Mouse Vascular Endothelial Cells Research Articles

A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

VEGF-induced Nrdp1 deficiency in vascular endothelial cells promotes cancer metastasis by degrading vascular basement membrane.

Vascular endothelial cells (VECs) are key players in the formation of neovessels and tumor metastasis, the ultimate cause of the majority of cancer-related human death. However, the crosstalk between VECs and metastasis remain greatly elusive. Based on our finding that tumor-associated VECs present significant decrease of Nrdp1 protein which is closely correlated with higher metastatic probability, herein we show that the conditional medium from hypoxia-incubated cancer cells induces extensive Nrdp1 downregulation in human and mouse VECs by vascular endothelial growth factor (VEGF), which activates CHIP, followed by Nrdp1 degradation in ubiquitin-proteasome-dependent way. More importantly, lung metastases of cancer cells significantly increase in conditional VECs Nrdp1 knockout mice. Mechanically, Nrdp1 promotes degradation of Fam20C, a secretory kinase involved in phosphorylating numerous secreted proteins. Reciprocally, deficiency of Nrdp1 in VECs (ecNrdp1) results in increased secretion of Fam20C, which induces degradation of extracellular matrix and disrupts integrity of vascular basement membrane, thus driving tumor metastatic dissemination. In addition, specific overexpression of ecNrdp1 by Nrdp1-carrying adeno-associated virus or chemical Nrdp1 activator ABPN efficiently mitigates tumor metastasis in mice. Collectively, we explore a new mechanism for VEGF to enhance metastasis and role of Nrdp1 in maintaining the integrity of vascular endothelium, suggesting that ecNrdp1-mediated signaling pathways might become potential target for anti-metastatic therapies.

Endothelial deubiquinatase YOD1 mediates Ang II-induced vascular endothelial-mesenchymal transition and remodeling by regulating β-catenin.

Hypertension is a prominent contributor to vascular injury. Deubiquinatase has been implicated in the regulation of hypertension-induced vascular injury. In the present study we investigated the specific role of deubiquinatase YOD1 in hypertension-induced vascular injury. Vascular endothelial endothelial-mesenchymal transition (EndMT) was induced in male WT and YOD1-/- mice by administration of Ang II (1 μg/kg per minute) via osmotic pump for four weeks. We showed a significantly increased expression of YOD1 in mouse vascular endothelial cells upon Ang II stimulation. Knockout of YOD1 resulted in a notable reduction in EndMT in vascular endothelial cells of Ang II-treated mouse; a similar result was observed in Ang II-treated human umbilical vein endothelial cells (HUVECs). We then conducted LC-MS/MS and co-immunoprecipitation (Co-IP) analyses to verify the binding between YOD1 and EndMT-related proteins, and found that YOD1 directly bound to β-catenin in HUVECs via its ovarian tumor-associated protease (OTU) domain, and histidine at 262 performing deubiquitination to maintain β-catenin protein stability by removing the K48 ubiquitin chain from β-catenin and preventing its proteasome degradation, thereby promoting EndMT of vascular endothelial cells. Oral administration of β-catenin inhibitor MSAB (20 mg/kg, every other day for four weeks) eliminated the protective effect of YOD1 deletion on vascular endothelial injury. In conclusion, we demonstrate a new YOD1-β-catenin axis in regulating Ang II-induced vascular endothelial injury and reveal YOD1 as a deubiquitinating enzyme for β-catenin, suggesting that targeting YOD1 holds promise as a potential therapeutic strategy for treating β-catenin-mediated vascular diseases.

PDCD4 knockdown ameliorates lipopolysaccharide-induced endothelial cell damage by improving mitochondrial dynamics

