Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury.

Kinase non-catalytic C-lobe domain containing 1 (KNDC1) exists in dendrites, guanine nucleotide exchange factor complexes and neuronal cell bodies as a putative protein‑protein interaction module that regulates a number of signaling pathways. Previous studies have demonstrated that the knockdown of KNDC1 delays human umbilical vein endothelial cell (HUVEC) senescence. However, the effect of KNDC1 overexpression on HUVEC function remains unclear. In the present study, an adenovirus vector carrying KNDC1 was constructed and then transfected into endothelial cells to observe cell senescence. Furthermore, the effect of KNDC1 overexpression on HUVEC senescence was investigated in vitro and the underlying molecular mechanism was investigated. Senescence‑associated β‑galactosidase staining was used to determine cellular senescence and reactive oxygen species (ROS) were monitored to detect the level of cell oxidative stress. The mRNA transcription level and protein expression were analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that KNDC1 overexpression possibly inhibited HUVEC activity and function and promoted HUVEC senescence. Mechanistic studies demonstrated that KNDC1 triggered a p53‑ROS positive feedback loop, which serves a crucial role in regulating senescence. In conclusion, to the best of the authors' knowledge, this is the first time that KNDC1‑adenovirus vector inhibition of HUVEC proliferation by activating the p53 signaling pathway has been reported. Theoretically, the results of the present study also support KNDC1 as a therapeutic target for future anti-senescence.

Objective: Bevacizumab is a recombinant humanized monoclonal antibody that blocks vascular endothelial growth factor (VEGF) with clear clinical benefits. However, overall survival of some cancer types remains low owing to resistance to bevacizumab therapy. While resistance is commonly ascribed to tumor cell invasion induced by hypoxia-inducible factor (HIF), less attention has been paid to the potential involvement of endothelial cells (ECs) in vasculature activated by anti-angiogenic drugs. Methods: Human umbilical vein ECs (HUVECs), bEnd.3 cells, and mouse retinal microvascular ECs (MRMECs) were treated with bevacizumab under conditions of hypoxia and effects on biological behaviors, such as migration and tube formation, examined. Regulatory effects on TGFβ1 and CD105 (endoglin) were established via determination of protein and mRNA levels. We further investigated whether the effects of bevacizumab could be reversed using the receptor tyrosine kinase inhibitor anlotinib. Results: Bevacizumab upregulated TGFβ1 as well as CD105, a component of the TGFβ receptor complex and an angiogenesis promoter. Elevated CD105 induced activation of Smad1/5, the inflammatory pathway and endothelial-mesenchymal transition. The migration ability of HUVECs was enhanced by bevacizumab under hypoxia. Upregulation of CD105 was abrogated by anlotinib, which targets multiple receptor tyrosine kinases including VEGFR2/3, FGFR1-4, PDGFRα/β, C-Kit, and RET. Conclusions: Bevacizumab promotes migration and tube formation of HUVECs via activation of the TGFβ1 pathway and upregulation of CD105 expression. Anlotinib reverses the effects of bevacizumab by inhibiting the above signals.

Although SDF-1/CXCR7 plays an important role in angiogenesis, the function and the pathway of the SDF-1/CXCR7 axis might depend on the cell type or tissue origin and not fully understood. In this study, we investigated the effect of CXCR7 in SDF-1-induced proliferation, migration, apoptosis, tube formation, and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), and the potential pathway of SDF-1/CXCR7. We confirmed that the silencing of CXCR7 inhibited the proliferation of HUVECs and contributed the apoptosis, while overexpressed CXCR7 increased SDF-1-induced HUVECs migration and tube formation. However, upregulated CXCR7 inhibited the expression of α-SMA, suggesting that CXCR7 might attenuate EndMT. In addition, overexpressed CXCR7 activated AKT and ERK signaling pathways but suppressed Wnt/β-catenin pathways in HUVECs. The inhibition of Wnt/β-catenin pathways decreased the expression of α-SMA. Altogether, these results suggest that CXCR7 might inhibit fibrosis via Wnt/β-catenin pathways during the process of angiogenesis.

