Endothelial Cell Viability Research Articles

MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs' in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3′UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.MicroRNA-432-5p expression in osteosarcoma cells regulates sprouting and intussusceptive angiogenesis by targeting PDGFB genes. MicroRNA-432-5p/PDGFB signaling can be targeted to treat angiogenesis in osteosarcoma along with other strategies which will further strengthen the effectiveness of antiangiogenic therapy.

Sesamol Epigenetically Induces Estrogen Receptor α Re-expression by Upregulating miR-370-3p in Estrogen Receptor α-Negative Breast Cancer.

Due to lack of estrogen receptor α (ERα, gene name: ESR1), ERα-negative breast carcinoma is insensitive to endocrine therapy, and restoration of ERα has become a promising strategy for ERα-negative breast cancer treatment. Sesamol, a naturally occurring phenolic compound, is usually extracted from sesame seeds. Previous investigations have unmasked its anti-oxidant and anti-inflammation properties. In this study, sesamol induced ERα functional re-expression followed by upregulation of its downstream pS2 and GREB1 genes in ERα-negative breast carcinoma. Moreover, it endowed responsiveness of ERα-negative breast carcinoma to the endocrine treatment drug 4-hydroxytamoxifen without influencing the viability of normal human umbilical vein endothelial cells. Mechanistically, sesamol induced ESR1 gene promoter demethylation by downregulating the expression of the DNA methyltransferases DNMT3A and DNMT3B, without affecting DNMT1. Moreover, the non-coding RNA miR-370-3p directly targeted DNMT3A and DNMT3B mRNA, and its expression increased upon treatment with sesamol. Artificial abrogation of miR-370-3p expression with an antagomir abolished the inhibition of DNMT3A and DNMT3B expression by sesamol, resulting in a fallback in ERα reactivation. In mice, sesamol significantly induced ERα re-expression via miR-370-3p-mediated downregulation of DNMT3A and DNMT3B. Sesamol may be a safe and effective option for clinical adjuvant therapy in patients with ERα-negative breast cancer.

Effect of Bevacizumab on the Viability and Metabolism of Human Corneal Epithelial and Endothelial Cells: An In Vitro Study.

PurposeTo examine the cytotoxic effects of bevacizumab on the viability and metabolism of human corneal epithelial cells (HCEpCs) and human corneal endothelial cells (HCEnCs), as well as human retinal pigment epithelial (ARPE-19) cells for comparison.MethodsImmortalized cell lines of HCEpCs, HCEnCs, and ARPE-19 cells were exposed to clinically relevant concentrations of bevacizumab (0.313–5.00 mg/mL). The ApoTox-Glo Triplex Assay was used to assess cell viability, cytotoxicity, and apoptosis, and the Mitochondrial ToxGlo Assay was used to assess cell membrane integrity and adenosine triphosphate (ATP) levels after a 24-hour treatment period.ResultsAcross all three cell types, we observed similar results of a decrease in cell viability at 5.00 mg/mL (P < 0.05) and an increase in cytotoxicity at 5.00 mg/mL (P < 0.05), whereas apoptotic activity remained unchanged (P > 0.05), which is a profile consistent with cells undergoing primary necrosis at high concentrations. Additionally, cell membrane integrity was compromised at 5.00 mg/mL (P < 0.05), whereas no decrease in ATP levels were observed (P > 0.05). Thus, no interference with mitochondrial oxidative phosphorylation in ATP production was seen, and the cells were able to maintain normal metabolic levels at high concentrations of bevacizumab.ConclusionsHCEpCs, HCEnCs, and ARPE-19 cells experience a decrease in viability and undergo primary necrosis when exposed to bevacizumab at a concentration of 5.00 mg/mL; however, they are able to maintain normal metabolism and mitochondrial function at the high concentrations used for the treatment of corneal neovascularization.Translational RelevanceThis study provides safety data on the concentrations of bevacizumab injected intravitreally and complements clinical data showing toxicity of topical bevacizumab on corneal epithelial and endothelial cells.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma

The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated invivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit invitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Exosome circCMTM3 promotes angiogenesis and tumorigenesis of hepatocellular carcinoma through miR-3619-5p/SOX9.

