Viability Of HUVECs Research Articles

Antioxidant peptides from protein hydrolysate of skipjack tuna milt: Purification, identification, and cytoprotection on H2O2 damaged human umbilical vein endothelial cells

To make full use of tuna by-products to prepare high-value products, skipjack tuna (Katsuwonus pelamis) milt was degreased, pretreated with microwave and separately hydrolyzed using trypsin, Neutrase, papain, and pepsin. Neutrase hydrolysate with the highest degree of hydrolysis (29.54 ± 1.19 %) and antioxidant activities were separated by ultrafiltration and a series of chromatographic methods, and thirteen antioxidant oligopeptides were prepared and identified as GHHAAA, PHPR, AKHQ, GRVPRV, ADMYW, VDDDD, SVTEV, VKIYI, VRDQY, IRDDY, YREY, AQRPR, and SMDV with molecular weights of 562.6, 505.6, 482.7, 654.8, 685.1, 578.0, 534.2, 634.6, 679.2, 681.2, 630.0, 626.4, and 450.0 Da, respectively. Moreover, GHHAAA, PHPR, SVTEV, VRDQY, and SMDV showed the highest Ferric-reducing power and radical scavenging activity on DPPH (EC50 values of 1.09 ± 0.08, 1.19 ± 0.07, 0.89 ± 0.08, 0.88 ± 0.11, and 1.24 ± 0.09 mg/mL, respectively) and ABTS (EC50 values of 1.12 ± 0.06, 1.14 ± 0.11, 1.02 ± 0.07, 1.04 ± 0.12, and 1.16 ± 0.07 mg/mL, respectively). In addition, GHHAAA, PHPR, SVTEV, VRDQY, and SMDV presented significant protective function on H2O2 oxidative damaged HUVECs by increasing the HUVECs viability and antioxidant enzyme activity, and lower the contents of ROS and MDA. This study suggested that GHHAAA, PHPR, SVTEV, VRDQY, and SMDV could be natural antioxidant ingredients used in pharmaceutical and functional products.

Exosomal lncRNA ATB Derived from Ovarian Cancer Cells Promotes Angiogenesis via Regulating miR-204-3p/TGFβR2 Axis.

BackgroundOvarian cancer is a life-threatening disease with a high mortality rate in women. Our previous work presented that long non-coding RNA (lncRNA) activated by transforming growth factor beta (TGF-β) (lncRNA ATB) played a role of oncogene in ovarian cancer. However, whether exosomal lncRNA ATB from ovarian cancer cells could regulate the tumorigenesis of ovarian cancer remains unclear.MethodsRT-qPCR assay was performed to evaluate the level of lncRNA ATB in cancer cells (SKOV3 and A2780). In addition, ovarian cancer cells-secreted exosomes were collected with ultracentrifugation. CCK8 assay was performed to detect the viability of ovarian cells and HUVECs. Meanwhile, Western blot was performed to detect the expression of mechanism related protein and tube formation assay was used to observe the angiogenesis of HUVECs. Finally, xenograft mice model was used to verify the role of ovarian cancer cell-derived exosomes in vivo.ResultsOvarian cancer cells-derived exosomes promoted the viability, angiogenesis and migration of HUVECs; however, knockdown of lncRNA ATB in HUVECs reversed these phenomena. In addition, exosomal lncRNA ATB promoted the tumorigenesis of ovarian cancer via regulating miR-204-3p/TGFβR2 axis. Furthermore, ovarian cancer cells-secreted exosomal lncRNA ATB increased tumor growth in vivo.ConclusionExosomal lncRNA ATB derived from ovarian cancer cells could improve tumor microenvironment via regulating miR-204-3p/TGFβR2 axis. Thus, this study might provide new knowledge for the treatment of ovarian cancer.

Tumor perivascular cell-derived extracellular vesicles promote angiogenesis via the Gas6/Axl pathway.