Lysyl oxidase (LOX) is an enzyme that oxidizes lysine residues in collagens and elastin. It stabilizes or remodels the extracellular matrix and basement membrane of blood vessels. Current oncology studies have revealed that LOX is upregulated in invasive cancer cells and bolstered cell movement, and LOX was observed to promote the angiogenesis and migration of endothelial cells. In the present study, angiogenesis and migration were examined in human umbilical vein endothelial cells (HUVECs). Following cell treatment with 0.1-0.4 mM β-aminoproprionitrile (BAPN), a specific inhibitor of LOX, angiogenesis was analyzed with a fibrin gel in vitro angiogenesis assay kit and migration was examined via a Boyden Chamber assay. Angiogenesis-associated gene expression was investigated with a microarray assay and confirmed with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results showed that HUVEC angiogenesis substantially increased in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and phorbol 12-myristate 13-acetate (PMA). In addition, LOX inhibition blocked the angiogenesis stimulated by VEGF bFGF and PMA, and the inhibition of LOX reduced the migration of HUVECs. Furthermore, the microarray and RT-qPCR revealed that BAPN downregulated myeloid progenitor inhibitory factor 1, and western blot analysis demonstrated that BAPN decreased the phosphorylation of MAPK and Akt, suggesting that the specific inhibitor of LOX, BAPN, may serve as an alternative strategy for preventing angiogenesis.

The oxLDL-based bioactive lipid lysophosphatidylcholine (LPC) is a key regulator of physiological processes including endothelial cell adhesion marker expression. This study explored the relationship between LPC and the human umbilical vein endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with a particular focus on the regulation of the LPC-G2A-ICAM-1/VCAM-1 pathway in this context. We explored the LPC-inducible role of orphan G protein receptor 2 (G2A) in associated regulatory processes by using human kidney epithelial (HEK293) cells that had been transfected with pET-G2A, human umbilical vein endothelial cells (HUVECs) in which an shRNA was used to knock down G2A, and western blotting and qPCR assays that were used to confirm changes in gene expression. For in vivo studies, a rabbit model of atherosclerosis was established, with serum biochemistry and histological staining approaches being used to assess pathological outcomes in these animals. The treatment of both HEK293 cells and HUVECs with LPC promoted ICAM-1 and VCAM-1 upregulation, while incubation at a pH of 6.8 suppressed such LPC-induced adhesion marker expression. Knocking down G2A by shRNA and inhibiting NF-κB activity yielded opposite outcomes. The application of a Gi protein inhibitor had no impact on LPC-induced ICAM-1/VCAM-1 expression. Atherosclerotic model exhibited high circulating LDL and LPC levels as well as high aortic wall ICAM-1/VCAM-1 expression. Overall, these results suggested that the LPC-G2A-ICAM-1/VCAM-1 pathway may contribute to the atherogenic activity of oxLDL, with NF-κB antagonists representing potentially viable therapeutic tools for the treatment of cardiovascular disease.

Oxidative stress and inflammation are considered as two key factors that contribute to the development of atherosclerosis. This study was to investigate the antioxidant capacity of huskless barley and to explore its protective functions through the regulation of the antioxidant defense and inflammatory response in human umbilical vein endothelial cells (HUVEC). The oxygen radical absorbance capacity (ORAC), ferric-reducing antioxidant power (FRAP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) scavenging capacity of water and alkali extracts of the polysaccharides from nine huskless barley varieties were investigated in vitro. The antioxidant properties of the alkaline extracts were more pronounced than those of the water extracts. The results from the cell model showed that pretreatment of HUVEC with the water or alkaline extracts of the polysaccharides from the huskless barley cultivars QHH and NLGL decreased the levels of reactive oxygen species (ROS), monocyte chemotactic protein 1 (MCP-1), and vascular cell adhesion molecule 1 (VCAM-1) but increased the level of superoxide dismutase (SOD) and maintained cell viability. Huskless barley polysaccharide extracts exhibited the vasodilatory effect of inhibiting angiotensin-converting enzyme (ACE) production. These discoveries revealed the potent protective functions of barley in oxidative damage and a potential role for barley in preventing chronic inflammation in cardiovascular diseases.

