induce oxidative stress, inflammation, and apoptosis of endothelial cells. To assess endothelial health following ferric carboxymaltose (FCM) administration, we analyzed the profile of circulating endothelial microvesicles (EMVs) and endothelial progenitor cells (EPCs) in a cohort of 23 HF patients using flow cytometry. Compared to healthy subjects, baseline levels of CD31+/CD41- EMVs were higher and EMVs featured a more apoptotic phenotype in HF patients. Following FCM administration, EMV levels showed a rapid but transient increase and displayed an altered phenotype profile with dominant augmentation of EMVs expressing inducible markers CD62E and CD54, indicating endothelial inflammatory activation and injury. Levels of circulating vasoregenerative CD45lowCD34+KDR+ EPCs were lower in HF patients and FCM application resulted in an early decrease of EPCs followed by substantial mobilization into the circulation after one week. Levels of EMVs and EPCs returned to baseline values within two and four weeks, respectively. HF patients with additional chronic kidney disease showed an elevated EMV/EPC ratio and diminished EPC mobilization, suggesting impaired vascular repair capacity. Providing a mechanistic link, in vitro experiments with cultured endothelial cells revealed that FCM dose-dependently promotes endothelial apoptosis, increases expression of adhesion molecules and CXCL12, and triggers generation of EMVs. Intravenous iron supplementation with FCM in HF patients induces a biphasic response with initial increased release of CD62E+ and CD54+ enriched EMVs and subsequent mobilization of EPCs, indicating endothelial dysfunction upon FCM and suggesting consecutive engagement of a defense program aimed to reconstitute vascular health.

Potential roles of NLRP3 inflammasome in the pathogenesis of Kawasaki disease.

There is a heterogeneous group of rare illnesses that fall into the vasculitis category and are characterized mostly by blood vessel inflammation. Ischemia and disrupted blood flow will cause harm to the organs whose blood arteries become inflamed. Kawasaki disease (KD) is the most prevalent kind of vasculitis in children aged 5years or younger. Because KD's cardiovascular problems might persist into adulthood, it is no longer thought of as a self-limiting disease. KD is a systemic vasculitis with unknown initiating factors. Numerous factors, such as genetic predisposition and infectious pathogens, are implicated in the etiology of KD. As endothelial cell damage and inflammation can lead to coronary endothelial dysfunction in KD, some studies hypothesized the crucial role of pyroptosis in the pathogenesis of KD. Additionally, pyroptosis-related proteins like caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), proinflammatory cytokines like IL-1 and IL-18, lactic dehydrogenase, and Gasdermin D (GSDMD) have been found to be overexpressed in KD patients when compared to healthy controls. These occurrences may point to an involvement of inflammasomes and pyroptotic cell death in the etiology of KD and suggest potential treatment targets. Based on these shreds of evidence, in this review, we aim to focus on one of the well-defined inflammasomes, NLRP3, and its role in the pathophysiology of KD.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi's sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Hypoxia activates GPR146 which participates in pulmonary vascular remodeling by promoting pyroptosis of pulmonary artery endothelial cells

BackgroundHypoxia is a risk factor of pulmonary hypertension (PH) and may induce pulmonary artery endothelial cells (PAECs) injury and inflammation. Pyroptosis is a form of cell death through maturation and secretion of inflammatory mediators. However, the mechanistic association of pyroptosis, PAECs injury, and inflammation remain unknown. Here, we explored in detail the effects of hypoxia on pyroptosis of PAECs. Experimental approachUsing RNA sequencing, we screened differentially expressed genes in pulmonary artery tissue of a Sugen5416/hypoxia-induced (SuHx) rat PH model. We examined the role of the differentially expressed gene G-protein coupled receptor 146 (GPR146) in PAECs through immunohistochemistry, immunofluorescence, CCK-8 assays, western blotings, real-time PCR, detection of reactive oxygen species, and lactate dehydrogenase release experiments. Key resultsAccording to RNA sequencing, GPR146 was 11.64-fold increased in the SuHx-induced PH model, compared to the controls. Further, GPR146 was highly expressed in pulmonary arterial hypertension human lung tissue and SuHx-induced rat PH lung tissues. Our results suggested that the expression of pyroptosis-related proteins was markedly increased under hypoxia, both in vivo and in vitro, which was inhibited by silencing GPR146. Moreover, inhibiting NLRP3 or caspase-1 effectively suppressed cleavage of caspase-1, production of interleukin (IL)-1β, IL-6, and IL-18 in PAECs by hypoxia and overexpression of GPR146. ConclusionOur results indicated that GPR146 induced pyroptosis and inflammatory responses through the NLRP3/caspase-1 signaling axis, thus triggering endothelial injury and vascular remodeling. Hypoxia may promote PAECs pyroptosis through upregulation of GPR146 and thereby facilitate the progression of PH. Taken together, these insights may help identify a novel target for the treatment of PH.