To elucidate the role of programmed cell death factor 4 (PDCD4) in mitochondrial dysfunction caused by sepsis-related vascular endothelial damage. Cultured human umbilical vein endothelial cells (HUVECs) and mouse vascular endothelial cells (C166 cells) were transfected with a small interfering RNA targeting PDCD4 followed by treatment with lipopolysaccharide (LPS) alone or in combination with carbonyl cyanide 3-chlorophenylhydrazone (FCCP). The proteomic changes in the cells after PDCD4 knockdown were analyzed using LC-MS/MS technique. The mRNA expressions of PDCD4 and the genes associated with cell inflammation and apoptosis were detected with RT-PCR, and the expressions of FIS1, DRP1 and OPA1 proteins key to mitochondrial fission and fusion were determined using Western blotting. JC-1 and MitoSOX fluorescent probes were used to observe the changes in mitochondrial membrane potential and mitochondrial reactive oxygen species levels under by a laser confocal microscope. LPS stimulation of the cells significantly increased the mRNA expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1) and enhanced the cellular expression of PDCD4 (P < 0.05). Proteomic analysis suggested a correlation between PDCD4 knockdown and changes in mitochondrial dynamics in the cells. LPS treatment significantly increased the expressions of mitochondrial fission proteins FIS1 and DRP1 and lowered the expression of the fusion protein OPA1 in the cells (P < 0.05), causing also mitochondrial oxidative stress and reduction of the mitochondrial membrane potential (P < 0.05). In HUVECs, treatment with FCCP significantly attenuated the protective effect of PDCD4 knockdown, which inhibited LPS-induced inflammation and oxidative stress and restored the balance between mitochondrial fission and fusion. PDCD4 knockdown protects vascular endothelial cells against LPS-induced damages by repressing mitochondrial fission and oxidative stress, promoting mitochondrial fusion, and maintaining normal mitochondrial function.

Exosomes derived from human umbilical cord mesenchymal stem cells loaded with RVG-Lamp2b and Netrin-1 promotes Schwann cell invasion and migration

BackgroundNetrin-1 has a neuroprotective effect by regulating angiogenesis, autophagy, apoptosis, and neuroinflammation. This study investigated the effects of netrin-1 delivery to mouse Schwann cells and vascular endothelial cells using exosomes modified with rabies virus glycoprotein (RVG) peptides. Materials and methodsRVG-Lamp2b and/or Netrin-1 were overexpressed in human umbilical cord mesenchymal stem cells to obtain exosomes modified with RVG-Lamp2b and/or loaded with Netrin-1. Then, exosomes were labeled with carboxyfluorescein diacetate succinimidyl ester and co-cultured with mouse Schwann cells and endothelial cells. Netrin-1 expression in Schwann cells and endothelial cells was measured using quantitative polymerase chain reaction and immunoblotting. Moreover, methyl thiazolyl tetrazolium assays and Transwell assays were used to detect proliferation, migration, and invasion of Schwann cells and endothelial cells. ResultsExosomes with RVG-Lamp2b entered Schwann cells more readily compared with the exosomes without RVG-Lamp2b. Meanwhile, this was not the case in endothelial cells. Netrin-1-loaded exosomes significantly promoted Netrin-1 expression, cell proliferation, migration, invasion, and epithelial-mesenchymal transition in Schwann cells and endothelial cells. These effects were further enhanced by Netrin-1-loaded exosomes modified with RVG-Lamp2b in Schwann cells, but not in endothelial cells. ConclusionHucMSC-derived exosomes loaded with RVG-Lamp2b and Netrin-1 promote proliferation, migration, and invasion of Schwann cells.

475-P: Intermittent Hypoxia Increased the Expression of Intercellular Adhesion Molecule-1 and Endothelial Cell–Specific Molecule-1 in Vascular Endothelial Cells via the Downregulation of MicroRNA-181a1

Sleep apnea syndrome (SAS), which is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]), is a risk factor for vascular dysfunction, as well as hypertension, and insulin resistance/Type 2 diabetes. We have reported correlations between IH and insulin resistance/Type 2 diabetes, and hypertension. However, the question of why vascular dysfunction is induced by IH has remained unanswered. Here, we investigated the effects of IH on the expression of endothelial inflammatory transcripts such as intercellular adhesion molecule-1 (ICAM-1), interleukin (IL)-8, C-C motif chemokine ligand 5 (CCL5), endothelial nitric oxide synthase (NOS3), vascular cell adhesion molecule-1 (VCAM-1), endothelial cell specific molecule 1 (ESM1), and endothelin-1 (EDN-1) using an in vitro IH system. Human and mouse vascular endothelial cells (HUEhT-1 and UV2) were exposed to IH or normoxia for 24 hours. Real-time RT-PCR revealed that IH significantly increased the mRNA levels of ICAM-1 and ESM1 in both HUEhT-1 (P=0.0436 and P=0.0145, respectively) and UV2 cells (P&amp;lt;0.0001 and P=0.0422, respectively). ELISA showed that ICAM-1 and ESM1 were significantly increased (P=0.0105 and P&amp;lt;0.0001, respectively) in the UV2 cell medium by IH. Reporter assays revealed that the promoter activities of ICAM-1 and ESM1 were not increased by IH. The microRNA (miR)-181a1 level of IH-treated cells was significantly decreased relative to that of normoxia-treated cells (P=0.0374). The IH-induced upregulation of ICAM-1 and ESM1 was abolished by the introduction of miR-181a1 mimic, indicating that IH stress increased levels of ICAM-1 and ESM1 via the inhibition of miR-181a1-mediated mRNA degradation, leading SAS patients to develop atherosclerosis. Disclosure S.Takasawa: None. M.Makino: None. A.Yamauchi: None. S.Sakuramoto-tsuchida: None. Y.Takeda: None. T.Uchiyama: None. R.Shobatake: None.

