Gene Expression In Human Umbilical Vein Endothelial Cells Research Articles

IPSC-derived megakaryocytes and platelets accelerate wound healing and angiogenesis

BackgroundPlatelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications.MethodsThe growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined.ResultMultiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model.ConclusionOur study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

Exploratory Investigation of Zinc-Modified Borosilicate Bioactive Glass: A New Methodology for Its Biocompatibility, Immunoregulation, and Pro-Angiogenic Property Evaluation.

The assessment of biodegradable materials, such as bioactive glass, under the existing ISO 10993 standard test methods poses a significant challenge due to potential cell viability impairment caused by the accumulation of degraded products in a static environment. Therefore, innovative methodologies are urgently needed to tailor the unique biodegradation characteristics of these materials, providing more precise and scientific insights into biosafety and efficacy verification. Motivation by its bidirectional regulation of angiogenesis and immunity, zinc (Zn) was incorporated into sol-gel-derived borosilicate bioactive glasses (SBSGs) to fabricate Zn-incorporated borosilicate bioactive glasses (SBSG-Zn) to complement the tissue repair capabilities of bioactive glasses. Both SBSG and SBSG-Zn glasses consist of nanosized particles, slit mesoporous pores, high specific surface areas, and bioreactivity. In vitro comparative analysis, conducted according to ISO 10993 standards, demonstrates that only at suitable dilution rates─such as the 8-fold dilution employed in this study─do extracts of SBSG and SBSG-Zn glasses exhibit low cytotoxicity when cultured with human umbilical vein endothelial cells (HUVECs). Notably, SBSG-Zn glasses show optimal promotion of angiogenic gene expression in HUVECs. Furthermore, within an appropriate concentration range of released ions, SBSG-Zn glass extracts not only promote cell survival but also modulate the expression of anti-inflammatory genes while simultaneously inhibiting pro-inflammatory genes concurrently. After being implanted in rat subcutaneous defect models, both SBSG and SBSG-Zn glasses demonstrated the local immunoregulation and angiogenic effects. SBSG-Zn stands out by demonstrating superior modulation of M1/M2 polarization in macrophages as validated by altered secretion of key factors in macrophages and expression of relevant growth factors in HUVECs. These findings underscore the potential for convenient manipulation of localized angiogenic and immunoregulation through the incorporation of zinc into bioactive glass, emphasizing the importance of ensuring the appropriate ion doses are applied for achieving optimal therapeutic efficiency.

DOT1-like histone lysine methyltransferase is critical for adult vessel maintenance and functions.

Disruptor of telomeric silencing 1-like (DOT1L) is the only known histone H3K79 methyltransferase essential for the development of the embryonic cardiovascular system, including the heart, blood vessels, and lymphatic vessels, through transcriptional regulation. Our previous study demonstrated that Dot1l deletion results in aberrant lymphatic development and function. However, its precise function in the postnatal cardiovascular system remains unknown. Using conditional and inducible Dot1l knockout (KO) mice, along with a reporter strain carrying the Geo gene at the Dot1l locus, DOT1L expression and its function in the vascular system during postnatal life were investigated. To assess vessel morphology and vascular permeability, we administered Latex or Evans blue dye to KO mice. In addition, in vitro tube formation and cell migration assays were performed using DOT1L-depleted human umbilical vein endothelial cells (HUVECs). Changes in the expression of vascular genes in HUVECs were measured by quantitative polymerase chain reaction. Our findings demonstrate that conditional Dot1l knockout in the Tg (Tie2-cre) strain results in abnormal blood vessel formation and lymphatic anomalies in the intestine. In a mouse model of Rosa26-creER-mediated inducible Dot1l knockout, we observed vascular phenotypes, including increased vascular permeability and brain hemorrhage, when DOT1L was deleted in adulthood. Additionally, DOT1L depletion in cultured HUVECs led to impaired cell migration and tube formation, likely due to altered gene transcription. These findings highlight the essential role of DOT1L in maintaining vascular integrity and function during embryonic development and postnatal life. Our study revealed that DOT1L is required for the maintenance of adult vascular function through the regulation of gene expression.

