Activity Of Human Umbilical Vein Endothelial Cells Research Articles

Cancer-associated Fibroblasts-derived Exosomes with HOXD11 Overexpression Promote Ovarian Cancer Cell Angiogenesis Via FN1.

Cancer-associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote metastasis in patients with ovarian cancer (OC). Here, we try to further understand the mechanism by which CAFs-derived exosomes (CAFs-Exo) promoted angiogenesis in OC. We intersected differentially expressed genes in OC cells after CAFs-Exo treatment in the GSE147610 dataset with a list of transcription factors to identify homeobox protein hox-D11 (HOXD11) as a possible cargo of CAFs-Exo. HOXD11 encapsulated by CAFs-Exo enhanced colony formation, migration, and invasion of OC cells. HOXD11 bound to the promoter of fibronectin (FN1) and promoted its transcription. HOXD11 knockdown from CAFs-Exo significantly repressed the VEGF and CD31 protein expression and tube formation, viability, and migration of human umbilical vein endothelial cells (HUVEC) and slowed angiogenesis and tumor growth in mice. Furthermore, we found that overexpression of FN1 increased the expression of angiogenic factors and activity of HUVEC in the presence of HOXD11 knockdown. These results verify the significant contribution of CAFs-Exo to angiogenesis in OC, which could be partially due to the promotion of FN1 mediated by HOXD11.

Liraglutide ameliorates high glucose-induced vascular endothelial injury through TRIB3/NF-κB signaling pathway.

As one of the most commonly used antidiabetic medications clinically, liraglutide is involved in the protection of vascular endothelium, and whether it can relieve high glucose-induced vascular endothelial damage was unknown. This study aims to address the response of liraglutide (LIRA) on human umbilical vein endothelial cells, as well as to elucidate its possible underlying mechanism. We established a vascular endothelial cell injury model by exposing human umbilical vein endothelial cells (HUVECs) to high glucose, and used LIRA pretreatment before HG treatment to address the endothelial protective effect of LIRA. Our results suggest that LIRA prevented HG-induced HUVEC apoptosis, oxidative stress, inflammasome activation, and pyroptosis. Furthermore, silencing of tribbles homolog 3 (TRIB3) could markedly reduce HG-induced HUVEC apoptosis, ROS level, the expressions of TXNIP, cleaved caspase3, NLRP3, and caspase1, indicating TRIB3 inhibition protected HUVECs against HG-induced vascular endothelial injury. In addition, LIRA restrained NF-κB/IκB-α signaling pathway activation in HUVECs. Thus, LIRA appears to mitigate HG-induced apoptosis, oxidative stress, inflammasome activation, and pyroptosis in HUVECs via regulating the TRIB3/NF-κB/IκB-α signaling pathway. Our study provides new insight into the mechanisms underlying the protective activity of LIRA against the vascular endothelial injury in diabetic vascular complication.

Erastin promotes random-pattern skin flaps survival by inducing mTORC1-TFEB mediated autophagy

Random-pattern skin flaps are important method for skin reconstruction after defect; however, the distal end of flaps is not easily viable due to inadequate nutrient supply. Erastin is a well-established ferroptosis inducer, but our study found that low-dose of erastin (2 μM) may reduce nutrient deficiency induced cell death in human umbilical vein endothelial cells (HUVECs). RNA-seq analysis suggested that its role was related to autophagy regulation. Follow-up studies have shown that the use of autophagy inhibitors or the knockdown of TFEB in HUVECs can both reduce the anti-apoptotic effect of erastin in HUVECs. Mechanism study demonstrated that erastin can suppress mTORC1 and promote TFEB activity in HUVECs, suggesting that the effect of erastin on the survival of HUVECs under nutrient deprivation conditions is regulated by mTORC1/TFEB. Subsequently, we evaluated the effect of erastin on the survival of random-pattern skin flaps in mice in vivo. On the postoperative day 7, we observed a significant increase in flap survival area, blood perfusion, and microvascular density after erastin treatment; also, erastin treatment showed enhanced autophagy within the ischemic region. In summary, our study demonstrates that low-dose of erastin may suppress cell death in endothelial cells under nutrient deficiency condition, and its effects may relate to the mTORC1-TFEB medicated autophagy regulation, erastin treatment may be a potential therapy for random-pattern skin flaps.