As one of the most common and lethal carcinomas, hepatocellular carcinoma (HCC) is a global health concern and affects millions of people worldwide. Current treatments for HCC are very limited due to its unclear pathogenesis. Here, we aim to further investigate the role of circCMTM3/microRNA (miR)-3619-5p in HCC. Human blood samples were collected from HCC patients and healthy people. Quantitative reverse transcription-polymerase chain reaction and western blot analysis were undertaken to measure levels of circCMTM3, miR-3619-5p, SOX9, and exosome markers. The MTT, colony formation, and Transwell assays were used to examine the viability, migration, and invasion of human umbilical vein endothelial cells (HUVECs), respectively. Tube formation assay was used to assess angiogenesis. Dual luciferase assay was used to validate circCMTM3/miR-3619-5p and miR-3619-5p/SOX9 interactions. A nude mouse xenograft model was used to test the role of circCMTM3 in HCC in vivo. Levels of circCMTM3 in exosomes from HCC patients and cells were elevated. Knockdown of circCMTM3 greatly decreased viability, migration, and invasion of HUVECs, as well as angiogenesis. CircCMTM3 acted as a miR-3619-5p sponge and miR-3619-5p inhibitor reversed the effects of si-circCMTM3 on angiogenesis. MiR-3619-5p directly targeted SOX9 and modulated angiogenesis through SOX9. Furthermore, knockdown of circCMTM3 suppressed angiogenesis and HCC tumor growth in vivo. The exosome circCMTM3/miR-3619-5p/SOX9 axis from HCC cells promotes angiogenesis and thus contributes to HCC tumorigenesis.

Uremic Serum Damages Endothelium by Provoking Excessive Neutrophil Extracellular Trap Formation

Background: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Methods: Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, we differentiated HL-60 cells into neutrophil-like cells (dHL-60) by applying retinoic acid and determined the effect of uremic serum on these dHL-60 and human umbilical vein EC (HUVEC). Findings: The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality or vascular access failure. In vitro, HD-derived serum significantly increased NET formation by dHL-60, and these NETs decreased EC viability and induced their apoptosis. In addition, the ICAM-1 level in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Interpretation: Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction. Funding: This research was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1A2C110138611). Declaration of Interests: No conflicts of interest in this study. Ethics Approval Statement: This study was approved by the Institutional Ethics Committee and informed consent was obtained from the HD patients and HVs.

Dual l-Carnosine/Aloe vera Nanophytosomes with Synergistically Enhanced Protective Effects against Methylglyoxal-Induced Angiogenesis Impairment.

Microvascular complications are among the major outcomes of patients with type II diabetes mellitus, which are the consequences of impaired physiological functioning of small blood vessels and angiogenic responses in these patients. Overproduction and accumulation of methylglyoxal (MGO), a highly reactive dicarbonyl byproduct of glycolysis pathway, has been acclaimed as the main inducer of impaired angiogenic responses and microvascular dysfunction in diabetic patients with uncontrolled hyperglycemia. Hence, an effective approach to overcome diabetes-associated microvascular complications is to neutralize the deleterious activity of enhanced the concentration of MGO in the body. Owing to the glycation inhibitory activity of Aloe vera whole extract, and capability of l-carnosine, an endogenous dipeptide, in attenuating MGO's destructive activity, we examined whether application of a combination of l-carnosine and A. vera could be an effective way of synergistically weakening this reactive dicarbonyl's impaired angiogenic effects. Additionally, overcoming the poor cellular uptake and internalization of l-carnosine and A. vera, a nanophytosomal formulation of the physical mixture of two compounds was also established. Although l-carnosine and A. vera at whole studied combination ratios could synergistically enhance viability of human umbilical vein endothelial cells (HUVECs) treated with MGO, the 25:1 w/w ratio was the most effective one among the others (27 ± 0.5% compared to 12 ± 0.3 to 18 ± 0.4%; F (4, 15) = 183.9, P < 0.0001). Developing dual nanophytosomes of l-carnosine/A. vera (25:1) combination ratio, we demonstrated superiority of the nanophytosomal formulation in protecting HUVECs against MGO-induced toxicity following a 24-72 h incubation period (17.3, 15.8, and 12.4% respectively). Moreover, 500 μg/mL concentration of dual l-carnosine/A. vera nanophytosomes exhibited a superior free radical scavenging potency (63 ± 4 RFU vs 83 ± 5 RFU; F (5, 12) = 54.81, P < 0.0001) and nitric oxide synthesizing capacity (26.11 ± 0.19 vs 5.1 ± 0.33; F (5, 12) = 2537, P < 0.0001) compared to their physical combination counterpart. Similarly, 500 μg/mL dual l-carnosine/A. vera nanophytosome-treated HUVECs demonstrated a superior tube formation capacity (15 ± 3 vs 2 ± 0.3; F (5, 12) = 30.87, P < 0.001), wound scratch healing capability (4.92 ± 0.3 vs 3.07 ± 0.3 mm/h; F (5, 12) = 39.21, P < 0.0001), and transwell migration (586 ± 32 vs 394 ± 18; F (5, 12) = 231.8, P < 0.001) and invasion (172 ± 9 vs 115 ± 5; F (5, 12) = 581.1, P < 0.0001) activities compared to the physical combination treated ones. Further confirming the proangiogenic activity of the dual l-carnosine/A. vera nanophytosomes, a significant shift toward expression of proangiogenic genes including HIF-1α, VEGFA, bFGF, KDR, and Ang II was reported in treated HUVECs. Overall, dual l-carnosine/A. vera nanophytosomes could be a potential candidate for attenuating type II DM-associated microvascular complications with an impaired angiogenesis background.