Aberrant angiogenesis is a hallmark of cancer and is critically associated with tumor progression. Perivascular cells are essential components of blood vessels, and the role of tumor perivascular cell-derived extracellular vesicles (TPC-EVs) in angiogenesis remains elusive. In the present study, using genetic mouse models and pharmacological inhibitors, we found that ablation of perivascular cells inhibited angiogenesis in allografted colorectal cancer tumors. Further studies demonstrated that TPC-EVs promoted the proliferation, migration, invasion, viability, and tube formation of HUVECs. They also facilitated vessel spouting in rat aortic rings and induced neovascularization in chick chorioallantoic membranes (CAMs). Silencing of Gas6 or blockade of the Axl pathway suppressed TPC-EV-induced angiogenesis in vitro and ex vivo. Moreover, inhibition of the Gas6/Axl signaling pathway impaired TPC-EV-mediated angiogenesis in vivo. Our findings present a deeper insight into the biological functions of TPCs and TPC-EVs in tumor angiogenesis and demonstrate that TPC-EV-derived Gas6 could be an attractive and innovative regulator of tumor angiogenesis.

Astaxanthin suppresses End MT by LOX-1 pathway in ox-LDL-induced HUVECs

Introduction Astaxanthin (ASX) is carotenoid with the highest antioxidant activity in various cell types and reverse atherosclerosis. However, the roles and detailed mechanisms of ASX in atherosclerosis associated endothelial injury remains unclear. Methods In vitro atherosclerosis model was established in HUVECs by incubation with oxidized low-density lipoprotein (ox-LDL). Cell viability and oxidative stress were measured. The mRNA and protein expressions of lectin-like ox-LDL receptor (LOX-1) and other related genes were determined. Results ox-LDL reduced cell viability of HUVECs, and induced oxidative stress, as evidenced by elevated cellular malondialdehyde (MDA) and decreased superoxide dismutase (SOD). Pretreatment with ASX (50, 100, 200, and 400 μM) markedly reversed the reduction in cell viability and an increase in migration of HUVECs induced by ox-LDL (50 μg/mL). ASX attenuated the increase in the endothelial-to-mesenchymal transition (EndMT) process, as evidenced by increased CD31 and decreased α-SMA and vimentin proteins by ASX treatment in HUVECs. Furthermore, ASX attenuated the increase in MDA and decrease in SOD induced by ox-LDL, increased supernatant NO production, attenuated the increase in iNOS and decrease in eNOS in HUVECs with ox-LDL. ASX enhanced mRNA and protein expressions of the lectin-like ox-LDL receptor (LOX-1), which was dependent on ASX’s antioxidant activity. The inhibitory effect of ASX on EndMT could be abolished by overexpression of LOX-1 in HUVECs induced by ox-LDL. Conclusions Our data speculate that ASX prevents ox-LDL-induced endothelial cell injury and EndMT by inducing antioxidant property (SOD and NO) and decreasing LOX-1 expression.

Berberine Protects against TNF-α-Induced Injury of Human Umbilical Vein Endothelial Cells via the AMPK/NF-κB/YY1 Signaling Pathway.

Endothelial injury, characterized by an inflammatory response and increased permeability, is an initial stage of atherosclerosis (AS). Adenosine 5′-monophosphate (AMP), activated protein kinase (AMPK), and Nuclear Factor kappa B (NF-κB)/Yin Yang 1(YY1) signaling pathways play important roles in the process of endothelial injury. Berberine (BBR), a bioactive alkaloid isolated from several herbal substances, possesses multiple pharmacological effects, including anti-inflammatory, antimicrobial, antidiabetic, anticancer, and antioxidant activities. Previous studies showed a protective effect of berberine against endothelial injury. However, the underlying mechanism remains unclear. We explored the potential effect of BBR on TNF- (tumor necrosis factor-) α-induced injury of human umbilical endothelial cells (HUVECs) and studied its possible molecular mechanism. In the present study, HUVECs were divided into three groups. HUVEC viability was measured with Cell Counting Kit-8 assay. Extracellular lactic dehydrogenase (LDH) concentration was measured with LDH leakage assay. Endothelial microparticle (EMP) numbers were evaluated by flow cytometry analysis assay. The expression of proinflammatory cytokines was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). The mRNA expression of NF-κB and YY1 was detected by Real-Time PCR (RT-PCR). The protein expression of NF-κB, YY1, and AMPK was detected by immunofluorescence microscopy assay or western blot analysis. The results showed that LDH concentration, EMPs numbers, and the expression of proinflammatory cytokines (IL-6, IL-8, and IL-1β) increased in TNF-α-induced injured HUVECs, but ameliorated by BBR pretreatment. BBR pretreatment upregulated the expression of phosphorylated AMPK and downregulated the expressions of NF-κB and YY1 in injured HUVECs induced by TNF-α, which were offset by the AMPK inhibitor Compound C (CC). The results indicated that BBR protected against TNF-α-induced endothelial injury via the AMPK/NF-κB/YY1 signaling pathway.