Enhancing angiogenesis in peri-implant soft tissue with bioactive silk fibroin microgroove coatings on zirconia surfaces.

Zirconia abutments and restorations have improved the aesthetic appeal of implant restoration, yet peri-implantitis poses a significant threat to long-term success. The soft tissue surrounding implants is a crucial biological barrier against inflammation and subsequent bone loss. Peri-implantitis, akin to periodontitis, progresses rapidly and causes extensive tissue damage. Variations in tissue structure significantly influence disease progression, particularly the lower vascular density in peri-implant connective tissue, compromising its ability to combat infection and provide essential nutrients. Blood vessels within this tissue are vital for healing, with angiogenesis playing a key role in immune defense and tissue repair. Enhancing peri-implant soft tissue angiogenesis holds promise for tissue integration and inflammation control. Microgroove surfaces have shown potential in guiding vessel growth, but using subtractive technologies to carve microgrooves on zirconia surfaces may compromise mechanical integrity. In this study, we utilized inkjet printing to prepare bioactive silk fibroin microgrooves (SFMG) coating with different sizes on zirconia surfaces. SFMG coating, particularly with 90 µm width and 10 µm depth, effectively directed human umbilical vein endothelial cells (HUVECs) along microgrooves, promoting their proliferation, migration, and tube formation. The expression of vascular endothelial growth factor A and fibroblast growth factor in HUVECs growing on SFMG coating was upregulated. Additionally, the SFMG coating activated the PI3K-AKT pathway and increased glycolytic enzyme gene expression in HUVECs. In conclusion, SFMG coating enhances HUVEC growth and angiogenesis potential by activating the PI3K-AKT pathway and glycolysis, showing promise for improving tissue integration and mitigating inflammation in zirconia abutments and restorations.

FUS regulates the alternative splicing of cell proliferation genes related to atherosclerosis.

FUS plays a significant role as an RNA-binding protein in several cellular processes, including RNA splicing, DNA repair, and transcriptional regulation. However, the RNA-binding capacity of FUS in atherosclerosis is unclear. We aimed to study the functions of FUS in inflammatory regulation through the role of the splicing factor. We knocked down FUS with siRNA to further study the overall transcriptional level and select alternative splicing (AS) of FUS regulation in human umbilical vein endothelial cells (HUVECs) by RNA sequencing. The results suggested that the knockdown of FUS significantly affected gene expression in HUVECs. In addition, the knockdown of FUS resulted in 200 differentially expressed genes (DEGs) that were highly related to apoptotic process, signal transduction, multicellular organism development, cell adhesion and regulation of transcription, and DNA-templated pathways. Importantly, FUS extensively regulated 2870 AS events with a significant difference. Functional analysis of its modulated AS genes revealed they were highly enriched in cell cycle and cell population proliferation pathways. The qRT-PCR and RNA-seq data showed consistent results. Our findings suggested new knowledge of the mechanisms of FUS associated with atherosclerosis.

Vascularized polypeptide hydrogel modulates macrophage polarization for wound healing