Ocular pharmacological and biochemical profiles of 6-thioguanine: a drug repurposing study.

The purine analog 6-thioguanine (6TG), an old drug approved in the 60s to treat acute myeloid leukemia (AML), was tested in the diabetic retinopathy (DR) experimental in vivo setting along with a molecular modeling approach. A computational analysis was performed to investigate the interaction of 6TG with MC1R and MC5R. This was confirmed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose (25mM) for 24h. Cell viability in HUVECs exposed to high glucose and treated with 6TG (0.05-0.5-5µM) was performed. To assess tube formation, HUVECs were treated for 24h with 6TG 5µM and AGRP (0.5-1-5µM) or PG20N (0.5-1-5-10µM), which are MC1R and MC5R antagonists, respectively. For the in vivo DR setting, diabetes was induced in C57BL/6J mice through a single streptozotocin (STZ) injection. After 2, 6, and 10weeks, diabetic and control mice received 6TG intravitreally (0.5-1-2.5mg/kg) alone or in combination with AGRP or PG20N. Fluorescein angiography (FA) was performed after 4 and 14weeks after the onset of diabetes. After 14weeks, mice were euthanized, and immunohistochemical analysis was performed to assess retinal levels of CD34, a marker of endothelial progenitor cell formation during neo-angiogenesis. The computational analysis evidenced a more stable binding of 6TG binding at MC5R than MC1R. This was confirmed by the tube formation assay in HUVECs exposed to high glucose. Indeed, the anti-angiogenic activity of 6TG was eradicated by a higher dose of the MC5R antagonist PG20N (10µM) compared to the MC1R antagonist AGRP (5µM). The retinal anti-angiogenic effect of 6TG was evident also in diabetic mice, showing a reduction in retinal vascular alterations by FA analysis. This effect was not observed in diabetic mice receiving 6TG in combination with AGRP or PG20N. Accordingly, retinal CD34 staining was reduced in diabetic mice treated with 6TG. Conversely, it was not decreased in diabetic mice receiving 6TG combined with AGRP or PG20N. 6TG evidenced a marked anti-angiogenic activity in HUVECs exposed to high glucose and in mice with DR. This seems to be mediated by MC1R and MC5R retinal receptors.

Elucidating the role of Rhodiola rosea L. in sepsis-induced acute lung injury via network pharmacology: emphasis on inflammatory response, oxidative stress, and the PI3K-AKT pathway

Context Sepsis-induced acute lung injury (ALI) is associated with high morbidity and mortality. Rhodiola rosea L. (Crassulaceae) (RR) and its extracts have shown anti-inflammatory, antioxidant, immunomodulatory, and lung-protective effects. Objective This study elucidates the molecular mechanisms of RR against sepsis-induced ALI. Materials and methods The pivotal targets of RR against sepsis-induced ALI and underlying mechanisms were revealed by network pharmacology and molecular docking. Human umbilical vein endothelial cells (HUVECs) were stimulated by 1 μg/mL lipopolysaccharide for 0.5 h and treated with 6.3, 12.5, 25, 50, 100, and 200 μg/mL RR for 24 h. Then, the lipopolysaccharide-stimulated HUVECs were subjected to cell counting kit-8 (CCK-8), enzyme-linked immunosorbent, apoptosis, and Western blot analyses. C57BL/6 mice were divided into sham, model, low-dose (40 mg/kg), mid-dose (80 mg/kg), and high-dose (160 mg/kg) RR groups. The mouse model was constructed through caecal ligation and puncture, and histological, apoptosis, and Western blot analyses were performed for further validation. Results We identified six hub targets (MPO, HRAS, PPARG, FGF2, JUN, and IL6), and the PI3K-AKT pathway was the core pathway. CCK-8 assays showed that RR promoted the viability of the lipopolysaccharide-stimulated HUVECs [median effective dose (ED50) = 18.98 μg/mL]. Furthermore, RR inhibited inflammation, oxidative stress, cell apoptosis, and PI3K-AKT activation in lipopolysaccharide-stimulated HUVECs and ALI mice, which was consistent with the network pharmacology results. Discussion and conclusion This study provides foundational knowledge of the effective components, potential targets, and molecular mechanisms of RR against ALI, which could be critical for developing targeted therapeutic strategies for sepsis-induced ALI.