Abstract 10791: Regeneration-Associated Cells Derived Exosomes Beneficial Than Mesenchymal Stromal Cells Derived Exosome in the Context of Myocardial Ischemia-Injury

Introduction: Vasculogenic conditioning of pro-inflammatory cells such as macrophages type 1, T cells, and primitive EPCs shifted their phenotype to pro-regenerative cells such as vasculogenic EPCs, M2 macrophages, and reg T cells, also known as regeneration-associated cells (RACs). Here, we investigated therapeutic efficacy of RAC-derived extracellular vesicles (RACev) in comparison with MSC-derived EVs (MSCev) in the context of myocardial ischemia-reperfusion injury (IRI). Methods: RACev and MSCev were isolated via ultracentrifugation. EVs quantity and quality were characterized by nanotracking analysis and CD9, CD63, Alix expression. EVs miR was sequenced and regenerative responsible miRs were validated using TargetScan and miRBase. Allogeneic immune response to the RACev and MSCev were evaluated. The function of RACev and MSCev were evaluated using HUVECs proliferation/cell-cycle assays in vitro, and repetitive (30min, d1, and d3 after IRI) systemic infusion with either RACev or MSCev in a myocardial IRI model. Results: Nanotracking analysis showed significant differences in quantity RACev vs. MSC (P= 0.03). In vitro , RACev markedly enhanced cell viability, proliferation, and migration of HUVECs in a dose-dependent manner compared to MSCev. Systemic injection of RACs (5x10 5 ) derived EVs improved cardiac functions such as ejection fraction (P= 0.005) than MSCev treated group. In histology, RACev transplanted group showed less interstitial fibrosis and enhanced capillary densities (P =0.05) compared to MSCev. These beneficial effects are coupled with significant expression of angiogenesis, anti-inflammation, cardiomyogenesis, and anti-fibrosis-related miRs in RACev but not in MSCev. In vivo bioluminescence and labeled EVs histology tracking analyses depicted preferential accumulation of RACev into the IRI myocardium (P=0.01) than MSCev. Immune phenotyping analysis confirmed similar immunomodulatory effects of MSCev and RACev. Conclusion: Repetitive systemic transplantation of RACev is superior to MSCev in terms of cardiac function enhancements via crucial angiogenesis, anti-fibrosis, anti-inflammation, cardiomyogenesis, and anti-apoptosis-related miRs delivery to the ischemic tissue.

Exosomal microRNA-125a-3p from human adipose-derived mesenchymal stem cells promotes angiogenesis of wound healing through inhibiting PTEN.

Angiogenesis plays a key in the process of tissue repair and wound healing. Human adipose-derived mesenchymal stem cells (HADSCs) have been found to act a promotion role during angiogenesis. Moreover, miR-125a-3p in HADSCs could promote the angiogenesis of HUVECs, but their specific mechanism in wound healing needs further study. Western blotting and qRT-PCR were used for detecting the protein and mRNA level, respectively. Exosomes were isolated successfully, and transmission electron microscope was used to identify exosomes. Angiogenesis, cell migration, and proliferation were detected with tube formation, wound healing, and MTT assays. The interactions of miR-125a-3p and PTEN were validated using dual-luciferase reporter assay. Animal model was used to evaluate the effect of miR-125a-3p on wound healing. HADSCs-exosome remarkably promoted the viability, migration, and angiogenesis of HUVECs. Knockdown of miR-125a-3p in HADSCs could inhibit the effect of HADSCs-exosome, while overexpression of miR-125a-3p could further promote the effect of HADSCs-exosome on HUVECs. MiR-125a-3p from HADSCs-exosome inhibited the expression of PTEN in HUVECs. Knockdown of PTEN promoted the viability, migration, and angiogenesis of HUVECs and reversed the effect of miR-125a-3p knockdown on HUVECs. Finally, miR-125a-3p from HADSCs-exosome could promote wound healing and angiogenesis in mice by inhibiting PTEN in mice wound granulation tissues. MiR-125a-3p from the HADSCs-exosome promoted the wound healing and angiogenesis, and these effects were achieved through regulating PTEN. This study may provide a new thought for the treatment and prevention of tissue repair.