A ketogenic diet improves vascular hyperpermeability in type 2 diabetic mice by downregulating vascular pescadillo1 expression.

The role of pescadillo1 (PES1) in regulating vascular permeability has been unknown. This study probes the role of PES1 and its mediated molecular mechanism in modulating vascular hyperpermeability in diabetic mice. Male C57BL/6J and db/db mice were fed a standard diet and a ketogenic diet (KD). Meanwhile, mouse vascular endothelial cells (MVECs) were treated with β-hydroxybutyric acid (β-HB), Pes1 siRNA or a Pes1 overexpression plasmid. Additionally, knockout (KO) of Pes1 in mice was applied. After 12 weeks of feedings, enhanced vascular PES1 expression in diabetic mice was inhibited by the KD. The suppression of PES1 was also observed in β-HB-treated MVECs. In mice with Pes1 KO, the levels of vascular VEGF and PES1 were attenuated, while the levels of vascular VE-cadherin, Ang-1 and Occludin were upregulated. Similar outcomes also occurred after the knockdown of Pes1 in cultured MVECs, which were opposite to the effects induced by PES1 overexpression in MVECs. In vitro and in vivo experiments showed that high glucose concentration-induced increases in vascular paracellular permeability declined after MVECs were treated by β-HB or by knockdown of Pes1. In contrast, increases in vascular permeability were induced by overexpression of Pes1, which were suppressed by coadministration of β-HB in cultured endothelial cells. Similarly declines in vascular permeability were found by Pes1 knockdown in diabetic mice. Mechanistically, β-HB decreased PES1-facilitated ubiquitination of VE-cadherin. The KD suppressed the diabetes-induced increase in PES1, which may result in vascular hyperpermeability through ubiquitination of VE-cadherin in type 2 diabetic mice.

Capsaicin Alleviates Vascular Endothelial Dysfunction and Cardiomyopathy via TRPV1/eNOS Pathway in Diabetic Rats.

Background Endothelial dysfunction and cardiomyopathy are considered to be important vascular complications associated with diabetes. This study was designed to investigate whether capsaicin (CAP), a selective TRPV1 agonist, could prevent diabetes-induced endothelial dysfunction and cardiomyopathy. Methods Male Sprague Dawley rats aged 8 weeks were injected intraperitoneally with streptozotocin (STZ, 50 mg/kg) to establish the diabetes model. The diabetic rats were randomly divided into the untreated diabetes group (DM, 10/group) and diabetes plus CAP treatment group (DM+CAP, 10/group); meanwhile, the nondiabetic healthy rats were used as normal controls (10/group). DM+CAP group were treated with CAP by gavage for 8 weeks. The cultured mouse vascular endothelial cells were exposed to different concentrations of glucose in the presence or absence of CAP treatment. The TRPV1 inhibitor capsazepine (CPZ) and eNOS inhibitor L-NAME were used in vivo and in vitro experiment. Results CAP treatment significantly decreased the serum total cholesterol (TC) and total triglyceride (TG) and ameliorated the pathogenesis and fibrosis in the heart, while did not significantly improve plasma glucose level and the body weights of diabetic rats. In addition, CAP enhanced the expression of TRPV1 and eNOS in the heart and normalized the vascular permeability under diabetic state. Similarly, CAP treatment also increased nitric oxide and reduced reactive oxygen species. The same results were observed in cultured mouse vascular endothelial cells by CAP treatment. These beneficial effects of CAP were abolished by either CPZ or L-NAME. Conclusions CAP might protect against hyperglycemia-induced endothelial dysfunction and diabetic cardiomyopathy through TRPV1/eNOS pathway.