Effects of conditioned medium of alveolar epithelial cells on vascular endothelial cell damage

To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism. The LPS induced type II alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting. Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2 438.95±64.89, 3 036.41±96.69 vs. 1 736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67 205.60±3 430.40 vs. 56 272.67±7 650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.

A new look at angiogenesis inhibition of geniposide in experimental arthritis by blocking angiopoietin-2 exocytosis.

Angiogenesis is a key player in the pathogenesis of rheumatoid arthritis. Exocytosis from Weibel-Palade bodies is a prerequisite for angiopoietin-2 (Ang-2) to activate endothelial cells and initiate angiogenesis. Geniposide (GE) was previously reported to exert anti-angiogenic effects. The aim of this study was to shed light on whether and how GE regulates Ang-2 exocytosis. A rat model of adjuvant arthritis (AA) was established to evaluate the therapeutic effect of GE (60 and 120 mg/kg) especially in synovial angiogenesis. In addition, the Matrigel plug assay was used to detect the effect of GE (120 and 240 mg/kg) on angiogenesis in AA mice. In vitro, sphingosine-1-phosphate (S1P)-stimulated human umbilical vein endothelial cells (HUVECs) were used to investigate the effect and mechanism of GE on Ang-2 exocytosis. It was found that GE improved the symptoms of AA rats and inhibited angiogenesis in AA, which may be related to the down-regulation of S1P receptors 1, 3 (S1PR1, S1PR3), phospholipase Cβ3 (PLCβ3), inositol 1,4,5-trisphosphate receptor (IP3 R) and Ang-2 expression. The results of in vitro experiments showed that S1P induced rapid release of Ang-2 from HUVECs with multigranular exocytosis. Suppression of the S1P/S1PR1/3/PLCβ3/Ca2+ signal axis by the S1PR1/3 inhibitor VPC23019 and the IP3 R inhibitor 2-APB blocked Ang-2 exocytosis, accompanied by diminished angiogenesis in vitro. GE dose-dependently weakened S1P/S1PR1/3/PLCβ3/Ca2+ signal axis activation, Ang-2 exocytosis and angiogenesis in HUVECs (p < 0.05, p < 0.01). Overall, these findings revealed that angiogenesis inhibition of GE was partly attributed to the intervention of Ang-2 exocytosis through negatively modulating the S1P/S1PR1/3/PLCβ3/Ca2+ signal axis, providing a novel strategy for rheumatoid arthritis anti-angiogenic therapy.

Extensive preclinical evaluation of combined mangiferin and glycyrrhizic acid for restricting synovial neovascularization in rheumatoid arthritis