Assessment of structural, biological and drug release properties of electro-sprayed poly lactic acid-dexamethasone coating for biomedical applications.

The efficacy of an implant is highly depends on its coating characteristics mainly determined by polymer properties and coating technique. Electro-spraying is an inexpensive and versatile coating technique with various advantages for biomedical application. In this study, the efficacy of electro-sprayed (ES) poly lactic acid (PLA)-dexamethasone (DEX) coatings for medical implants was evaluated and compared with spin-coated samples as control. Structural properties of coatings were investigated using X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Confocal and scanning electron microscopy (SEM), contact angle measurement and nanoindentation tests were used to study surface properties. Coating degradation rate and drug release profile were studied for 40days. Cell viability experiments were also performed on human endothelial (HUVEC) and smooth muscle cells (HUASMC) using MTT assay and SEM. XRD and DSC analysis showed electro-spraying significantly reduce PLA and DEX crystallinity. Surface studies showed ES coatings has significantly higher hydrophobicity and roughness with microbead-nanofiber morphology vs. micro-nanoporous structure of spin-coated samples. Initial burst release of DEX was 22% and 10% after 6h and total release was 71% and 46% after 40days for ES and spin-coated samples, respectively. HUVEC viability of ES samples was higher than spin-coated ones after 1 and 4days. However, dexamethasone release profile reduced HUASMC proliferation in ES PLA-DEX samples in comparison to spin-coated after 1 and 3days. In conclusion, in vitro results showed potential of ES PLA-DEX as a biocompatible and efficient anti-inflammatory coating with suitable drug release profile for future applications such as coronary drug eluting stents.

Retraction

Retraction: "Propranolol suppresses HUVEC viability, migration, VEGF expression, and promotes apoptosis by downregulation of miR-4295", by Feng Zhao, Xiaoliang Yang, Guangqi Xu, Jianhai Bi, Renrong Lv, and Ran Huo, J Cell Biochem. 2019; 6614-6623: The above article, published online on 28 October 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27957) has been retracted by agreement between the authors, the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed after the authors stated that a part of the experimental data in the article could not be reproduced. Thus, the conclusions are considered to be invalid.

Anti-growth and pro-apoptotic effects of dasatinib on human oral cancer cells through multi-targeted mechanisms.

Dasatinib is an inhibitor of Src that has anti‐tumour effects on many haematological and solid cancers. However, the anti‐tumour effects of dasatinib on human oral cancers remain unclear. In this study, we investigated the effects of dasatinib on different types of human oral cancer cells: the non‐tumorigenic YD‐8 and YD‐38 and the tumorigenic YD‐10B and HSC‐3 cells. Strikingly, dasatinib at 10 µM strongly suppressed the growth and induced apoptosis of YD‐38 cells and inhibited the phosphorylation of Src, EGFR, STAT‐3, STAT‐5, PKB and ERK‐1/2. In contrast, knockdown of Src blocked the phosphorylation of EGFR, STAT‐5, PKB and ERK‐1/2, but not STAT‐3, in YD‐38 cells. Dasatinib induced activation of the intrinsic caspase pathway, which was inhibited by z‐VAD‐fmk, a pan‐caspase inhibitor. Dasatinib also decreased Mcl‐1 expression and S6 phosphorylation while increased GRP78 expression and eIF‐2α phosphorylation in YD‐38 cells. In addition, to its direct effects on YD‐38 cells, dasatinib also exhibited anti‐angiogenic properties. Dasatinib‐treated YD‐38 or HUVEC showed reduced HIF‐1α expression and stability. Dasatinib alone or conditioned media from dasatinib‐treated YD‐38 cells inhibited HUVEC tube formation on Matrigel without affecting HUVEC viability. Importantly, dasatinib's anti‐growth, anti‐angiogenic and pro‐apoptotic effects were additionally seen in tumorigenic HSC‐3 cells. Together, these results demonstrate that dasatinib has strong anti‐growth, anti‐angiogenic and pro‐apoptotic effects on human oral cancer cells, which are mediated through the regulation of multiple targets, including Src, EGFR, STAT‐3, STAT‐5, PKB, ERK‐1/2, S6, eIF‐2α, GRP78, caspase‐9/3, Mcl‐1 and HIF‐1α.