More than 70 human adenoviruses with type-dependent pathogenicity have been identified but biological information about the majority of these virus types is scarce. Here we employed multiple sequence alignments and structural information to predict receptor usage for the development of an adenoviral vector with novel biological features. We report the generation of a cloned adenovirus based on human adenovirus type 17 (HAdV17) with high sequence homology to the well characterized human adenovirus type 37 (HAdV37) that causes epidemic keratoconjunctivitis (EKC). Our study revealed that human CD46 (CD46) is involved in cell entry of HAdV17. Moreover, we found that HAdV17 infects endothelial cells (EC) in vitro including primary cells at higher efficiencies compared to the commonly used human adenovirus type 5 (HAdV5). Using a human CD46 transgenic mouse model, we observed that HAdV17 displays a broad tropism in vivo after systemic injection and that it transduces ECs in this mouse model. We conclude that the HAdV17-based vector may provide a novel platform for gene therapy.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been widely used due to their multipotency, a broad range of sources, painless collection, and compliance with standard amplification. Cell sheet technology is a tissue engineering methodology requiring scaffolds free, and it provides an effective method for cell transplantation. To improve the therapeutic efficacy, we combined hUC-MSCs with cell sheet technology to evaluate the safety and efficacy of hUC-MSC sheets in preclinical studies using appropriate animal models.

Aberrant vascular network is a hallmark of cancer. However, the role of vascular endothelial cells (VECs)-expressing PD-L1 in tumor immune microenvironment and antiangiogenic therapy remains unclear. In this study, we used the specimens of cancer patients for immunohistochemical staining to observe the number of PD-L1+ CD34+ VECs and infiltrated immune cells inside tumor specimens. Immunofluorescence staining and flow cytometry were performed to observe the infiltration of CD8+ T cells and FoxP3+ T cells in tumor tissues. Here, we found that PD-L1 expression on VECs determined CD8+ T cells', FoxP3+ T cells' infiltration, and the prognosis of patients with lung adenocarcinoma. Anlotinib downregulated PD-L1 expression on VECs through the inactivation of AKT pathway, thereby improving the ratio of CD8/FoxP3 inside tumor and remolding the immune microenvironment. In conclusion, our results demonstrate that PD-L1 high expression on VECs inhibits the infiltration of CD8+ T cells, whereas promotes the aggregation of FoxP3+ T cells into tumor tissues, thus becoming an "immunosuppressive barrier". Anlotinib can ameliorate the immuno-microenvironment by downregulating PD-L1 expression on VECs to inhibit tumor growth.

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway.

Endothelial cell apoptosis induced by oxidative stress is an early event in the development of atherosclerosis. Several antioxidant enzymes which can cope with oxidative stress are up-regulated by the anti-atherogenic laminar blood flow often seen in straight or unbranched regions of blood vessels. However, the molecular mechanism responsible for flow-induced beneficial effects is incompletely understood. Here we report the role of glutaredoxin 1 (Grx1), an antioxidant enzyme, in flow-mediated protective effect in endothelial cells. Specifically, we found that Grx1 is markedly up-regulated by the steady laminar flow. Increasing Grx1 reduces the pro-apoptotic protein Bim expression through regulating Akt-FoxO1 signaling and also attenuates H2O2-induced Bim activation via inhibiting JNK phosphorylation, subsequently preventing the apoptosis of endothelial cells. Grx1 knockdown abolishes the inhibitory effect of steady laminar flow on Bim. The inhibitory effect of Grx1 on Bim is dependent on Grx1's thioltransferase activity. These findings indicate that Grx1 induction plays a key role in mediating the protective effect of laminar blood flow and suggest that Grx1 may be a potential therapeutic target for atherosclerosis.

Sirtuin1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including endothelial function. Caveolin1 (Cav1) is also an important determinant of endothelial function. We asked if Sirt1 governs endothelial Cav1 and endothelial function by regulating miR-204 expression and endoplasmic reticulum (ER) stress. Knockdown of Sirt1 in endothelial cells, and in vivo deletion of endothelial Sirt1, induced endothelial ER stress and miR-204 expression, reduced Cav1, and impaired endothelium-dependent vasorelaxation. All of these effects were reversed by a miR-204 inhibitor (miR-204 I) or with overexpression of Cav1. A miR-204 mimic (miR-204 M) decreased Cav1 in endothelial cells. In addition, high-fat diet (HFD) feeding induced vascular miR-204 and reduced endothelial Cav1. MiR-204-I protected against HFD-induced downregulation of endothelial Cav1. Moreover, pharmacologic induction of ER stress with tunicamycin downregulated endothelial Cav1 and impaired endothelium-dependent vasorelaxation that was rescued by overexpressing Cav1. In conclusion, Sirt1 preserves Cav1-dependent endothelial function by mitigating miR-204-mediated vascular ER stress.