BackgroundSynovial neovascularization promotes rheumatoid arthritis (RA) progression. Baihu guizhi decoction (BHGZD) has a potential in restricting this pathological change of RA.PurposeTo identify bioactive compounds (BACs) of BHGZD and to elucidate the underlying mechanisms in restricting synovial neovascularization of RA.MethodThrough transcriptomic profiling, the chemical profiling of BHGZD and its effective transcriptomic profiling against RA were identified. Then, candidate targets and the corresponding BACs against synovial neovascularization were screened by “disease gene-drug target” interaction network analysis and in silico molecular docking. The binding affinities of candidate BAC-target pairs were verified using surface plasmon resonance, and the pharmacokinetic characteristics of BACs in vivo after BHGZD administration at different time points were detected by Ultra Performance Liquid Chromatography-Mass spectrum/Mass spectrum. After that, in vivo experiments based on adjuvant-induced arthritis (AIA-M) rats, and in vitro experiments based on human umbilical vein endothelial cells (HUVEC) and arthritic synovial fibroblasts (MH7A) were carried out to evaluate the pharmacological effects of BHGZD and the two-BACs-combination, and to verify the associated mechanisms.ResultVEGFA/VEGFR2/SRC/PI3K/AKT signal axis was screened as one of the key network targets of BHGZD against synovial neovascularization in RA. Mangiferin (MG) and glycyrrhizic acid (GA) were identified as the representative BACs of BHGZD for their strong binding affinities with components of the VEGFA/VEGFR2/SRC/PI3K/AKT signal axis, and their high exposed quantity in vivo. Both BHGZD and the two-BAC combination of MG and GA were demonstrated to be effective in restricting disease severity, reducing synovial inflammation and decreasing the formation of vascular opacities in AIA-M rats, and also reducing the migrative and invasive activities of HUVEC and MH7A cells and attenuating the lumen formation ability of HUVEC cells significantly. Mechanically, both BHGZD and the two-BAC combination markedly reduced the expression of VEGFA in synovial tissues, the serum levels of VEGF and NO, and the enzymatic activity of eNOS, increased the content of endostatin, and also reversed the abnormal alterations in the VEGFA/VEGFR2/SRC/PI3K/AKT signal axis in vivo and in vitro.ConclusionMG and GA may be the representative BACs of BHGZD for restricting excessive synovial vascularization in RA via regulating VEGFA/VEGFR2/SRC/PI3K/AKT signal axis.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 μg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Race and sex differences in ROS production and SOD activity in HUVECs.

Black individuals and men are predisposed to an earlier onset and higher prevalence of hypertension, compared with White individuals and women, respectively. Therefore, the influence of race and sex on reactive oxygen species (ROS) production and superoxide dismutase (SOD) activity following induced inflammation was evaluated in female and male human umbilical vein endothelial cells (HUVECs) from Black and White individuals. It was hypothesized that HUVECs from Black individuals and male HUVECs would exhibit greater ROS production and impaired SOD activity. Inflammation was induced in HUVEC cell lines (n = 4/group) using tumor necrosis factor-alpha (TNF-α, 50ng/ml). There were no between group differences in ROS production or SOD activity in HUVECs from Black and White individuals, and HUVECs from Black individuals exhibited similar SOD activity at 24hr compared with 4hr of TNF-α treatment (p>0.05). However, HUVECs from White individuals exhibited significantly greater SOD Activity (p<0.05) at 24hr as compared to 4hr in the control condition but not with TNF-α treatment (p>0.05). Female HUVECs exhibited significantly lower ROS production than male HUVECs in the control condition and following TNF-α induced inflammation (p<0.05). Only female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF-α induced inflammation (p<0.05). HUVECs from White individuals alone exhibit blunted SOD activity when comparing control and TNF-α conditions. Further, compared to female HUVECs, male HUVECs exhibit a pro-inflammatory state.

Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2)

Article Oxidized LDL Regulates Endothelin-1 and Oxidative Stress in Vascular Endothelial Cells: Role of Extracellular Regulated Kinase1/2 (ERK1/2) Haishan Xu 1,#, Jinhong Duan 1,#, Jun Tao 2, Wen Wang 3, Yunqing Wu 1,^, Shunling Dai 1,*, and Jun Ren 4,5, 1 Faculty of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005 China 2 Department of Cardiovascular Surgery, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510000 China 3 Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China 4 Department of Cardiology, Zhongshan Hospital Fudan University, Shanghai 200032, China 5 National Clinical Research Center for Interventional Medicine, Shanghai 200032, China # These two authors contributed equally to this work ^ Deceased * Correspondence: daishunling@aliyun.com (Shunling Dai); corresponding author:jren_aldh2@outlook.com (Jun Ren) Abstract: It is perceived that oxidized low density lipoprotein (oxLDL) perturbs endothelial function and fosters endothelin-1 (ET-1) secretion although the underlying mechanism remains elusive. This study was designed to decipher potential mechanisms underscoring oxLDL-evoked regulation of ET-1 and signaling pathways involved in endothelial cells. ET-1 mRNA expression, secretion and promoter function were determined using RT-PCR, enzyme immunometric and luciferase assays, respectively. GO and GSEA bioinformatics analyses depicted differentially expressed genes (DEGs) mainly associated with cell proliferation, cell division, cellular structure, energy supply, and apoptosis in oxLDL-challenged endothelial cells. Incubation of oxLDL overtly increased ROS production, apoptosis, mRNA level, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), the effects were mitigated by N-Acetyl Cysteine (NAC). Moreover, oxLDL challenge evoked phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) in HUVECs, the effect was reversed by NAC and MEK inhibitor PD98059. NAC and PD98059 nullified oxLDL- induced rises in mRNA expression, secretion and promoter activity of ET-1. Truncation of 5’-flanking sequence of ET-1 (–566 bpLuc to –250 bpLuc) displayed elevated luciferase activity with 24-h oxLDL incubation. Fusion plasmid from –233 and –185 bp Luc drastically dampened luciferase activity in basal and oxLDL-challenged HUVECs. Transfection of reporter construct –250 bp Luc with a 2 bp mutation at AP-1 locus, removed basal and oxLDL- evoked rises in ET-1 promoter activity. Collectively, our findings support that oxLDL evoked activation of ERK1/2 signaling likely through ROS production, en route to upregulation of endothelial transcriptional factor AP-1, resulting in expression and secretion of ET-1.