Circ_0068087 Silencing Ameliorates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Vascular Endothelial Cells Depending on miR-186-5p-Mediated Regulation of Roundabout Guidance Receptor 1.

Background: Circular RNAs (circRNAs) are endogenous non-coding RNAs involved in the progression of atherosclerosis (AS). We investigated the role of circ_0068087 in AS progression and its associated mechanism.Methods: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) were performed to analyze the viability, apoptosis, and inflammatory response of HUVECs, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the Western blot assay were performed to measure the expression of RNA and protein. Cell oxidative stress was analyzed using commercial kits. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the interaction between microRNA-186-5p (miR-186-5p) and circ_0068087 or roundabout guidance receptor 1 (ROBO1).Results: Oxidized low-density lipoprotein (ox-LDL) exposure upregulated the circ_0068087 level in HUVECs. ox-LDL-induced dysfunction in HUVECs was largely attenuated by the silence of circ_0068087. Circ_0068087 negatively regulated the miR-186-5p level by interacting with it in HUVECs. Circ_0068087 knockdown restrained ox-LDL-induced injury in HUVECs partly by upregulating miR-186-5p. ROBO1 was a downstream target of miR-186-5p in HUVECs. Circ_0068087 positively regulated ROBO1 expression by sponging miR-186-5p in HUVECs. MiR-186-5p overexpression exerted a protective role in ox-LDL-induced HUVECs partly by downregulating ROBO1.Conclusion: Circ_0068087 interference alleviated ox-LDL-induced dysfunction in HUVECs partly by reducing ROBO1 expression via upregulating miR-186-5p.

MiR-34b-3p Impaired HUVECs Viability and Migration via Targeting PDK1 in an In Vitro Model of Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) leads to poor pregnancy outcomes. The methods for GDM early diagnosis and treatment are still unknown. This study aimed to investigate the expression and diagnostic potential of miR-34b-3p in GDM patients and further analyzed the effects of miR-34b-3p on HUVECs viability and migration. The expression of miR-34b-3p was detected in HUVECs of GDM and normal pregnant women by qRT-PCR. Then the HUVECs were isolated from normal pregnant women. High glucose (HG) was used to treat the HUVECs to mimic the GDM in vitro. The cell viability and migration were determined by MTT, wound healing assay, and transwell assay. The interaction between miR-34b-3p and PDK1 was evaluated by luciferase activity assay. Our results showed that miR-34b-3p was up-regulated in HUVECs of GDM patients. Then the HUVECs were isolated from normal pregnant women and they were treated with HG to mimic the GDM in vitro. Interestingly, knockdown of miR-34b-3p restored the impairment of HG treatment-induced effects in HUVECs. More importantly, PDK1 was proved to be a potential target of miR-34b-3p. Finally, the rescue experiments confirmed that miR-34b-3p impaired cell viability and migration ability in HUVECs by targeting PDK1. These findings concluded that miR-34b-3p impaired HUVECs viability and migration in GDM by targeting PDK1, which might provide a novel perspective for the pathogenesis and underlying therapeutic target for GDM.

Irigenin alleviates angiotensin II-induced oxidative stress and apoptosis in HUVEC cells by activating Nrf2 pathway.