Wound repair involves a sophisticated process that includes angiogenesis, immunoregulation and collagen deposition. However, weak revascularization performance and the lack of biochemical cues to trigger immunomodulatory function currently limit biomaterial applications for skin regeneration and tissue engineering. Herein, we fabricate a new bioactive polypeptide hydrogel (QK-SF) constituted by silk fibroin (SF) and a vascular endothelial growth factor mimetic peptide KLTWQELYQLKYKGI (QK) for tissue regeneration by simultaneously promoting vascularization and macrophage polarization. Our results showed that this QK-SF hydrogel can be prepared via an easy manufacturing process, and exhibited good gel stability and low cytotoxicity to cultured human umbilical vein endothelial cells (HUVECs) via both live/dead and cell counting kit-8 assays. Importantly, this QK-SF hydrogel triggered macrophage polarization from M1 into M2, as exemplified by the enhanced expression of the M2 marker and decreased expression of the M1 marker in RAW264.7 cells. Furthermore, the QK-SF hydrogel showed high capacity for inducing endothelial growth, migration and angiogenesis, which were proved by increased expression of angiogenesis-related genes in HUVECs. Consistent with in vitro findings, in vivo data show that the QK-SF hydrogel promoted M2 polarization, keratinocyte differentiation, and collagen deposition in the mouse skin wound model in immunohistochemistry assay. Furthermore, this QK-SF hydrogel can reduce inflammation, induce angiogenesis and promote wound healing as exemplified by the increased vessel formation and decreased wound area in the mouse skin wound model. Altogether, these results indicate that the bioactive QK-SF hydrogel plays dual functional roles in promoting angiogenesis and immunoregulation for tissue regeneration. Statement of significanceThe QK-SF hydrogel plays dual functional roles in promoting angiogenesis and immunoregulation for tissue repair and wound healing. The QK-SF hydrogel can be prepared via an easy manufacturing process, and exhibited good gel stability and low cytotoxicity to cultured HUVECs. The QK-SF hydrogel triggered macrophage polarization from M1 into M2. The QK-SF hydrogel showed high capacity for inducing endothelial growth, migration and angiogenesis. The QK-SF hydrogel promoted M2 polarization, keratinocyte differentiation, and collagen deposition.

Exosomes from human adipose-derived stem cells promoted the expression of angiogenic factors in endothelial cells

Introduction: In regenerative medicine, human adipose tissue–derived mesenchymal stem cells (hadMSCs) have exhibited tremendous promise as an efficient approach to chronic cardiovascular diseases. Noble effects of mesenchymal stem cells (MSCs) have been acknowledged to contribute to not only cellular engraftment and response to the site of injury but also paracrine action via the release of extracellular vesicles, notably exosomes. This study aimed to investigate the effects of exosomes from hadMSCs (hadMSC-Exo) on the angiogenesis of endothelial cells. Methods: Firstly, hadMSCs were characterized and cultured at a density of 500,000 cells in exosome-free medium for 48 hours. The conditioned medium was ultracentrifuged for exosome isolation. Obtained exosomes were characterized with the expression of CD9, CD63, and CD81 markers by flow cytometry and imaged with a TEM microscope. Exosomes were added to a culture medium of human umbilical vein endothelial cells (HUVECs) to understand the effects of exosomes on HUVECs. The expression of some angiogenic genes in HUVECs was identified by RT-qPCR. Results: The concentration of total protein in isolated exosomes by Bradford assay was 0.51 ± 0.04 mg/mL. Exosomes were successfully isolated and characterized by their morphology and immunophenotype. The results showed that exosomes significantly increased the mRNA expression of TGF (11.56 ± 2.03, P < 0.05), Flk-1 (4.04 ± 0.01, P < 0.05), VE-cadherin (7.25 ± 2.30, P < 0.05), and CD31 (3.02 ± 1.13, P < 0.05). Conclusion: This study suggested that exosomes promoted the angiogenesis of endothelial cells in vitro.

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function.

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin–Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor—alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties.

Effects of quercetin and metabolites on uric acid biosynthesis and consequences for gene expression in the endothelium

BackgroundUric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. MethodsInhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 μmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. ResultsIn human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3′-sulfate and the gut microbial metabolite 3′,4′-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. ConclusionsWe propose that the main mechanism by which quercetin, as quercetin-3′-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.