NEDD4 attenuates oxidized low‑density lipoprotein‑induced inflammation and dysfunction in vascular endothelial cells via regulating APEX1 expression.

Atherosclerosis chiefly results from inflammation as well as vascular endothelial cell dysfunction. Methylation levels of neuronally expressed developmentally downregulated 4 (NEDD4) were found to be fortified in atherosclerosis patients and NEDD4 deficiency enhanced vascular calcification. However, the exact function of NEDD4 in inflammation and vascular endothelial dysfunction remains to be elucidated. In the present study, CCK-8 assay was used to estimate cell viability. Reverse transcription-quantitative PCR was adopted to examine the expression of NEDD4, inflammation-associated enzymes and apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1). Western blotting was used to test NEDD4, endothelial nitric oxide synthase, inducible nitric oxide synthase and APEX1 protein levels. Cytotoxicity was detected by a lactate dehydrogenase (LDH) kit. Reactive oxygen species level was tested by a corresponding kit. Vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 contents were examined with ELISA. Cell adhesion assays evaluated the adhesion of endothelial cells. Co-immunoprecipitation assay was used to test the relationship between NEDD4 and APEX1. The data revealed that NEDD4 expression rapidly declined in oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Following NEDD4 overexpression, the active damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were attenuated. After co-transfection of APEX1 interference plasmids and NEDD4 overexpression plasmids, cell damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were improved again. Taken together, NEDD4 attenuated ox-LDL-induced inflammation and endothelial dysfunction by regulating APEX1 expression.

TRIM59 attenuates ox-LDL-induced endothelial cell inflammation, apoptosis, and monocyte adhesion through AnxA2.

Atherosclerosis (AS), a chronic inflammatory vascular disease, is a cause of heart attack and ischemic stroke. Tripartite motif-containing protein 59 (TRIM59), a member of the tripartite motif family, has been reported to be involved in inflammatory diseases. This study was to investigate the role of TRIM59 in oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells and examine the mechanism of TRIM59. To simulate a cellular model of AS in vitro, varying concentrations of ox-LDL (i.e., 20, 40, 60, 80, and 100 µg/mL) were used to treat the human umbilical vein endothelial cells (HUVECs) for 24 h. The messenger ribonucleic acid (RNA) and protein levels of TRIM59, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and annexin 2 (AnxA2) were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficacy of overexpression (Ov)-TRIM59 and small-interfering RNA-AnxA2 was examined by RT-qPCR and western blot. Cell counting kit-8 assays, lactate dehydrogenase (LDH) assays, enzyme-linked immunosorbent assays, and terminal-deoxynucleotidyl transferase mediated nick end labeling staining were used to examine viability, LDH expression, inflammation, and apoptosis in HUVECs. The protein levels of B-cell lymphoma 2, Bcl-2-associated X (BAX), cleaved caspase3, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were assessed by western blot. Additionally, the adhesion of THP-1 to ox-LDL-induced HUVECs was detected using monocyte adhesion assays and the binding of TRIM59 and AnxA2 was verified by co-immunoprecipitation. This study showed that TRIM59 expression was decreased in the ox-LDL-induced HUVECs while LOX-1 expression was increased. After transfection with Ov-TRIM59, TRIM59 in ox-LDL-induced HUVECs was increased, and TRIM59 overexpression alleviated the viability damage, inflammation, and apoptosis of the ox-LDL-induced HUVECs. In addition, THP-1 adhesion to the ox-LDL-induced HUVECs was also suppressed by TRIM59 overexpression. This study also showed that TRIM59 could bind to AnxA2 and promote AnxA2 expression in ox-LDL-stimulated HUVECs. Moreover, the rescue experiments revealed that TRIM59 suppressed the viability damage, inflammation, apoptosis, and monocyte adhesion of the ox-LDL-induced HUVECs via AnxA2. TRIM59 protected against ox-LDL-induced AS by binding to AnxA2.