Endothelial dysfunction is closely related to various cardiovascular diseases. Oxidative stress and apoptosis are involved in the progress of endothelial dysfunction. Irigenin (IR) has antioxidative properties. We investigated IR as a novel therapy for angiotensin II (Ang II)-induced endothelial dysfunction and explored the potential mechanisms of IR. After human umbilical vein endothelial cell lines (HUVECs) were treated with Ang II (100, 200, 300 and 400 nmol/L) alone, IR (2.5, 5, 10, 20 and 40 μmol/L) alone or Ang II plus IR for 24 h, HUVECs viability, lactate dehydrogenase (LDH), apoptosis, oxidative stress, apoptosis-related protein and nuclear factor E2-related factor 2 (Nrf2) levels were detected by Cell Counting Kit (CCK)-8 assay, enzyme-linked immunosorbent assay, flow cytometry and western blot. Transfection rate of Nrf2 was detected by western blot. In the next rescue experiment, we used silent Nrf2 (siNrf2) to verify the previous experimental results. Different concentrations' Ang II repressed HUVECs viability and increased LDH release, and different concentrations' IR did not affect HUVECs viability or LDH release. Furthermore, IR elevated cell viability and Nrf2 level, inhibited LDH release, apoptosis, oxidative stress and apoptosis-related protein levels in Ang II-induced HUVECs. More important, siNrf2 suppressed the expression of Nrf2, and siNrf2 abrogated the protective effect of IR on Ang II-induced Nrf2 expression, cell viability, LDH activity, oxidative stress generation and apoptosis-related protein in HUVECs. IR protected HUVECs from Ang II-induced oxidative stress and apoptosis injury by activating Nrf2 pathway.

Angiogenic Effects of Secreted Factors from Periodontal Ligament Stem Cells.

Periodontal disease is a chronic inflammation of tooth-supporting tissues, and the destruction of these tissues results in tooth loss. Regeneration of periodontal tissues is the ultimate goal of periodontal treatment. We previously reported that transplantation of conditioned medium (CM) of periodontal ligament stem cells (PDLSCs) demonstrated the enhancement of periodontal tissue regeneration, compared to CM from fibroblasts (Fibroblast-CM). We hypothesized that the angiogenic effects of PDLSC-CM might participate in the enhanced wound healing of periodontal tissues. The aim of this study was to investigate the effect of PDLSC-CM on the functions of endothelial cells. PDLSCs were cultured from periodontal ligament tissues obtained from healthy volunteers. Human gingival epithelial cells, dermal fibroblasts, osteoblasts, and umbilical vein endothelial cells (HUVECs) were purchased from commercial sources. The functions of endothelial cells were examined using immunostaining of Ki67, observation of nuclear fragmentation and condensation (apoptosis), and network formation on Matrigel. Vascular endothelial cell growth factor (VEGF) level was measured using an ELISA kit. HUVECs demonstrated higher cell viability in PDLSC-CM when compared with those in Fibroblast-CM. HUVECs demonstrated a higher number of Ki67-positive cells and lower apoptosis cells in PDLSC-CM, compared to Fibroblast-CM. Additionally, HUVECs formed more capillary-like structures in PDLSC-CM than Fibroblast-CM. PDLSC-CM contained higher levels of angiogenic growth factor, VEGF, than Fibroblast-CM. Our results showed that PDLSC-CM increased cell viability, proliferation, and capillary formation of HUVECs compared to Fibroblast-CM, suggesting the angiogenic effects of PDLSC-CM, and the effect is a potential regenerative mechanism of periodontal tissues by PDLSC-CM.

Astragaloside IV alleviates atherosclerosis through targeting circ_0000231/miR-135a-5p/CLIC4 axis in AS cell model in vitro.