High fructose induces dysfunctional vasodilatation via PP2A-mediated eNOS Ser1177 dephosphorylation

BackgroundProcessed foods are popular and contain large amounts of industrial fructose, which changes people’s diet and exacerbates the negative health effects of high fructose. Several studies have shown that excessive intake of fructose has a major impact on vascular disease. However, the mechanism of the effect of high fructose on blood vessels is currently unclear.MethodsThe effect of fructose on the vasodilatation of isolated thoracic aortic rings was observed by using wire myography in wild-type (WT) mice. Cell viability and nitric oxide (NO) production were assessed by the corresponding kits in mouse vascular endothelial cells. The effect of fructose on endothelial nitric oxide synthase (eNOS) and protein phosphatase 2A (PP2A) and their changes in phosphorylation were detected by using Western blots. Moreover, a PP2A inhibitor (okadaic acid, OA) was used to evaluate the relationship between fructose and PP2A. Furthermore, PP2ACα endothelial-specific knockout (PP2A cKO) mice were used to detect the vasodilatation of in vitro fructose-incubated thoracic aortic rings by using wire myography.ResultsHigh fructose induced endothelium-dependent dysfunctional vasodilatation. High fructose reduced acetylcholine (Ach)-induced vasodilation but did not affect sodium nitroprusside (SNP)-induced vasodilation. Accordingly, NO production and the phosphorylation level of eNOS at serine (Ser) 1177 (P-eNOS) in vascular endothelial cells were remarkably reduced without changes in cell viability. The expression of protein phosphatase 2A catalytic subunit (PP2AC) was increased and the expression of phosphorylated PP2AC (P-PP2A, tyrosine [Tyr] 307) was significantly decreased. Nevertheless, these effects were reversed by OA. Moreover, knockout of the PP2A gene could recover the response of vessels to Ach under high fructose stimulation.ConclusionsOur observations demonstrate an underlying mechanism of fructose-induced dysfunctional vasodilatation. Fructose could activate PP2A, which leads to decrease in the phosphorylation of eNOS at Ser1177 and the reduction of NO release, thus leading to the occurrence of endothelium-dependent dysfunctional vasodilatation.

Characterization of \u03b3-H2AX foci formation under alpha particle and X-ray exposures for dose estimation

DNA double-strand break (DSB) induction is one of the phenotypes of cellular damage from radiation exposure and is commonly quantified by γ-H2AX assay with the number of excess fluorescent foci per cell as the main component. However, the number of foci alone may not fully characterize the state of DNA damage following exposures to different radiation qualities. This study investigated the feasibility of utilizing the focus size distribution and dephosphorylation rate of γ-H2AX to identify the type of causative radiation and dose. Human lung epithelial cells and mouse vascular endothelial cells were used to observe the expression changes of γ-H2AX foci due to alpha particle and X-ray exposures. Results showed that the average number of excess foci per cell linearly increased with the dose. The focus size distribution showed a consistent pattern depending on the causative radiation type. Three criteria for the identification of causative radiation type were derived from experimental focus size distributions and were validated in blind testing with correct identification of 27 out of 32 samples. The dose could be estimated based on the proportionality constant specific to the identified radiation type with a difference of less than 15% from the actual value. The different dephosphorylation rates of γ-H2AX produced from alpha particle and X-ray exposures were effectively utilized to determine the individual dose contributions of alpha particles and X-rays under mixed beam exposure. Individual doses were estimated to have differences of less than ~ 12% from actual values.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

CD226 deficiency promotes glutaminolysis and alleviates mitochondria damage in vascular endothelial cells under hemorrhagic shock.