The effect of simvastatin treatment on TNF-alpha-induced inflammation in human vascular endothelial cells (HUVECs)

The effect of simvastatin treatment on TNF-alpha-induced inflammation in human vascular endothelial cells (HUVECs)

SIRT3 mediates the effects of PCSK9 inhibitors on inflammation, autophagy, and oxidative stress in endothelial cells.

Background: Proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors (i) are a class of lipid-lowering drugs suggested to hold a plethora of beneficial effects independent of their LDL cholesterol-lowering properties. However, the mechanism underlying such observations is debated. Methods: Human aortic endothelial cells (TeloHAEC) were pre-treated with 100 µg/mL of the PCSK9i evolocumab and then exposed to 20 ng/mL of IL-6, a major driver of cardiovascular diseases (CVD), in both naïve state and after siRNA-mediated suppression of the NAD-dependent deacetylase sirtuin-3 (SIRT3). Inflammation, autophagy, and oxidative stress were assessed through Western Blots, ELISAs, and/or immunofluorescence coupled by flow cytometry. To explore the human relevance of the findings, we also evaluated the expression of IL-6, SIRT3, IL-1β, the ratio LC3B II/I, and PCSK9 within the plaques of patients undergoing carotid endarterectomy (n=277), testing possible correlations between these proteins. Results: PCSK9i improved a range of phenotypes including the activation of inflammatory pathways, oxidative stress, and autophagy. Indeed, treatment with PCSK9i was able to counteract the IL-6 induced increase in inflammasome activation, the accrual of autophagic cells, and mitochondrial ROS accumulation. Of note, silencing of SIRT3 reverted the beneficial effects observed with PCSK9i treatment on all these phenomena. In atheroma specimens, the expression of PCSK9 was inversely related to that of SIRT3 while positively correlating with IL-6, IL-1β, and the ratio LC3B II/I. Conclusions: Overall, these data suggest that PCSK9i bear intrinsic anti-inflammatory, anti-autophagic, and antioxidant properties in endothelial cells, and that these pleiotropic effects might be mediated, at least in part, by SIRT3. These results provide an additional mechanism supporting the emerging knowledge relative to the benefit of PCSK9i on CVD beyond LDL-lowering and uncover SIRT3 as a putative mediator of such pleiotropy.

PS-B01-5: TISSUE-SPECIFIC REGULATION OF LRG1 EXPRESSION IN MULTIPLE HYPERTENSION MODELS OF MOUSE

Object: Leucine-rich alpha-2-glycoprotein-1 (LRG1), which is firstly isolated from human plasma, has been reported to be related to several diseases as a biomarker or a pathogenic factor. Serum LRG1 is associated with the prognosis of diabetic nephropathy and cardiovascular diseases, and we previously demonstrated that LRG1 especially plays a pivotal role in the initial development of diabetic nephropathy. In addition, a previous study showed that increased plasma LRG1 levels in patients with type 2 diabetes mellitus was associated with increased systolic blood pressure (BP), arterial stiffness estimated by pulse wave velocity, and decreased endothelium-dependent vasodilation estimated by laser doppler flowmetry. However, the relationship between LRG1 and BP have remained substantially unknown. With respect to the function of LRG1, LRG1 is expressed in endothelial cells and promotes pathological angiogenesis by modulating TGF-beta signaling. LRG1 switches TGF-beta signaling from the Smad2/3 pathway to the Smad1/5/8 pathway. In addition, the activation of Smad2/3 signaling enhanced endothelial nitric oxide synthase (eNOS) production and promoted endothelium-dependent vasodilation. Suppressing eNOS activity reduces NO levels and leads to endothelial dysfunction and hypertension. Moreover, LRG1 is increased in response to inflammation and oxidative stress in endothelial cells. However, little is known about the relationship between LRG1 in endothelial cells and eNOS. Based on the previous findings, we hypothesized that LRG1 may inhibit eNOS production by potentially blocking the Smad2/3 pathway, which will promote to form a vicious cycle that progresses vascular endothelial damage and hypertension. As the first step to elucidate this hypothesis, we investigated LRG1 expression in various tissues including vascular endothelium in some hypertension models with endothelial dysfunction. Design and method: Male 10–12 weeks-aged C57/BL mice were divided into control and hypertension with endothelial dysfunction. BP was measured using a tail-cuff method during the experiment, and mice were sacrificed and their organs were collected at the end of the experiment. We examined the expression of LRG1, Smad2/3, Smad1/5/8 and NOS in the vascular endothelium, kidney, heart, adipose tissue by PCR, Western blot and immunohistochemical analyses. Results: We are currently conducting the experiments and will present our findings at this conference. Conclusion: We plan to present the results of tissue-specific regulation of LRG1 expression in some hypertension models of mice at this conference.