Non-coding RNAs (ncRNAs) have shown to act as crucial mediators in atherosclerosis (AS) development. The purpose of our study was to explore the role of Astragaloside IV (ASV) and circular RNA_0000231 (circ_0000231) in AS using AS cell model. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to analyze cell viability and apoptosis. Migration ability was assessed by transwell migration assay and wound healing assay. The inflammatory response was evaluated via enzyme-linked immunosorbent assay (ELISA). Oxidative status was assessed via matching commercial kits. Western blot assay was conducted to detect the expression of monocyte chemoattractant protein 1 (MCP1), intercellular adhesion molecule 1 (ICAM1), and chloride intracellular channel 4 (CLIC4). The levels of circ_0000231, its linear form Rho GTPase activating protein 12 (ARHGAP12), microRNA-135a-5p (miR-135a-5p), and CLIC4 messenger RNA (mRNA) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Circ_0000231-miRNA interactions were established using Starbase and Circbank softwares, while the targets of miR-135a-5p were explored by Starbase software. Dual-luciferase reporter assay and RNA-pull down assay were used to verify these target interactions. ASV suppressed the apoptosis, inflammation, and oxidative stress while recovered the viability and migration ability of HUVECs which were mediated by oxidized low-density lipoprotein (ox-LDL). Circ_0000231 overexpression antagonized the protective role of ASV in ox-LDL-induced HUVECs. MiR-135a-5p was verified as a direct target of circ_0000231, and circ_0000231 contributed to ox-LDL-induced cell injury of HUVECs through down-regulating miR-135a-5p. MiR-135a-5p directly interacted with the 3' untranslated region (3'-UTR) of CLIC4 mRNA in HUVECs, and miR-135a-5p protected HUVECs against ox-LDL-induced injury through down-regulating CLIC4. ASV protected HUVECs against ox-LDL-induced injury through targeting circ_0000231/miR-135a-5p/CLIC4 axis. Targeting circ_0000231/miR-135a-5p/CLIC4 axis might provide a novel insight to develop effective strategy for AS treatment.

AGEs/RAGE blockade downregulates Endothenin-1 (ET-1), mitigating Human Umbilical Vein Endothelial Cells (HUVEC) injury in deep vein thrombosis (DVT)

ABSTRACT This study is aimed at identifying the roles of AGE/RAGE and ET-1 in deep vein thrombosis (DVT). Advanced glycation end products (AGEs) in glycated human serum albumin (M-HSA) were detected by ELISA. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis, followed by ELISA for the detection of inflammatory cytokine level and oxidative stress level in HUVECs. Immunofluorescence was performed to detect ET-1 and eNOS expression. The expression of specific proteins was assayed by western blot. As a result, decreased HUVEC viability was observed after stimulation with M-HSA, whereas RAGE inhibitor improved it. Cell apoptosis showed the opposite trend. Additionally, M-HSA-induced inflammatory cytokine release and oxidative stress of HUVECs were both alleviated by RAGE inhibitor. RAGE inhibitor also increased the levels of NO and eNOS while decreasing the level of ET-1 in M-HSA-stimulated HUVECs. Furthermore, decreased protein expression of Bax, cleaved-caspase3, RAGE, p65, ET-1 and iNOS was observed after treatment with RAGE inhibitor, in addition to increased protein expression of Bcl-2 and eNOS. In conclusion, blocking AGE/RAGE pathway downregulates ET-1, thereby mitigating HUVEC damage in DVT.

Kirenol Inhibits B[a]P-Induced Oxidative Stress and Apoptosis in Endothelial Cells via Modulation of the Nrf2 Signaling Pathway.

Atherosclerosis is a persistent inflammatory disorder specified by the dysfunction of the arteries, the world's leading cause of cardiovascular diseases. We sought to determine the effectiveness of KRL in B[a]P-induced oxidative stress and programmed cell death in endothelial cells. Western blotting, real-time PCR, DCFH2-DA, and TUNEL staining were performed to detect pPI3K, pAKT, Nrf2, HO-1, NQO-1, Bcl2, Bax, and caspase-3 on the HUVECs. Through the pretreatment of KRL, a drastic enhancement was observed in the cell viability of HUVECs, whereas DNA damage and generation of reactive oxygen species induced by B[a]P was suppressed. KRL's potential use as an antioxidant was observed to have a direct correlation with an antioxidant gene's augmented expression and the nuclear translocation activation of Nrf2, even during the event when B[a]P was found to be absent. In addition, this study proved that the signaling cascades of PI3K/AKT mediated Nrf2 translocation. Activation of suppressed nuclear Nrf2 and reduced antioxidant genes across cells interacting with an LY294002 confirmed this phenomenon. In addition, knockdown of Nrf2 by Nrf2-siRNA transfection abolished the protective effects of KRL on HUVECs cells against oxidative damage. Finally, the expression of apoptotic proteins also supported the hypothesis that KRL may inhibit endothelial dysfunction. This study showed that KRL potentially prevents B[a]P-induced redox imbalance in the vascular endothelium by inducing the Nrf2 signaling via the PI3K/AKT pathway.