Evaluation of fine particulate matter on vascular endothelial function in vivo and in vitro

Ambient fine particulate matter (PM2.5) and high-fat diet (HFD) are linked to the development of atherosclerosis. However, there is still unknown about the PM2.5-induced atherosclerosis formation on vascular endothelial injury after co-exposed to PM2.5 and HFD. Thus, the aim of this study was to evaluate the effects of PM2.5 on atherogenesis in C57BL/6 mice and endothelial cells, as well as the co-exposure effect of PM2.5 and HFD. In vivo study, C57BL/6 mice exposed to PM2.5 and fed with standard chow diet (STD) or HFD for 1 month. PM2.5 could increase vascular stiffness accessed by Doppler ultrasound, and more serious in co-exposure group. PM2.5 impaired vascular endothelial layer integrity, exfoliated endothelial cells, and inflammatory cells infiltration through H&E staining. PM2.5 reduced the expression of platelet/endothelial cell adhesion molecule-1 (PECAM-1) in vessel. Moreover, PM2.5 could induce systemic inflammation detected by Mouse Inflammation Array. In vitro study, PM2.5 triggered markedly mitochondrial damage by transmission electron microscope (TEM) and flow cytometer. Inflammatory cytokines were significantly increased in PM2.5-exposed group. The cell viability and migration of endothelial cells were significantly suppressed. In addition, PM2.5 remarkably declined the expression of vascular endothelial growth factor receptor 2 (VEGFR2) and increased the expression of somatostatin (SST) and its receptor. In conclusion, co-exposure of PM2.5 and HFD might induce systemic inflammation and endothelial dysfunction in normal mice. Moreover, PM2.5 could reduce vascular endothelial repair capacity through inhibiting the proliferation and migration of endothelial cells.

Lacidipine Ameliorates the Endothelial Senescence and Inflammatory Injury Through CXCR7/P38/C/EBP-β Signaling Pathway.

Background: Lacidipine, a third-generation calcium channel blocker, exerts beneficial effects on the endothelium of hypertensive patients in addition to blood pressure lowering. However, the detailed mechanism underlying Lacidipine-related endothelial protection is still elusive.Methods: Sixteen spontaneous hypertensive rats (SHRs) were randomly divided into two groups: Lacidipine-treated SHR group and saline-treated control group. Tail systolic blood pressure was monitored for four consecutive weeks. Endothelial cells (ECs) were pretreated with Lacidipine prior to being stimulated with H2O2, bleomycin, or Lipopolysaccharides (LPS) in vitro. Then, cell activity, migration, and senescence were measured by Cell Counting Kit-8 assay, transwell assay, and β-galactosidase staining, respectively. The fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used to assess the intracellular reactive oxygen species (ROS). Related protein expression was detected by Western blotting and immunofluorescence.Results: Our data showed that Lacidipine treatment lowered the blood pressure of SHRs accompanied by the elevation of CXCR7 expression and suppression of P38 and CCAAT/enhancer-binding protein beta (C/EBP-β) compared with the control group. In vitro experiments further demonstrated that Lacidipine increased the cell viability and function of ECs under oxidative stress, cell senescence, and inflammatory activation via the CXCR7/P38/signaling pathway.Conclusions: Our results suggested that Lacidipine plays a protective role in EC senescence, oxidative stress, and inflammatory injury through the regulation of CXCR7/P38/C/EBP-β signaling pathway.