Hemorrhagic shock (HS) is common in clinical emergencies, leading to millions of deaths each year globally. CD226 is a costimulatory adhesion molecule expressed on both immune cells and endothelial cells (ECs) to regulate their metabolic activity and function. As endothelial dysfunction occurs after HS, the roles CD226 plays in vascular EC metabolism were investigated. CD226fl/fl Tekcre mice were adopted to achieve vascular EC-specific knockout of CD226, and subjected to HS modelling. Serum levels of crucial intermediate metabolites were evaluated through liquid chromatography-mass spectrometry analysis. Human umbilical vein ECs (HUVECs) were used to study the effects of CD226 under hypoxia in vitro. Seahorse analysis evaluated the cellular glycolysis and mitochondria bioenergetics. Results showed that CD226 deficiency in vascular ECs alleviated HS-induced intestinal damage and inflammatory response in mice. Animal studies indicated an improved energy metabolism when CD226 was knocked out in ECs after HS, as evidenced by enhanced glutamine-glutamate metabolism and decreased lactic acid levels. Glut-1 was upregulated in mouse vascular ECs after HS and HUVECs under hypoxia, combined with decreased CD226. Moreover, HUVECs with CD226 knockdown exhibited relieved mitochondrial damage and early apoptosis under hypoxia, whereas CD226 overexpression showed opposite effects. Seahorse analysis showed that downregulated CD226 significantly increased mitochondrial ATP production and glucose uptake in HUVECs under hypoxia. Additionally, Erk/PHD2 signaling-mediated HIF-1α/Glut-1 and HIF-2α/ASCT2 pathways were involved in CD226 regulation on HUVEC glutaminolysis after hypoxia. Hence, CD226 deficiency promotes bypass energy supply to vascular ECs under ischemic or hypoxic stress, to ameliorate the stress-mediated metabolic disturbance.

Retraction.

Retraction: "Effects of RNA interference-mediated E-selectin gene silencing on cell adhesion molecule expression and cell-cell adhesion in vascular endothelial cells in mice with immunologic contact urticaria," by Zu-Dong Meng, Xiao-Lan Wang, Tian-Ping Du, Yu Wang, Gui-Fang Qin, Hong-Bo Zhao, Yu-Jie Chen, Bo Tian, J Cell Biochem. 2019; 115-125: The above article, published online on 11 September 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27150) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context. Thus, the editors consider the conclusions of this article to be invalid.

CTLA-2 Alpha Is a Potent Inhibitor of Angiogenesis in Murine Ocular Tissue.

Cytotoxic T lymphocyte antigen-2 (CTLA-2) alpha has been reported to suppress the activities of cathepsin L (Cath L), which is deeply involved in angiogenesis. Therefore, we assessed whether CTLA-2 alpha plays a role in angiogenesis in ocular tissue. To establish models of corneal inflammation and experimental choroidal neovascularization (CNV), male C57BL/6J mice (n = 5) underwent corneal suture placement or laser-induced CNV, respectively. Mice were then injected with recombinant CTLA-2 alpha (1 µg) into the peritoneal cavity at day 0 and every 2 days after operation. In vitro experiments were performed to assess the inflammatory response by measuring TNF-alpha secretion in peritoneal cavity exudate cells (PECs) or the proliferation of mouse vascular endothelial cells (mVECs). CTLA-2 alpha treatment dramatically suppressed corneal angiogenesis, as well as laser-induced CNV. Moreover, CTLA-2 alpha inhibited the proliferation of mVECs in vitro, while CTLA-2 alpha abolishment was able to rescue proliferation. However, CTLA-2 alpha could not suppress cytokine secretion from inflammatory cells such as PECs. In summary, CTLA-2 alpha was able to suppress angiogenesis by suppressing endothelial cell proliferation. Further studies are needed to investigate its usefulness as a new antiangiogenic treatment for a variety of conditions, including age-related macular degeneration.

Silencing ICAM-1 reduces the adhesion of vascular endothelial cells in mice with immunologic contact urticaria.

Immunologic contact urticaria (ICU) is an immediate response of wheal caused by various contactants in vulnerable individuals, with undefined pathogenesis. In the present study, we aim to explore the effects of intercellular cell adhesion molecule-1 (ICAM-1) gene silencing by RNA inference (RNAi) on vascular endothelial cells (VECs) adhesion molecule expression and cell-cell adhesion in ICU mice. Sixty BALB/c mice were selected, among which 48 mice were used for establishment of ICU models. VECs from normal and ICU mice were grouped into different groups. Expressions of ICAM-1, eosinophilic cationic protein (ECP), total immunologlobulin E (tIgE), L-selectin (CD62L), integrin, alpha L (CD11a) in tissues and cells were evaluate by RT-qPCR and western blotting. Cell proliferation was evaluated by MTT assay and EdU staining and cell adhesive function by cell-cell adhesion assay. Compared with normal mice, ICU mice had increased expressions of ICAM-1, ECP, tIgE, CD62L, and CD11a.ICAM-1 gene silencing decreased expressions of ECP, tIgE, CD62L, and CD11a, enhanced cell proliferation, and more activity in cell adhesion. These findings indicate that RNAi-mediated gene silencing of ICAM-1 may decrease VECs adhesion expression and reduce cell-cell adhesion in mice with ICU.