Abstract 15183: Thrombopoietin Has a Protective Effect Against Apoptosis of Endothelial Cells Through Activating Pi3k/akt Pathway

Introduction: Thrombopoietin (TPO) is a hematopoietic growth factor for platelet lineage. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models and treatment with TPO reduced brain damage and improved sensorimotor functions (Li L et al, Aging-US, 2020). However, the underlying mechanism of TPO in this model, and its role in neural cells and endothelial cells were still unclear. Methods: C17.2 cells were divided into control (0.5% FCS), Normal (10% FCS), TPO and TPO + LY294002 groups. Human umbilical vein endothelial (HUVEC) cells were divided in to normal, CoCl 2 , TPO and TPO + CoCl2 groups. The expressed of TPO and c-mpl was tested by RT-PCR. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in PI3K/AKT pathway was detected by using Western blot. Results: Both TPO and c-mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. CoCl 2 significantly inhibited the growth of HUVECs. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl 2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl 2 (P<0.001). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl 2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusions: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP.

Thrombopoietin Protects Neural Cells and Endothelial Cells from Apoptosis Via PI3K/AKT Pathway

Background: Thrombopoietin (TPO) is a hematopoietic growth factor that regulates the production of platelets and stimulates production and differentiation. The expression of TPO and TPO receptor (c-mpl) in the central nervous system (CNS) has been identified. However, the role of TPO in neural cells and endothelial cells were not clear. Methods: C17.2 and human umbilical vein endothelial (HUVEC) cells were treated with CoCl2, TPO, or TPO + CoCl2. TPO was added into the culture medium 48 h before CoCl2 treatment. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in the PI3K/AKT pathway was detected by using Western blot. Results: TPO has a dose-dependent effect on the growth of C17.2 cells. LY-294002 pretreatment suppressed the TPO-induced AKT activation and abolished the prosurvival effect of TPO. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl2 (P<0.01). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusion: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

Optimization of cell seeding on electrospun PCL-silk fibroin scaffolds

The availability of cell growth-supporting materials for regenerative medicine is limited. Here, we aimed to improve the attachment and growth of endothelial cells (HUVECs) and fibroblasts on electrospun poly(ε-caprolactone) (PCL) mats with different fiber diameters and on newly-established PCL and silk fibroin (PCL/SF) blended fibers obtained by using benign solvents (mixture of acetic acid and formic acid) for electrospinning.PCL mats were produced in two different average fiber diameters, namely micro and nano range. The electrospinning method was optimized to produce PCL/SF blended fibers. Morphology (F-actin), viability (calcein) and metabolic activity (WST-8 assay) of HUVECs and fibroblasts cultured in 2D on the mats of different fiber size and composition were compared. Subsequently, cylindrical PCL/SF scaffolds were produced and colonized with cells using 3D radial magnetic cell seeding.Both PCL and PCL/SF nanofibers provided better support for initial cell adhesion (day 1) compared with PCL microfibers. At day 7, the highest metabolic activity in HUVECs and fibroblasts was observed on PCL/SF mats. Microscopic studies confirmed that on day 7, the best support for cell growth was observed on PCL/SF. Tubular PCL/SF scaffolds were successfully colonized using magnetic nanoparticles and 3D radial magnetic cell seeding.Taken together, the fibers obtained by blending SF with PCL combine the mechanical benefits of PCL with improved biological functionality of SF. Electrospun PCL/SF nanofibers represent a promising material to produce both flat and 3D structures with potential for enhanced cell attachment and tissue regeneration.