Abstract 2649: An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy; however, targeting pro-angiogenic pathways has shown significant clinical benefit. The development of targeted anti-angiogenetic agents has been hampered by the lack of clinically relevant models for screening. We have previously reported that the generation of 3D-tumoroids containing the unaltered tumor stroma could effectively be used to detect tumor response to small molecule inhibitors as well as antibody-based therapeutic agents. In this study we described a novel ex vivo platform to develop anti-angiogenic therapeutic strategies using fresh patient tumoroids of hepatocellular carcinoma and renal cell carcinoma. Materials and Methods: All fresh tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays, 3D tumoroids measuring 150 µm in size were treated with tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib or axitinib. Treatment-mediated changes in endothelial cell viability was assessed by the confocal-based high-content real time imaging platform using fluorescent labeled anti-CD31 and anti-VEGFR2 antibodies, combined with custom image analysis algorithms. Additionally, treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Results: Our data shows that endothelial cells and capillary structures could clearly be visualized within3D tumoroids by confocal microscopy. Furthermore, we were able to quantify apoptotic cell death in endothelial cells induced by TKI inhibitors. Flow cytometry analysis demonstrated significant changes in T-cell activation upon treatment with TKIs that closely correlated with increased proinflammatory cytokine release. Observations found that the combination of anti-angiogenic treatments and immuno-modulatory agents significantly improved treatment responses. Conclusion: The ex vivo platform described in this study allowed assessment of the effect of anti-angiogenetic TKIs on tumor capillary endothelial cells as well as on tumor resident immune cell populations in intact RCC tumoroids of fresh patient tumor samples. We believe this clinically relevant approach can be used to identify the most effective anti-angiogenic and immunotherapeutic drugs and drug combinations in RCC and may improve personalized therapy for individual patients in clinical studies. Citation Format: Jared Ehrhart, Mibel M. Pabón, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2649.

HBXIP accelerates glycolysis and promotes cancer angiogenesis via AKT/mTOR pathway in bladder cancer

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. Although HBXIP is known to be associated with a poor prognosis for bladder cancer (BC), its effects on glycolysis and angiogenesis in BC have not been investigated. BC prognosis and relative gene expression of HBXIP were analyzed using the GEPIA, UALCAN, and STRING databases. BC cell angiogenesis and glycolysis were assessed by vasculogenic mimicry and glycolysis assay. Human umbilical vein endothelial cell (HUVEC) viability, migration, and angiogenesis were assessed by CCK8, transwell, wound healing, and tube formation assays. The results showed that HBXIP was highly expressed in BC tissues and cells. Knockdown of HBXIP expression decreased the levels of glucose uptake, lactate production, and glycolytic enzyme expression in BC cells, and decreased cell viability and migration of HUVECs. Additionally, silencing HBXIP reduced the total length of tubes and number of intersections, and EPO and VEGF protein expression in BC cells and HUVECs. Furthermore, knockdown of HBXIP expression reversed cell viability, migration, tube formation, and vasculogenic mimicry under high glucose and lactate conditions. Mechanistically, silencing of HBXIP reduced the protein expression levels of pAKT-ser473 and pmTOR, and inhibition of HBXIP, AKT, and mTOR expression decreased glycolytic enzyme protein expression. Our findings suggest that HBXIP reduces glycolysis in BC cells via regulation of AKT/mTOR signaling, thereby blocking BC angiogenesis. Collectively, this study provides a potential strategy to target HBXIP and AKT/mTOR for regulating glycolysis progression concurrently with anti-angiogenesis effects, and thereby develop novel therapeutics for the treatment of BC.

Biological Effects of XyloCore, a Glucose Sparing PD Solution, on Mesothelial Cells: Focus on Mesothelial-Mesenchymal Transition, Inflammation and Angiogenesis.

Glucose-based solutions remain the most used osmotic agents in peritoneal dialysis (PD), but unavoidably they contribute to the loss of peritoneal filtration capacity. Here, we evaluated at a molecular level the effects of XyloCore, a new PD solution with a low glucose content, in mesothelial and endothelial cells. Cell viability, integrity of mesothelial and endothelial cell membrane, activation of mesothelial and endothelial to mesenchymal transition programs, inflammation, and angiogenesis were evaluated by several techniques. Results showed that XyloCore preserves mesothelial and endothelial cell viability and membrane integrity. Moreover XyloCore, unlike glucose-based solutions, does not exert pro-fibrotic, -inflammatory, and -angiogenic effects. Overall, the in vitro evidence suggests that XyloCore could represent a potential biocompatible solution promising better outcomes in clinical practice.