CLIC4 Regulates Endothelial Barrier Control by Mediating PAR1 Signaling via RhoA.

Endothelial CLICs (chloride intracellular channel proteins) CLIC1 and CLIC4 are required for the GPCRs (G-protein-coupled receptors) S1PR1 (sphingosine-1-phosphate receptor 1) and S1PR3 to activate the small GTPases Rac1 (Ras-related C3 botulinum toxin substrate 1) and RhoA (Ras homolog family member A). To determine whether CLIC1 and CLIC4 function in additional endothelial GPCR pathways, we evaluated CLIC function in thrombin signaling via the thrombin-regulated PAR1 (protease-activated receptor 1) and downstream effector RhoA. We assessed the ability of CLIC1 and CLIC4 to relocalize to cell membranes in response to thrombin in human umbilical vein endothelial cells (HUVEC). We examined CLIC1 and CLIC4 function in HUVEC by knocking down expression of each CLIC protein and compared thrombin-mediated RhoA or Rac1 activation, ERM (ezrin/radixin/moesin) phosphorylation, and endothelial barrier modulation in control and CLIC knockdown HUVEC. We generated a conditional murine allele of Clic4 and examined PAR1-mediated lung microvascular permeability and retinal angiogenesis in mice with endothelial-specific loss of Clic4. Thrombin promoted relocalization of CLIC4, but not CLIC1, to HUVEC membranes. Knockdown of CLIC4 in HUVEC reduced thrombin-mediated RhoA activation, ERM phosphorylation, and endothelial barrier disruption. Knockdown of CLIC1 did not reduce thrombin-mediated RhoA activity but prolonged the RhoA and endothelial barrier response to thrombin. Endothelial-specific deletion of Clic4 in mice reduced lung edema and microvascular permeability induced by PAR1 activating peptide. CLIC4 is a critical effector of endothelial PAR1 signaling and is required to regulate RhoA-mediated endothelial barrier disruption in cultured endothelial cells and murine lung endothelium. CLIC1 was not critical for thrombin-mediated barrier disruption but contributed to the barrier recovery phase after thrombin treatment.

Role of UDP-glucose ceramide glucosyltransferase in venous malformation.