MoS2 Nanoclusters-based biomaterials for disease- impaired wound therapy

Disease-impaired wound healing is one of the most challenging issues clinically, which requires the migration, proliferation and differentiation of multiple types of skin cells, and overcomes the disease-associated problems. To achieve this goal, we set out to firstly synthesize MoS2 (MS) nanoclusters and then to incorporate them into sodium alginate (SA) hydrogel to prepare SA-MS hydrogel as a kind of MoS2 nanoclusters-based biomaterial. The prepared SA-MS hydrogel demonstrates porous microstructure and uniform distribution of Mo elements. The released Mo4+ ions from SA-MS hydrogel exhibit distinct effects on the promotion of the migration and proliferation of human dermal fibroblasts cells (HDFs), human umbilical vein endothelial cells (HUVECs) and human hair dermal papilla cells (HhDPCs) and significantly up-regulated the expression of specific genes in HUVECs and HhDPCs in vitro. SA-MS hydrogel has significant effect on promoting angiogenesis around the wound bed and regeneration of hair follicles of diabetes-impaired wound in C57BL/6 mice diabetic model in vivo by the released Mo4+ ions. Besides, SA-MS hydrogel successfully suppressed skin cancer and realized the skin cancer-impaired wound healing of melanoma-bearing nude mice by the excellent photothermal performance of SA-MS hydrogel in combination of the stimulatory effect of the released Mo4+ ions. Therefore, the prepared SA-MS hydrogel may be one of the promising candidates for disease-impaired wound healing and provides a broad prospect for the development of skin regeneration in the future.The prepared SA-MS hydrogel releases Mo4+ ions to promote the migration and proliferation of HDFs, HVUECs, and HhDPCs. Moreover, the released Mo4+ ions have a significantly promoted role for the diabetes-impaired wound healing and combine with the photothermal performance of the SA-MS hydrogel to affect the healing of skin cancer impaired wound. Eventually, achieve the role of wound healing by promoting angiogenesis and hair follicle regeneration at the wound bed. Therefore, SA-MS hydrogels have an excellent ability of promoting healing for disease-impaired wound.

Glioblastoma Cells Do Not Affect Axitinib-Dependent Senescence of HUVECs in a Transwell Coculture Model.

Axitinib is an orally available inhibitor of tyrosine kinases, with high specificity for vascular endothelial growth factor receptors (VEGFRs) 1, 2, and 3. It is approved for the treatment of advanced renal cell carcinoma and is in phase II clinical trials for recurrent glioblastoma (GBM). GBM is a brain tumor peculiar in its ability to induce neoangiogenesis. Since both GBM tumor cells and endothelial cells of tumor vasculature express VEGFRs, Axitinib exerts its inhibitory action on both tumor and endothelial cells. We and others previously demonstrated that Axitinib triggers cellular senescence. In particular, Axitinib-dependent senescence of HUVECs (human umbilical vein endothelial cells) is accompanied by intracellular reactive oxygen species(ROS) increase and early ataxia telangiectasia mutated(ATM) activation. Here we wondered if the presence of glioblastoma tumor cells could affect the HUVEC senescence upon Axitinib exposure. To address this issue, we cocultured HUVECs together with GBM tumor cells in transwell plates. HUVEC senescence did not result in being affected by GBM cells, neither in terms of β galactosidase activity nor of proliferation index or ATM phosphorylation. Conversely, Axitinib modulation of HUVEC gene expression was altered by cocultured GBM cells. These data demonstrate that the GBM secretome modifies HUVECs’ transcriptomic profile upon Axitinib exposure, but does not prevent drug-induced senescence.

Investigation of the Mechanism Underlying Calcium Dobesilate-Mediated Improvement of Endothelial Dysfunction and Inflammation Caused by High Glucose.