Effect of hydroxychloroquine on antiphospholipid antibodies-inhibited endometrial angiogenesis

Background Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombotic events and/or pregnancy morbidity (≥3 recurrent early miscarriage or fetal death or a prematurity <34 weeks of gestation) with persistently positive antiphospholipid antibodies (aPLs). It is reported that aPLs damage the placental tissue by binding to β2-glycoprotein I (β2GPI) on the surface of trophoblast and endothelial cells. Hydroxychloroquine (HCQ) is considered to be beneficial in the treatment of obstetrical APS and shown to restore the aPL-inhibited invasion and differentiation of trophoblast. However, not enough evidence exists regarding the effect of HCQ on endometrial angiogenesis. The aim of our study was to assess whether HCQ has an effect on aPL-inhibited endothelial angiogenesis. Methods In this research, to explore the effect of HCQ for angiogenesis, we investigated: (1) Human umbilical vein endothelial cells (HUVECs) viability by CCK-8; (2) HUVECs migration by wound healing; (3) HUVEC angiogenesis by Matrigel assay in vitro; (4) mRNA expression of MMP-2 and VEGF by real-time quantitative Polymerase Chain Reaction (RT-PCR); (5) protein expression of VEGF, MMP-2 by western blot. Results We found that HCQ treatment significantly restored the expression of aPL-inhibited VEGF and MMP-2. HCQ restored aPL-inhibited HUVEC proliferation, migration, and angiogenesis in vitro. Conclusion In conclusion, aPLs inhibit HUVECs angiogenesis, however, HCQ can restore the effect of aPL-inhibited HUVECs migration and angiogenesis in vitro, demonstrating its beneficial therapeutic role in obstetrical APS.

In Vitro Effects of BCR-ABL Tyrosine Kinase Inhibitors on Endothelial Cells Survival and Functions

BackgroundBCR-ABL tyrosine kinase inhibitors (TKIs) are the mainstay of the treatment from chronic myeloid leukemia (CML). However, new generation TKIs (dasatinib, nilotinib, bosutinib and ponatinib) increase the risk of arterial occlusive events (e.g. ischemic stroke, peripheral artery disease, myocardial infarction) in patients with CML compared with the first generation (imatinib). To date, the mechanism(s) of these side effects is poorly understood but clinical data suggest that new generation TKIs might accelerate atherogenesis, in which endothelial cells play major function in its initiation (e.g. lipid deposition, leukocyte recruitment).AimsThis research aims to determine the effect of BCR-ABL TKIs on endothelial cell survival and major functions (endothelial cell migration and expression of adhesion molecules) on in vitro models.MethodsThe 5 BCR-ABL TKIs were tested at 3 clinically relevant concentrations. Effects of BCR-ABL TKIs on endothelial cells were assessed on the EA.hy926 cell line and on endothelial cells derived from human umbilical vein (HUVEC). Viability after 24h of treatment with BCR-ABL TKIs was assessed using MTS and LDH assays following standard protocols. The impact of TKI on the expression of 3 adhesion molecules (i.e. ICAM-1, VCAM-1 and E-selectin) was measured by on-cell ELISA after 4-hour TNF-α activation (10ng/mL), cell fixation, blockade and staining with primary and secondary antibodies. Migration of endothelial cells was evaluated by a scratch assay. EA.hy926 and HUVEC cells were cultured in 24-well plates and pre-treated with BCR-ABL TKIs in serum-free condition. The confluent monolayer was then scratched with a pipette tip and the wound closure was observed through inverted microscope. Photography were taken at baseline, after 2h, 4h, 6h and 24h and analyzed using the Leica Application Suite software (version 4.7) to quantify the extent of cell migration into the gap.ResultsDasatinib, nilotinib and ponatinib at high concentration reduce cell metabolism indicating an inhibition of proliferation or induction of apoptosis by the 3 TKIs. Additionally, high-dose ponatinib (0.5µM) induces necrosis as demonstrated by increased LDH release. Cell death within the arterial wall has already been recognized in atherosclerosis, and it appears plausible that dasatinib, nilotinib and ponatinib facilitate atherosclerosis development through alteration of endothelial cell viability. Leukocyte recruitment is also an important process in atherogenesis and requires the expression of adhesion molecules by activated endothelial cells. We assessed the expression of 3 adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in HUVEC and demonstrate a decreased of ICAM-1, VCAM-1 and E-selectin expression with dasatinib, nilotinib and ponatinib at high concentration (0.5µM, 2µM and 0.5µM respectively). This diminution correlates with the decreased viability of endothelial cell with these 3 treatments. Oppositely, imatinib and bosutinib have no or little impact on adhesion molecule expression. Finally, we evaluate the migration ability of endothelial cell after treatment. Scratch assay results showed that high dose dasatinib inhibits endothelial cell migration whereas other TKIs do not have any impact. Therefore, dasatinib might induce arterial occlusive events through impaired vascular wound healing.ConclusionsOver the 5 commercialized BCR-ABL TKIs, dasatinib, nilotinib and ponatinib possess the most impact on endothelial cells. They reduce viability by inducing apoptosis, necrosis or inhibiting cell proliferation, which possibly help to the development of atherosclerosis through impaired endothelium permeability, enabling migration and trapping of lipoprotein into the intima. Additionally, dasatinib reduces endothelial cell migration, which might contribute to arterial thrombosis formation. DisclosuresNo relevant conflicts of interest to declare.