MiR-155 inhibits oxidized low-density lipoprotein-induced apoptosis in different cell models by targeting the p85α/AKT pathway.

The apoptosis of vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and macrophages directly causes the instability or rupture of atherosclerotic plaques. Accumulating evidence suggests that oxidized low-density lipoprotein (OxLDL) could induce apoptosis via endogenous or exogenous pathways. Interestingly, it has been reported that microRNA155 (miR-155) plays a pivotal role in the regulation of apoptosis. Here, we hypothesized that overexpression of miR-155 could inhibit OxLDL-induced apoptosis by targeting the p85α/AKT pathway. In this study, we established models of OxLDL-induced apoptosis in mouse VECs, VSMCs, and macrophages. Furthermore, we explored the effects of miR-155 expression on the apoptosis of different cells, and ultimately revealed whether miR-155 regulated apoptosis by targeting the p85α/AKT pathway. The results demonstrated that miR-155 inhibited p85α expression and attenuated VEC, VSMC, and macrophage apoptosis, at least in part by suppressing the expression of p85α-activated AKT to inhibit apoptosis. Our findings collectively suggested that miR-155 attenuated OxLDL-mediated apoptosis in different cells by targeting p85α, supporting its possible therapeutic role in atherosclerosis.

NDRG1 activates VEGF-A-induced angiogenesis through PLC\u03b31/ERK signaling in mouse vascular endothelial cells

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.

Genetic and Functional Variants Analysis of the GATA6 Gene Promoter in Acute Myocardial Infarction.

Background: Acute myocardial infarction (AMI) which is a specific type of coronary artery disease (CAD), is caused by the combination of genetic factors and acquired environment. Although some common genetic variations have been recorded to contribute to the development of CAD and AMI, more genetic factors and potential molecular mechanisms remain largely unknown. The GATA6 gene is expressed in the heart during embryogenesis and is also detected in vascular smooth muscle cells (VSMCs), different human primary endothelial cells (ECs), and vascular ECs in mice. To date, no studies have directly linked GATA6 gene with regulation of the CAD. Methods: In this study, we used a case-control study to investigate and analyze the genetic variations and functional variations of the GATA6 gene promoter region in AMI patients and controls. A variety of statistical analysis methods were utilized to analyze the association of single nucleotide polymorphisms (SNPs) with AMI. Functional analysis of DNA sequence variants (DSVs) was performed using a dual luciferase reporter assay. In vitro, electrophoretic mobility shift assay (EMSA) was selected to examine DNA-protein interactions. Results: A total of 705 subjects were enrolled in the study. Ten DSVs were found in AMI patients (n = 352) and controls (n = 353), including seven SNPs. One novel heterozygous DSV, (g.22168409 A > G), and two SNPs, [g.22168362 C > A(rs1416421760) and g.22168521 G > T(rs1445501474)], were reported in three AMI patients, which were not found in controls. The relevant statistical analysis, including allele and genotype frequencies between AMI patients and controls, five genetic models, linkage disequilibrium (LD) and haplotype analysis, and SNP–SNP interactions, suggested no statistical significance (P > 0.05). The transcriptional activity of GATA6 gene promoter was significantly increased by the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)]. The EMSA revealed that the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)] evidently influenced the binding of transcription factors. Conclusion: In conclusion, the DSV (g.22168409 A > G) and SNP [g.22168362 C > A(rs1416421760)] may increase GATA6 levels in both HEK-293 and H9c2 cell lines by affecting the binding of transcription factors. Whether the two variants identified in the GATA6 gene promoter can promote the development and progression of human AMI by altering GATA6 levels still requires further studies to verify.