Background/Aims Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease. Calcium dobesilate (CaD) is widely used to treat diabetic retinopathy. Recent studies have demonstrated that CaD exerts protective effects against diabetic nephropathy. The aim of this study was to elucidate the molecular and cellular mechanisms underlying the protective effects of CaD. Methods Human umbilical vein endothelial cells (HUVECs) were cultured with different D-glucose concentrations to determine the effects of high glucose on HUVEC gene expression. HUVECs were also incubated with CaD (25 μM, 50 μM, and 100 μM) for 3 days to determine the effects of CaD on HUVEC viability. db/db mice were treated with CaD. 2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) blocked the nuclear factor-κB (NF-κB) pathway in HUVECs. A pentraxin 3 (PTX3) small interfering RNA (siRNA) intervention experiment was performed in the cells. An adenovirus-encapsulated PTX3 siRNA intervention experiment was performed in db/db mice. Western blot and real-time PCR analyses were used to detect PTX3, p-IKBa/IKBa (I-kappa-B-alpha), and p-eNOS/eNOS (endothelial nitric oxide synthase) expression in mice and HUVECs. Hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining were used to observe renal tissue damage in mice. PTX3 expression was observed by immunohistochemical staining. Results CaD downregulated the expression of PTX3 and p-IKBa/IKBa and upregulated the expression of p-eNOS/eNOS in vitro. When TPCA-1 was used, high glucose induced high PTX3 expression, and the expression of p-eNOS/eNOS increased. After PTX3 gene silencing, the expression of p-eNOS/eNOS also increased. In vivo, CaD reduced the expression of PTX3 and p-IKBa/IKBa in the kidneys of db/db mice and increased the expression of p-eNOS/eNOS. After PTX3 gene silencing, the urine protein and renal function of db/db mice were ameliorated, the glomerular extracellular matrix was decreased, and the expression of p-eNOS/eNOS was increased. Conclusions Our results suggested that CaD may inhibit the expression of PTX3 by altering the IKK/IKB/NF-κB pathway, thereby improving endothelial dysfunction in HUVECs. PTX3 may be a potential therapeutic target for DKD.

Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling.

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3.

Development of MicroRNA-146a-Enriched Stem Cell Secretome for Wound-Healing Applications.

Secretome-based therapies have the potential to become the next generation of viable therapeutic wound repair treatments. However, precise strategies aimed to refine and control the secretome composition are necessary to enhance its therapeutic efficacy and facilitate clinical translation. In this study, we aim to accomplish this by transfecting human adipose-derived stem cells (hASCs) with microRNA-146a, which is a potent regulator of angiogenesis and inflammation. The secretome composition obtained from the transfected hASCs (secretome146a) was characterized and compared to nontransfected hASCs secretome to evaluate changes in angiogenic and anti-inflammatory growth factor, cytokine, and miRNA content. In vitro proliferation, migration, and tubular morphogenesis assays using human umbilical vein endothelial cells (HUVECs) were completed to monitor the proangiogenic efficacy of the secretome146a. Finally, the anti-inflammatory efficacy of the secretome146a was assessed using HUVECs that were activated to an inflammatory state by IL-1β. The resulting HUVEC gene expression and protein activity of key inflammatory mediators were evaluated before and after secretome treatment. Overall, the secretome146a contained a greater array and concentration of therapeutic paracrine molecules, which translated into a superior angiogenic and anti-inflammatory efficacy. Therefore, this represents a promising strategy to produce therapeutic secretome for the promotion of wound repair processes.

Flavin Oxidase-Induced ROS Generation Modulates PKC Biphasic Effect of Resveratrol on Endothelial Cell Survival.