Impact of Tyrosine Kinase Inhibitors Used in Chronic Myeloid Leukemia on Signaling and Epigenetics in Endothelial Cells

Background: Tyrosine kinase inhibitors (TKIs) have revolutionized treatment of chronic myeloid leukemia (CML) resulting in long term survival with chronic therapy. Vascular toxicities have emerged as complications of the second generation CML TKIs; dasatinib, ponatinib and nilotinib. Dasatinib is associated with increased risk of pulmonary hypertension, ponatinib with arterial ischemic events and nilotinib with peripheral arterial occlusive events and cardiovascular ischemia by unknown mechanisms. The first generation CML TKI imatinib is not associated with vascular toxicity.Hypothesis: We hypothesize that the CML TKIs- ponatinib, dasatinib and nilotinib, but not imatinib, cause vascular toxicity by affecting endothelial cell (EC) function in association with alterations in phospho-signaling and histone modifications.Methods To test this hypothesis, we studied the effect of CML TKIs imatinib, nilotinib, dasatinib and ponatinib on EC function using three in-vitro assays measuring EC survival, EC-leucocyte adhesion (a measure of vascular inflammation) and scratch wound closure (a measure of wound healing capacity). Cultured human umbilical vein ECs (HUVECs) were treated with vehicle, imatinib (4uM), dasatinib (200nM), nilotinib (3 uM) or ponatinib (100nM). Cell viability was measured at 18 hours using a Cell TiterGlo Luminescent Cell Viability assay (Promega). For the leucocyte adhesion assay, fluorescent labeled leucocytes (U937) were incubated with the ECs and adherent fluorescent cells were quantified. For the wound healing assay, a scratch “wound” was made with a plastic P1000 pipette tip and cells that migrated into the wound area were counted at 18 hours. To assess signaling, two novel, global, unbiased, high throughput proteomic assays developed at the Broad Institute were used. The P100 assay is a mass spectrometry-based targeted proteomics assay that quantifies 100 representative phosphopeptides. The global chromatin profiling (GCP) assay uses mass spectrometry to simultaneously quantify over 70 bulk histone modifications including phosphorylation, ubiquitination, methylation and acetylation. HUVECs were treated in triplicate with each drug for 3 hours for the P100 assay and for24 hours for the GCP assay. For each assay, the peptides were mixed with isotopically labeled internal standards and mass spectrometry was performed to quantify each phospho-peptide or modified histone peptide relative to the universal standard.Results: The CML TKIs that are associated with vascular toxicity (nilotinib, ponatinib and dasatinib) negatively impacted all the EC functions measured. Specifically, nilotinib, ponatinib and dasatinib significantly decreased endothelial cell viability and endothelial cell migration in response to a scratch wound and increased leucocyte adhesion to ECs. Imatinib did not have a negative effect on ECs in any of those assays. Using the mass spectrometry based P100 and GCP assays, we found that nilotinib affects histone epigenetics and protein phosphorylation in HUVECs. In particular, nilotinib treatment modulates ABL1, MAP4, BRD4 and JUND phosphorylation and also increases histone H3K9 trimethylation with H3S10 phosphorylation in HUVECS. We also compared the nilotinib profiles obtained in HUVECs to that of primary human aortic smooth muscle cells (HAoSMC) and several cancer cell lines including MCF7 (breast), A549 (lung), A375 (skin) and PC3 (prostate) (Figures 1a and 1b). While some changes are unique to ECs, others are similar to the effects in cancer cells and smooth muscle cells. GCP assay and P100 assays comparing the effects of all the CML TKIs in endothelial cells are in process.Conclusions: CML TKIs associated with vascular toxicity affect human EC function in vitro resulting in impaired endothelial survival and wound healing and increase inflammation. These changes are associated with alterations in signaling and epigenetic pathways in ECs in response to treatment with nilotinib. Increased vascular inflammation and impaired vascular healing are associated with cardiovascular thrombosis and ischemia. Further studies comparing the effects of all CML TKIs on ECs will be used to elucidate molecular mechanisms of vascular toxicity and help devise strategies to overcome these toxicities while maintaining anti-cancer efficacy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Effect of ABO Immune Complexes on Endothelial Cell Integrity As Measured By Electric Cell-Substrate Impedance Sensing (ECIS) and Confocal Microscopy