Enhanced brain delivery of hypoxia-sensitive liposomes by hydroxyurea for rescue therapy of hyperacute ischemic stroke.

Ischemic stroke is characterized by high morbidity, disability, and mortality. Unfortunately, the only FDA-approved pharmacological thrombolytic, alteplase, has a narrow therapeutic window of only 4.5 h. Other drugs like neuroprotective agents have not been clinically used because of their low efficacy. To improve the efficacy of neuroprotective agents and the effectiveness of rescue therapies for hyperacute ischemic stroke, we investigated and verified the variation trends of the blood-brain barrier (BBB) permeability and regional cerebral blood flow over 24 h in rats that had ischemic strokes. Hypoperfusion and the biphasic increase of BBB permeability are still the main limiting factors for lesion-specific drug distribution and drugbrain penetration. Herein, the nitric oxide donor hydroxyurea (HYD) was reported to downregulate the expression of tight junction proteins and upregulate intracellular nitric oxide content in the brain microvascular endothelial cells subjected to oxygen-glucose deprivation, which was shown to facilitate the transport of liposomes across brain endothelialmonolayer in an in vitro model. HYD also increased the BBB permeability and promoted microcirculation in the hyperacute phase of stroke. The neutrophil-like cell-membrane-fusogenic hypoxia-sensitive liposomes exhibited excellent performance in targeting the inflamed brain microvascular endothelial cells, enhancing cell association, and promoting rapid hypoxic-responsive release in the hypoxic microenvironment. Overall, the combined HYD and hypoxia-sensitive liposome dosing regimen effectively decreased the cerebral infarction volume and relieved neurological dysfunction in rats that had ischemic strokes; these therapies were involved in the anti-oxidative stress effect and the neurotrophic effect mediated by macrophage migration inhibitory factor.

PS-BPB01-1: SPARC IS INDUCED AT THE EARLY STAGE OF VASCULAR ENDOTHELIAL DYSFUNCTION TO COMPENSATE INFLAMMATION IN DOCA-SALT HYPERTENSIVE RATS

Objective: Extracellular matrix (ECM) regulates various cellular functions as well as provides structural support. SPARC (secreted protein acidic and rich in cysteine) is one of the ECM components, which participates in various cellular functions, especially during morphogenesis and tissue remodeling. We have reported that 1) SPARC, which increases in accordance with renal injury, causes collagen deposition through ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) production, and 2) renin-angiotensin system is placed in the upstream of SPARC overexpression in the hypertensive rat kidney. The present study investigated the roles for SPARC in vascular endothelial cell dysfunction and inflammation in the setting of hypertension. Design and method: Rats were treated with deoxycorticosterone acetate (DOCA) and 1% salt water for 0, 1, 2 or 3 weeks. Blood pressure was measured by tail-cuff methods, and thoracic aorta was isolated to examine acetylcholine-induced vasodilatory response, macrophage infiltration by immunohistochemistry, and protein expression of SPARC and ADAMTS1 by Western blotting. In the separate experiment, angiotensin II (10–7mol/L, 6hr) or SPARC recombinant protein (100nmol/L, 24hr) was treated to cultured rat aortic endothelial cells (RAECs) with/without siRNA targeting SPARC and ADAMTS1. MCP-1 and LOX-1 mRNA levels were measured by real-time RT-PCR. Result: DOCA-salt caused time-dependent increases in systolic blood pressure, impairment of endothelium-dependent vasodilation, and macrophage infiltration into the adventitial area of the aorta. The expression of SPARC in the DOCA-salt rat aorta was enhanced at week1 and week2 compared to week0. ADAMTS1 expression tended to increase from week1 and showed significant increases at week3. In RAECs, SPARC and ADAMTS1 gene knockdown aggravated angiotensin II-induced overexpression of LOX-1 and MCP-1 mRNA, indicating anti-inflammatory effects of SPARC. On the other hand, SPARC recombinant protein treatment induced the upregulation of LOX-1 and MCP-1. Conclusions: SPARC may be induced at the early stage of vascular injury to compensate inflammation under the existence of angiotensin II. We need further investigation into the diverse effects of SPARC and the relationship between SPARC and ADAMTS1.