Background: Dietary intake of natural antioxidants is thought to impart protection against oxidative-associated cardiovascular diseases. Despite many in vivo studies and clinical trials, this issue has not been conclusively resolved. Resveratrol (RES) is one of the most extensively studied dietary polyphenolic antioxidants. Paradoxically, we have previously demonstrated that high RES concentrations exert a pro-oxidant effect eventually elevating ROS levels leading to cell death. Here, we further elucidate the molecular determinants underpinning RES-induced oxidative cell death. Methods: Using human umbilical vein endothelial cells (HUVECs), the effect of increasing concentrations of RES on DNA synthesis and apoptosis was studied. In addition, mRNA and protein levels of cell survival or apoptosis genes, as well as protein kinase C (PKC) activity were determined. Results: While high concentrations of RES reduce PKC activity, inhibit DNA synthesis and induce apoptosis, low RES concentrations elicit an opposite effect. This biphasic concentration-dependent effect (BCDE) of RES on PKC activity is mirrored at the molecular level. Indeed, high RES concentrations upregulate the proapoptotic Bax, while downregulating the antiapoptotic Bcl-2, at both mRNA and protein levels. Similarly, high RES concentrations downregulate the cell cycle progression genes, c-myc, ornithine decarboxylase (ODC) and cyclin D1 protein levels, while low RES concentrations display an increasing trend. The BCDE of RES on PKC activity is abrogated by the ROS scavenger Tempol, indicating that this enzyme acts downstream of the RES-elicited ROS signaling. The RES-induced BCDE on HUVEC cell cycle machinery was also blunted by the flavin inhibitor diphenyleneiodonium (DPI), implicating flavin oxidase-generated ROS as the mechanistic link in the cellular response to different RES concentrations. Finally, PKC inhibition abrogates the BCDE elicited by RES on both cell cycle progression and pro-apoptotic gene expression in HUVECs, mechanistically implicating PKC in the cellular response to different RES concentrations. Conclusions: Our results provide new molecular insight into the impact of RES on endothelial function/dysfunction, further confirming that obtaining an optimal benefit of RES is concentration-dependent. Importantly, the BCDE of RES could explain why other studies failed to establish the cardio-protective effects mediated by natural antioxidants, thus providing a guide for future investigation looking at cardio-protection by natural antioxidants.

Association of tribbles homologue 1 gene expression in human umbilical vein endothelial cells with duration of intrauterine exposure to hyperglycaemia.

Maternal gestational diabetes mellitus (GDM) is considered to be an important factor that epigenetically predisposes offspring to metabolic and cardiovascular diseases. However, the mechanisms of how intrauterine hyperglycaemia affects offspring have not been thoroughly studied. The mammalian tribbles homologue 1 (TRIB1) gene is associated with plasma lipid concentrations and coronary artery disease (CAD). Our aim was to study the effect of GDM and its treatment terms on the level of TRIB1 gene expression in human umbilical vein endothelial cells (HUVECs) of newborns from women with and without GDM. The study included 50 women with GDM and 25 women without GDM (control group). Women with GDM were divided into three groups according to their gestational age when the treatment of GDM started: 24-28 weeks (GDM1, N=16), 29-32 weeks (GDM2, N=25) and >34 weeks (GDM3, N=9). The levels of TRIB1 gene expression in GDM3, GDM2, GDM1 and control groups were 2.8±1.1, 4.2±2.4, 6.0±3.4 and 8.1±6.1, respectively (p=0.001). After comparison in pairs the difference was significant for the following pairs: GDM2-control (p=0.004), GDM3-control (p=0.002), GDM1-GDM3 (p=0.012). Notably, if treatment had been started before the 28th week of gestation, the difference in TRIB1 gene expression in HUVECs was not significant (p=0.320 for comparison between GDM1 and control groups). Our findings support the hypothesis that TRIB1 gene expression in HUVECs depends on the duration of intrauterine exposure to hyperglycaemia.

Abstract 1802: Apoptosis pathway-focused gene expression profiling of a novel VEGFR2 inhibitor