Background: ABO non-identical transfusions have been associated with increased risk of hemolytic transfusion reactions. We previously reported that the ABO immune complexes (ABO-IC) formed in vitro as a result of ABO non-identical exposure affect platelet function, red blood cell integrity, and clot kinetics (Vox Sang. 2016;110:219-26). We developed an in vitro model as a means to investigate the interaction between ABO-IC and endothelial cells (EC).Methods: The ABO-IC were prepared by combining equal volumes of concentrated O plasma (high titers of anti-A and anti-B) with A plasma (high concentration of A antigens). Anti-A titers were measured before and after mixing to confirm the presence of ABO-IC. ABO-known human pulmonary vessel EC were then seeded onto gold microarray plates to prepare for electric cell-substrate impedance sensing (ECIS). The trans-endothelial electrical resistance (TER) was recorded for 48 hours after the addition of media, PBS, thrombin, O, A, B, and/or AB plasmas added 1:1 with media, as well as various concentrations of ABO-IC. Statistics were performed using the chi-square test to compare TER values at various timepoints to baseline. Human pulmonary EC were cultured on glass-bottomed petri dishes for confocal microscopy. The cells were stained with fluorescent green calcein AM dye and assessed for viability at 100x magnification at baseline and again 15 minutes following the addition of ABO-IC.Results: Substantial decreases in TER were observed over the course of 2 hours in O and A EC when treated with non-identical plasma, and sustained over 10 hours (p<0.05 for all). Significant decreases in TER were also observed in cells treated with various concentrations of ABO-IC, over 2 hours and were found to be dose-dependent and independent of ABO type (p<0.05 for all). Unlike the ABO-IC, exposure to thrombin revealed a marked decrease in TER after 2 hours, although the final TER measurement revealed complete recovery (Table 1a-c). Incubation with ABO-identical plasma showed slight decreases in TER over time that were not statistically significant when compared to incubation with cell media (Figure 1a-b). Physical damage to the EC membrane were observed via confocal microscopy after 15 minutes of exposure to ABO-IC (Figures 2a-b).Discussion: The exposure of ABO-IC to human pulmonary vessel EC revealed significantly decreased monolayer integrity via ECIS and confocal microscopy. The decreases seen in the TER of EC incubated with non-identical plasma and ABO-IC indicate the potential for capillary leak as a possible reaction to ABO non-identical transfusion. We have shown that the substantial decrease in TER when exposed to plasma is ABO type-dependent, and that the damage caused to the cells happens within a few hours and is irreversible over 48 hours. The confocal images reveal a significant change in the morphology of the EC membrane within 15 minutes of exposure to ABO-IC. These preliminary data suggest that anti-A and ABO-IC may significantly affect EC viability and function. This in vitro model provides potential novel mechanisms for the detrimental effects of ABO non-identical transfusion on EC. Further investigation of biomarkers for cell viability and integrity in response to exposure to ABO-IC is underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione.Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.