Endothelial dysfunction is a characteristic of many vascular related diseases such as hypertension. Peroxisome proliferator activated receptor gamma, coactivator 1α (PGC-1α) is a unique stress sensor that largely acts to promote adaptive responses. Therefore, we sought to define the role of endothelial PGC-1α in vascular function using mice with endothelial specific loss of function (PGC-1α EC KO) and endothelial specific gain of function (PGC-1α EC TG). Here we report that endothelial PGC-1α is suppressed in angiotensin-II (ATII)-induced hypertension. Deletion of endothelial PGC-1α sensitized mice to endothelial dysfunction and hypertension in response to ATII, whereas PGC-1α EC TG mice were protected. Mechanistically, PGC-1α promotes eNOS expression and activity, which is necessary for protection from ATII-induced dysfunction as mice either treated with an eNOS inhibitor (LNAME) or lacking eNOS were no longer responsive to transgenic endothelial PGC-1α expression. Finally, we determined that the orphan nuclear receptor, estrogen related receptor α (ERRα) is required to coordinate the PGC-1α -induced eNOS expression. In conclusion, endothelial PGC-1α expression protects from vascular dysfunction by promoting NO• bioactivity through ERRα induced expression of eNOS.

Endoplasmic reticulum (ER) stress has been implicated in vascular endothelial dysfunction of obesity, diabetes, and hypertension. MicroRNAs play an important role in regulating ER stress. Here we show that microRNA-204 (miR-204) promotes vascular ER stress and endothelial dysfunction by targeting the Sirtuin1 (Sirt1) lysine deacetylase. Pharmacologic ER stress induced by tunicamycin upregulates miR-204 and downregulates Sirt1 in the vascular wall/endothelium in vivo and in endothelial cells in vitro. Inhibition of miR-204 protects against tunicamycin-induced vascular/endothelial ER stress, associated impairment of endothelium-dependent vasorelaxation, and preserves endothelial Sirt1. A miR-204 mimic leads to ER stress and downregulates Sirt1 in endothelial cells. Knockdown of Sirt1 in endothelial cells, and conditional deletion of endothelial Sirt1 in mice, promotes ER stress via upregulation of miR-204, whereas overexpression of Sirt1 in endothelial cells suppresses miR-204-induced ER stress. Furthermore, increase in vascular reactive oxygen species induced by ER stress is mitigated by by miR-204 inhibition. Finally, nutritional stress in the form of a Western diet promotes vascular ER stress through miR-204. These findings show that miR-204 is obligatory for vascular ER stress and ER stress-induced vascular endothelial dysfunction, and that miR-204 promotes vascular ER stress via downregulation of Sirt1.

Aims: Dihydroartemisinin (DHA), a derivative of artemisinin, suppresses angiogenesis by regulating endothelial cell phenotypes. In this study, we investigated the effect of DHA on endothelial cell autophagy and the underlying mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with DHA. Formation of autophagosomes in HUVECs was observed by fluorescence microscope after pcDNA3.1-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) plasmids transfection. Dichlorofluorescein diacetate (DCFH-DA) staining was used to detect intracellular reactive oxygen species (ROS). Western blot was performed to detect the protein levels of LC3, p62, beclin 1, autophagy-related protein (Atg) 5, p-Akt (protein kinase B), p-mTOR (mammalian target of rapamycin), p-4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1), and p-p70S6K (p70 ribosomal S6 kinase). Results: DHA increased LC3-II and the number of fluorescent GFP-LC3 puncta in HUVECs. Silencing ATG5 by siRNA interference attenuated DHA-induced LC3-II elevation. DHA enhanced ROS production, but pretreatment with antioxidant N-acety-l-cysteine (NAC) failed to reduce DHA-induced autophagy in HUVECs. Pretreatment with PD98059, SP600125 and SB203580, the inhibitors of ERK, JNK, and p38 MAPK, did not reverse autophagy in DHA-treated HUVECs. DHA significantly reduced phosphorylation of Akt, mTOR, p70S6K, 4E-BP1 in HUVECs. Rapamycin, an mTOR antagonist, compromised DHA-induced autophagy. Conclusion: DHA induces autophagy in HUVECs by inhibition of the Akt/mTOR pathway.