Abstract Sprouting of the new blood vessel (angiogenesis) is fundamental to tumor growth, invasion, and metastatic dissemination. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes cell survival via opposing apoptosis. On the other hand, antagonizing VEGF pathway suppresses endothelial cell overgrowth and promotes apoptotic signals leading to the regression of the existing vasculature and prevents neovascularization of the tumors. In this respect, a new chemical entity, code named as F16, that was developed in our institute can effectively stop VEGF-driven angiogenesis by selectively blocking VEGFR2 and the downstream signals. Our previous studies have revealed that human umbilical vein endothelial cells (HUVECs) undergo apoptosis in response to F16 treatment. However, in the present study, our main focus was on investigating the status of the signal mediators of the apoptotic pathway after treating the HUVECs with F16. This was expected to outline the intracellular sequence of events involved in triggering apoptosis following F16 treatment. For this purpose, we analyzed apoptosis pathway-focused gene expression in HUVECs using the human apoptosis RT2 profiler which contains 84 key genes involved in regulation of programmed cell death. Our experimental results clearly identified up-regulated pro-apoptotic genes in response to F16 treatment which include tumor necrosis factor -8 and -10A (TNF-8 and TNF-10A), tumor necrosis factor superfamily member 9 (TNFRSF9), FAS cell surface receptor (FAS), FAS ligand (FASLG), lymphotoxin alpha (LTA), caspase 5 (CASP5), and cytochrome c (CYCS). Moreover, a few anti-apoptotic genes were also up-regulated which include B-cell lymphoma 2 related protein A1 (BCL2A1), caspase 14 (CASP14), Bcl2 associated athanogene 3 (BAG3), and interleukin 10 (IL-10). However, anti-apoptotic genes such as Bcl-2, Bcl-2 like 10 (BCL2L10), baculoviral IAP repeat containing -3 and -6 (BIRC-3, and BIRC-6), CASP8 and FADD like apoptosis regulator (CFLAR), and insulin like growth factor 1 receptor (IGF1R) were found to be significantly downregulated. Interestingly, no changes were found in the expression of genes related to DNA damage and repair pathways such as TP53, ABL1, CIDEA, and CIDEB. This indicates that F16 may not provoke DNA damage-induced apoptosis instead, it stimulates the intrinsic or the extrinsic pathway. In conclusion, the diverse gene expressions impacting apoptosis related factors shows that F16 can induce apoptosis via potentiation of TNF- and FAS- signaling. (This research was supported by the generous funds provided by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Mohammad Algahtani, Khalid Alhazzani, Thiagarajan Venkatesan, Appu Rathinavelu. Apoptosis pathway-focused gene expression profiling of a novel VEGFR2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1802. doi:10.1158/1538-7445.AM2017-1802

Trans Fatty Acids Suppress TNF‐α‐Induced Inflammatory Gene Expression in Endothelial (HUVEC) and Hepatocellular Carcinoma (HepG2) Cells

Trans fatty acids (TFA) intake has been linked to cardiovascular diseases and liver diseases; yet the effect of TFA on inflammation remains controversial. Accordingly, the objective of this paper was to determine the in vitro effects of TFA on inflammatory gene expression. Human umbilical vein endothelial cells (HUVEC) and human hepatocellular carcinoma (HepG2) cells were treated for 24h with either trans-vaccenic acid (tVA), trans-palmitoleic acid (tPA) or elaidic acid (EA) at concentrations of 5-150µM, or with a mixture of tVA and tPA (150/50µM). All TFA were highly incorporated into cell membranes, as determined by gas chromatography, representing 15-20% of total fatty acids in HUVEC and 3-8% in HepG2 cells. Incorporation of EA, a common industrial TFA, increased the ratio of the stearoyl-CoA desaturase (SCD-1), a key enzyme involved in fatty acid metabolism. Ruminant TFA, including tVA, tPA and the mixture of tVA and tPA, significantly reduced the TNF-α-induced gene expression of TNF, VCAM-1 and SOD2 in HUVEC, as well as TNF and IL-8 in HepG2 cells. EA also decreased inflammatory gene expression in HUVEC, but not in HepG2 cells. The inhibition of peroxisome proliferator-activated receptor (PPAR)-γ did not influence the effects of TFA on gene expression. Overall, physiological and supraphysiological concentrations of TFA, especially tVA and tPA, prevented inflammatory gene expression in vitro. This effect is independent of PPAR-γ activation and may be due to an alteration of fatty acid metabolism in cell membranes caused by the high incorporation of TFA.

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Platelet activity measurements and subsequent interleukin-1β-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE.

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

Background:Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis.Materials and Methods:Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured.Results:ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels (P = 0.09) and vWF (P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue.Conclusions:The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.