Adverse effect of polystyrene microplastics (PS-MPs) on tube formation and viability of human umbilical vein endothelial cells

Environmental contamination by microplastics (MPs) is an emerging concern in recent years due to associated adverse impacts of MPs on potential human health problems. Endothelial dysfunction is a condition in which the endothelial layer fails to form normally, and is associated with impaired vascular function. Despite the fact that MPs are known to enter the circulation system through intestinal epithelium, little has been known whether MPs impact the normal function of endothelial cells and the formation of vasculature. In the current study, we investigated the effect of polystyrene microplastics (PS-MPs) on tube formation and cytotoxicity in human umbilical vein endothelial cells (HUVECs). Our study showed that the treatment of HUVECs with PS-MPs significantly decreased cell viability, with intracellular accumulation occurring in a dose- and size-dependent manner. Moreover, significant dose-dependent inhibition of angiogenic tube formation was observed in HUVECs treated with 0.5 μm PS-MPs; this effect was accompanied by suppression of angiogenic signaling pathways and inhibitory activity against wound healing and cell migration. Regarding the mechanism of decreased viability, we observed increased autophagic and necrotic cell death. These results indicate that 6-h exposure of endothelial cells to PS-MPs represses tube-forming capacity, while 48-h exposure leads to autophagy and necrosis-mediated cytotoxicity.

Экспериментальное обоснование применения вископротекции эндотелия роговичного трансплантата для задней послойной кератопластики, сформированного при помощи фемтосекундного лазера

Purpose. To rationale experimentally the use of a viscoprotection of the corneal endothelium on the graft formed with a femtosecond laser for the descemet stripping endothelial keratoplasty. Material and methods. In our study, we used 12 pig's corneoscleral discs. The preservation time before the experiment averaged 12±4 hours. The corneas were divided into 2 groups. In the operating room, the graft was formed using an LDV Z8 femtosecond laser (Ziemer, Switzerland) from the endothelial side. Before applanation of the donor cornea and femtolaser head, a 1% solution of hydroxypropyl methylcellulose (HPMC) was applied to the endothelium - experimental group. The control group was appraised according to the standard technique, with the application of a few drops of a solution for storing the corneas. Then the applanation was monitored and evaluated by laser optical coherence tomography. Then the graft was separated from the bed and transferred to a conservation medium. Under laboratory conditions, to determine the viability of endothelial cells, the graft was stained with a «vital» dye with the commercial name Life and Dead (Abcam, UK) and placed in a confocal laser scanning microscope. Endothelial cells were counted using the ImageJ software. Results. In the experimental and control groups, the number of living endothelial cells (EC) was 91.06±1.49% and 83.86±2.14%, respectively (p&lt;0.001). The number of dead ECs in the control group was 7.2±0.65% more than in the experimental group and amounted to 16.14±2.14% and 8.94±1.49%, respectively (p&lt;0.001). Conclusion. The study demonstrated that the use of viscoprotection of the corneal graft endothelium for posterior lamellar keratoplasty is quite effective, and significantly reduces the loss of EC at the stage of cutting out the graft with a femtosecond laser. Key words: descemet stripping endothelial keratoplasty, femtosecond laser, vital dyes.

Antiangiogenic and antitumor potential of berbamine, a natural CaMKIIγ inhibitor, against glioblastoma

Glioblastoma (GBM) is one of the most malignant brain tumors and requires the formation of new blood vessels, called angiogenesis, for its growth and metastasis. Several proangiogenic factors, including vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF), stimulate GBM angiogenesis. Accordingly, blocking the angiogenesis induced by angiogenic factors represents a promising modality for the treatment of GBM. In this study, we evaluated the inhibitory effects of berbamine, a plant-derived compound, on the angiogenesis induced by VEGF and BDNF in human umbilical vein endothelial cells (HUVECs). Berbamine effectively inhibited the angiogenic features stimulated by VEGF (such as proliferation, adhesion, invasion, tube formation, and reactive oxygen species (ROS) generation in HUVECs) as well as those by BDNF, at concentrations that do not affect endothelial cell viability. The antiangiogenic effects of berbamine were associated with the downregulation of VEGF/VEGF receptor 2 (VEGFR2)/Ca2+/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and BDNF/tropomyosin receptor kinase B (TrkB)/CaMKIIγ signaling pathways. In addition, berbamine suppressed the expression of a key regulator of tumor angiogenesis, hypoxia-inducible factor-1α (HIF-1α), and its transcriptional target, VEGF, in U87MG GBM cells. Furthermore, berbamine significantly inhibited in vivo neovascularization as well as U87MG tumor growth in a chick embryo chorioallantoic membrane (CAM) model. All these findings suggest that berbamine may be utilized as a new antiangiogenic agent for the treatment of malignant brain tumors.