Hyperglycemia-mediated endothelial inflammation participates in the pathogenesis of cardiovascular complications in subjects with diabetes. Previous studies reported that phosphatase and tensin homolog deleted on chromosome ten (PTEN) and SET8 participate in high glucose-mediated endothelial inflammation. In this study, we hypothesize that SET8 regulates PTEN expression, thus contributing to high glucose-mediated vascular endothelial inflammation. Our data indicated that plasma soluble intercellular adhesion molecule-1 (sICAM-1) and endothelial selectin (e-selectin) were increased in patients with diabetes and diabetic rats. PTEN expression was augmented in the peripheral blood mononuclear cells of patients with diabetes and in the aortic tissues of diabetic rats. Our in vitro study indicated that high glucose increased monocyte/endothelial adhesion, endothelial adhesion molecule expression and p65 phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose led to endothelial inflammation via upregulation of PTEN. Furthermore, high glucose inhibited SET8 expression and histone H4 lysine 20 methylation (H4K20me1), a downstream target of SET8. SET8 overexpression reversed the effects of high-glucose treatment. shSET8-mediated endothelial inflammation was counteracted by siPTEN. Furthermore, SET8 was found to interact with FOXO1. siFOXO1 attenuated high glucose-mediated endothelial inflammation. FOXO1 overexpression-mediated endothelial inflammation was counteracted by siPTEN. H4K20me1 and FOXO1 were enriched in the PTEN promoter region. shSET8 increased PTEN promoter activity and augmented the positive effect of FOXO1 overexpression on PTEN promoter activity. Our in vivo study indicated that SET8 was downregulated and FOXO1 was upregulated in the peripheral blood mononuclear cells of patients with diabetes and the aortic tissues of diabetic rats. In conclusion, SET8 interacted with FOXO1 to modulate PTEN expression in vascular endothelial cells, thus contributing to hyperglycemia-mediated endothelial inflammation.

Mitochondrial dysfunction is considered to be a key contributor to the development of heart failure. Replacing injured mitochondria with healthy mitochondria to restore mitochondrial bioenergy in myocardium holds great promise for cardioprotection after infarction. This study aimed to investigate whether direct transplantation of exogenous mitochondria derived from mesenchymal stem cells (MSC-mt) is beneficial and superior in protecting cardiac function in a mouse model of myocardial infarction (MI) compared to mitochondria derived from skin fibroblast (FB-mt) and to explore the underlying mechanisms from their effects on the endothelial cells. The isolated MSC-mt presented intact mitochondrial morphology and activity, as determined by electron microscopy, JC-1 mitochondrial membrane potential assay, and seahorse assay. Direct injection of MSC-mt into the peri-infarct region in a mouse MI model enhanced blood vessel density, inhibited cardiac remodeling and apoptosis, thus improving heart function compared with FB-mt group. The injected MSC-mt can be tracked in the endothelial cells. In vitro, the fluorescence signal of MSC-mt can be detected in human umbilical vein endothelial cells (HUVECs) by confocal microscopy and flow cytometry after coculture. Compared to FB-mt, MSC-mt more effectively protected the HUVECs from oxidative stress-induced apoptosis and reduced mitochondrial production of reactive oxygen species. MSC-mt presented superior capacity in inducing tube formation, enhancing SCF secretion, ATP content and cell proliferation in HUVECs compared to FB-mt. Mechanistically, MSC-mt administration alleviated oxidative stress-induced endothelial senescence via activation of ERK pathway. These findings suggest that using MSCs as sources of mitochondria is feasible and that proangiogenesis could be the mechanism by which MSC-mt transplantation attenuates MI. MSC-mt transplantation might serve as a new therapeutic strategy for treating MI.

Unlike normal tissue-derived microvascular endothelial cells, tumor microvessel endothelial cells are highly reactive to growth factors and exhibit more adhesion molecules. Thus, vascular tumors are highly permeable and grow vigorously; this occurrence results in rapid growth and metastasis cancer cells. Therefore, understanding the characteristics of endothelial cells in the tumor microenvironment guides anti-angiogenic therapy. To this end, we explore the effect of the supernatant obtained from cultured Anip973 cells (high-metastatic human lung adenocarcinoma cells) on the biological behavior and on the cell surface markers of the human umbilical vein endothelial cell (HUVEC).

chA21 is a novel tumor-inhibitory antibody which recognized subdomain I of HER2 extracellular domain with an epitope distinct from other HER2 antibodies. Previously, we demonstrated that chA21 inhibits human ovarian carcinoma cell line SKOV-3 growth in vitro and in vivo study. In this study, we further investigated the anti-angiogenic efficacy combination of chA21 with trastuzumab in SKOV-3 xenograft model.