Venous malformation (VM) results from the abnormal growth of the vasculature; however, the detailed molecular mechanism remains unclear. As a glycosyltransferase, UDP-glucose ceramide glucosyltransferase (UGCG) is localized to the Golgi body and is a key enzyme in the first step of glycosphingolipid synthesis. Here, we aimed to explore the relationship between UGCG and the development of VM. First, investigations using RT-qPCR and Western blotting on the diseased vasculature of VM patients and normal vascular tissues revealed that UGCG expression was markedly elevated in the diseased vessels. Subsequently, immunofluorescence assay showed that UGCG was co-localized with CD31, an endothelial cell marker, in tissues from patients with VM and healthy subjects. Then, we established TIE2-L914F-mutant human umbilical vein endothelial cells (HUVECs) by lentivirus transfection. Next, Western blotting revealed that UGCG expression was considerably higher in HUVECsTIE2-L914F. In addition, we established a UGCG-overexpressing HUVECs line by plasmid transfection. With the CCK8 cell proliferation experiment, wound healing assay, and tube formation assay, we found that UGCG could promote the proliferation, migration, and tube formation activity of HUVECs, whereas the inhibition of UGCG could inhibit the proliferation, migration, and tube formation activity of HUVECsTIE2-L914F. Finally, Western blotting revealed that UGCG regulates the AKT/mTOR pathway in HUVECs. These data demonstrated that UGCG can affect the activity of vascular endothelial cells and regulate the AKT/mTOR signaling pathway; this is a potential mechanism underlying VM pathogenesis.

Metrnl deficiency retards skin wound healing in mice by inhibiting AKT/eNOS signaling and angiogenesis.

Meteorin-like (Metrnl) is a novel secreted protein with various biological activities. In this study, we investigated whether and how Metrnl regulated skin wound healing in mice. Global Metrnl gene knockout mice (Metrnl-/-) and endothelial cell-specific Metrnl gene knockout mice (EC-Metrnl-/-) were generated. Eight-mm-diameter full-thickness excisional wound was made on the dorsum of each mouse. The skin wounds were photographed and analyzed. In C57BL/6 mice, we observed that Metrnl expression levels were markedly increased in skin wound tissues. We found that both global and endothelial cell-specific Metrnl gene knockout significantly retarded mouse skin wound healing, and endothelial Metrnl was the key factor affecting wound healing and angiogenesis. The proliferation, migration and tube formation ability of primary human umbilical vein endothelial cells (HUVECs) were inhibited by Metrnl knockdown, but significantly promoted by addition of recombinant Metrnl (10 ng/mL). Metrnl knockdown abolished the proliferation of endothelial cells stimulated by recombinant VEGFA (10 ng/mL) but not by recombinant bFGF (10 ng/mL). We further revealed that Metrnl deficiency impaired VEGFA downstream AKT/eNOS activation in vitro and in vivo. The damaged angiogenetic activity in Metrnl knockdown HUVECs was partly rescued by addition of AKT activator SC79 (10 μM). In conclusion, Metrnl deficiency retards skin wound healing in mice, which is related to impaired endothelial Metrnl-mediated angiogenesis. Metrnl deficiency impairs angiogenesis by inhibiting AKT/eNOS signaling pathway.

Inhibition of circ_0000231 suppresses oxidized low density lipoprotein-induced apoptosis, autophagy and inflammation in human umbilical vein endothelial cells by regulating miR-590-5p/PDCD4 axis.

Circular RNAs (circRNAs) are the emerging informative RNAs, involved in cardiovascular diseases including atherosclerosis (AS). Endothelial injury is the initial qualitative change of AS. Thus, the objective of this study was to confirm the dysregulation and mechanism of circ_0000231 in cell model of AS at early stage in human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL). The expression of circ_0000231, miR-590-5p and programmed cell death 4 (PDCD4) was detected using real-time quantitative PCR and western blot. Cell injury was measured with MTT, flow cytometry, caspase-3 activity assay and enzyme-linked immunosorbent assay (ELISA). The interaction among circ_0000231, miR-590-5p and PDCD4 was validated by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and pull-down assays. Stress ox-LDL decreased cell viability, and increased apoptosis rate and caspase-3 activity in HUVECs in a dose- and time-dependent manner in concomitant with promotions of interleukin-6, interleukin-1β, tumor necrosis factor-α, LC3-II/I and Beclin-1 levels. Besides, circ_0000231 and PDCD4 expressions were upregulated, and miR-590-5p was downregulated in ox-LDL-stimulated HUVECs. Functionally, knockdown of circ_0000231 and overexpression of miR-590-5p could suppress ox-LDL-elicited above effects on apoptosis, autophagy and inflammatory response, accompanied with PDCD4 downregulation. Physically, miR-590-5p could directly interact with circ_0000231 and PDCD4. Downregulation of circ_0000231 suppresses HUVECs from ox-LDL-induced injury partially through regulating miR-590-5p/PDCD4 axis via competing endogenous RNA mechanism, showing a novel potential target for the pathology and treatment of endothelial injury in AS.