1438MicroRNA editing is integral for interleukin-6 trans-signalling and leukocyte trafficking to ischemic tissues

Abstract Background/Aim Adenosine to inosine RNA editing is an essential post-transcriptional RNA modification catalysed by adenosine deaminase acting on RNA-1 and -2 (ADAR1; ADAR2). Endothelial cells (ECs) attract and guide leukocytes to sites of ischemic tissue injury. Here we studied the role of RNA editing in ischemic disease. Methods Primary human and murine vascular endothelial cell cultures were used to assess the EC responses to interleukin-6 (IL-6) or ischemia. For the animal studies, the effect of ADAR2 in acute and chronic ischemic disease was evaluated in cremaster muscle microcirculation by intravital microscopy, in peritoneal cavity after sterile peritonitis and in gastrocnemius muscle after hind-limb ischemia by 8-colour flow cytometry and immunohistochemistry (IHC) studies of Adar2−/−/tg as well as of i(nducible)EC-ADAR2 knockout (KO) mice. For the mechanistic studies, deep RNA sequencing, qRT-PCR, western blot, confocal microscopy, target-specific microRNA (miRNA) editing studies, RNA-immunoprecipitation, miRNA/plasmid silencing/overexpression and luciferase reporter assays were used among others. For human studies, ischemic tissues derived from patients with acute or chronic ischemic heart disease were processed. Results ADAR2, but not ADAR1, expression is induced by &gt;2-fold in hypoxic ECs and in ischemic vascular ECs in mice and humans. Unbiased gene ontology analysis of the EC transcriptome indicated that ADAR2 controls inflammatory responses and predominantly the expression of interleukin-6-signal transducer (IL6ST), the co-receptor of IL-6. Subsequently, ADAR2 controls IL-6 trans-signalling in ECs as documented by the STAT3 phosphorylation and expression of the downstream leukocyte adhesion molecules, E-selectin and VCAM-1. IL-6-inflamed cremaster muscles showed that rolling and adhesion of leukocyte subsets to vascular wall were severely impaired in Adar2−/−/tg mice. Leukocyte transmigration was also diminished by &gt;2-fold in Adar2−/−/tg and in iEC-ADAR2 KO mice in response to IL-6 or ischemia. Similar results were obtained for leukocyte rolling, adhesion and infiltration after acute (4h) and chronic (3d; 21d) ischemia from iEC-ADAR2 KO mice and human ischemic muscle tissues. Next we studied how ADAR2 controls IL6ST expression. ADAR2-deficient vascular EC miRNAome revealed the upregulation of a conserved group of miRNAs targeting the IL6ST mRNA including miR-199a-5p and miR-335-3p. At a single-nucleotide level, ADAR2-induced RNA editing of the stem loops of the primary miR-199a1/2 and miR-335 directly disrupted Drosha recruitment to both and thus inhibited their maturation process. Accordingly, rescue experiments using miRNA-inhibitors restored IL6ST levels after ADAR2 deficiency. Conclusion Taking together, inhibition of the microRNA maturation process by ADAR2-mediated RNA editing is integral for IL-6 trans-signalling in vascular endothelium and subsequent leukocyte trafficking to ischemic tissues in mice and humans.

Analysis of cardiovascular disease-related NF-κB-regulated genes and microRNAs in TNFα-treated primary mouse vascular endothelial cells.

Activated nuclear factor-κB (NF-κB) plays an important role in the development of cardiovascular disease (CVD) through its regulated genes and microRNAs (miRNAs). However, the gene regulation profile remains unclear. In this study, primary mouse vascular endothelial cells (pMVECs) were employed to detect CVD-related NF-κB-regulated genes and miRNAs. Genechip assay identified 77 NF-κB-regulated genes, including 45 upregulated and 32 downregulated genes, in tumor necrosis factor α (TNFα)-treated pMVECs. Ten of these genes were also found to be regulated by NF-κB in TNFα-treated HeLa cells. Quantitative real-time PCR (qRT-PCR) assay confirmed the up-regulation of Egr1, Tnf, and Btg2 by NF-κB in the TNFα-treated pMVECs. The functional annotation revealed that many NF-κB-regulated genes identified in pMVECs were clustered into classical NF-κB-involved biological processes. Genechip assay also identified 26 NF-κB-regulated miRNAs, of which 21 were upregulated and 5 downregulated, in the TNFα-treated pMVECs. Further analysis showed that nine of the identified genes are regulated by seven of these miRNAs. Finally, among the identified NF-κB-regulated genes and miRNAs, 5 genes and 12 miRNAs were associated with CVD by miRWalk and genetic association database analysis. Taken together, these findings show an intricate gene regulation network raised by NF-κB in TNFα-treated pMVECs. The network provides new insights for understanding the molecular mechanism underlying the progression of CVD.