Stimulation of soluble guanylate cyclase activity with riociguat promotes angiogenesis and improves neovascularization after limb ischemia

Background and aimsThe NO-cGMP pathway is essential for angiogenesis, vasculogenesis and post-natal neovascularization. The key enzyme responsible for the synthesis of cGMP following binding of NO is soluble guanylate cyclase (sGC). Riociguat is the first member of a novel class of compounds known as sGC stimulators. We tested the hypothesis that stimulation of sGC with riociguat might improve neovascularization in response to ischemia. MethodsIn vitro, the angiogenic effect of riociguat was tested in human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization was investigated in a mouse model of limb ischemia. C57Bl/6 mice were treated by gavage with 3 mg/kg/day of riociguat for a total of 28 days. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. ResultsIn a matrigel assay in vitro, riociguat dose-dependently stimulates tubule formation in HUVECs. Cell migration (scratch assay) is also increased in HUVECs treated with riociguat. At the molecular level, riociguat treatment leads to rapid activation of the p44/p42 MAP kinase pathway in HUVECs. Inhibition of protein kinase G (PKG) activity supresses both p44/p42 MAP kinase activation and angiogenesis in HUVECs treated with riociguat. In vivo, treatment with riociguat improves blood flow recovery after ischemia (Laser Doppler imaging), and increases capillary density in ischemic muscles (CD31 immunostaining). Clinically, this is associated with a significant decrease of ambulatory impairment and ischemic damages. Interestingly, mice treated with riociguat also show a 94% increase in the number of bone marrow-derived pro-angiogenic cells (PACs) compared to control mice. Moreover, riociguat treatment is associated with a significant improvement of PAC functions including migratory capacity, adhesion to an endothelial monolayer, and integration into endothelial tubular networks. ConclusionsThe sGC stimulator riociguat promotes angiogenesis and improves neovascularization after ischemia. The mechanism involves PKG-dependent activation of p44/p42 MAP kinase pathway, together with an improvement of PAC number and functions. sGC stimulation could constitute a novel therapeutic strategy to reduce tissue ischemia in patients with severe atherosclerotic diseases.

Cathepsin S Knockdown Suppresses Endothelial Inflammation, Angiogenesis, and Complement Protein Activity under Hyperglycemic Conditions In Vitro by Inhibiting NF-κB Signaling

Hyperglycemia plays a key role in the development of microvascular complications, endothelial dysfunction (ED), and inflammation. It has been demonstrated that cathepsin S (CTSS) is activated in hyperglycemia and is involved in inducing the release of inflammatory cytokines. We hypothesized that blocking CTSS might alleviate the inflammatory responses and reduce the microvascular complications and angiogenesis in hyperglycemic conditions. In this study, we treated human umbilical vein endothelial cells (HUVECs) with high glucose (HG; 30 mM) to induce hyperglycemia and measured the expression of inflammatory cytokines. When treated with glucose, hyperosmolarity could be linked to cathepsin S expression; however, many have mentioned the high expression of CTSS. Thus, we made an effort to concentrate on the immunomodulatory role of the CTSS knockdown in high glucose conditions. We validated that the HG treatment upregulated the expression of inflammatory cytokines and CTSS in HUVEC. Further, siRNA treatment significantly downregulated CTSS expression along with inflammatory marker levels by inhibiting the nuclear factor-kappa B (NF-κB) mediated signaling pathway. In addition, CTSS silencing led to the decreased expression of vascular endothelial markers and downregulated angiogenic activity in HUVECs, which was confirmed by a tube formation experiment. Concurrently, siRNA treatment reduced the activation of complement proteins C3a and C5a in HUVECs under hyperglycemic conditions. These findings show that CTSS silencing significantly reduces hyperglycemia-induced vascular inflammation. Hence, CTSS may be a novel target for preventing diabetes-induced microvascular complications.

Attenuation of indoxyl sulfate–induced cell damage by cinchonidine—a Cinchona alkaloid—through the downregulation of p53 signaling pathway by promoting MDM2 cytoplasmic-nuclear shuttling in endothelial cells

Renocardiac syndromes are a critical concern among patients with chronic kidney disease (CKD). High level of indoxyl sulfate (IS), a protein-bound uremic toxin, in plasma is known to promote the pathogenesis of cardiovascular diseases by impairing endothelial function. However, the therapeutic effects of the adsorbent of indole, a precursor of IS, on renocardiac syndromes is still debated. Therefore, novel therapeutic approaches should be developed to treat IS-associated endothelial dysfunction. In the present study, we have found that cinchonidine, a major Cinchona alkaloid, exhibited superior cell-protective effects among the 131 test compounds in IS-stimulated human umbilical vein endothelial cells (HUVECs). IS-induced cell death, cellular senescence, and impairment of tube formation in HUVECs were substantially reversed after treatment with cinchonidine. Despite the cinchonidine did not alter reactive oxygen species formation, cellular uptake of IS and OAT3 activity, RNA-Seq analysis showed that the cinchonidine treatment downregulated p53-modulated gene expression and substantially reversed IS-caused G0/G1 cell cycle arrest. Although the mRNA levels of p53 were not considerably downregulated by cinchonidine in IS-treated HUVECs, the treatment of cinchonidine promoted the degradation of p53 and the cytoplasmic-nuclear shuttling of MDM2. Cinchonidine exhibited cell-protective effects against the IS-induced cell death, cellular senescence, and impairment of vasculogenic activity in HUVECs through the downregulation of p53 signaling pathway. Collectively, cinchonidine may be a potential cell-protective agent to rescue IS-induced endothelial cell damage.

Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thrombotic events and/or pregnancy morbidity in the presence of antiphospholipid antibodies detected either as anti-cardiolipin, anti-β2 Glycoprotein I (anti-β2GPI) or Lupus anticoagulant (LA). Endothelial deregulation characterizes the syndrome. To address gene expression changes accompanying the development of autoimmune phenotype in endothelial cells in the context of APS, we performed transcriptomics analysis in Human Umbilical Vein Endothelial Cells (HUVECs) stimulated with IgG from APS patients and β2GPI, followed by intersection of RNA-seq data with published microarray and ChIP-seq results (Chromatin Immunoprecipitation). Our strategy revealed that during HUVEC activation diverse signaling pathways such as TNF-α, TGF-β, MAPK38, and Hippo are triggered as indicated by Gene Ontology (GO) classification and pathway analysis. Finally, cell biology approaches performed side-by-side in naïve and stimulated cultured HUVECs, as well as, in placenta specimens derived from Healthy donors (HDs) and APS-patients verified the evolution of an APS-characteristic gene expression program in endothelial cells during the initial stages of the disease's development.

Extracellular histones trigger oxidative stress-dependent induction of the NF-kB/CAM pathway via TLR4 in endothelial cells

Extracellular histones have been reported to aggravate different pathophysiological processes by increasing vascular permeability, coagulopathy, and inflammation. In the present study, we elucidate how extracellular histones (10–100 µg/mL) concentration dependently increase cytosolic reactive oxygen species (ROS) production using human umbilical vein endothelial cells (HUVECs). Furthermore, we identify cyclooxygenase (COX) and NADPH oxidase (NOX) activity as sources of ROS production in extracellular histone-treated HUVEC. This COX/NOX-mediated ROS production is also involved in enhanced NF-kB activity and cell adhesion molecules (VCAM1 and ICAM1) expression in histone-treated HUVEC. Finally, by using different toll-like receptor (TLR) antagonists, we demonstrate the role of TLR4 in CAMs overexpression triggered by extracellular histones in endothelial cells. In conclusion, our data suggest that through TLR4 signaling, extracellular histones increase endothelial cell activation, a mechanism involving increased COX- and NOX-mediated ROS. These findings increase our understanding on how extracellular histones enhance systemic inflammatory responses in diseases in which histone release occurs as part of the pathological processes.