Cell Proliferation Of Human Umbilical Vein Endothelial Cells Research Articles

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing invivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

The requirement of the mitochondrial protein NDUFS8 for angiogenesis

Mitochondria are important for the activation of endothelial cells and the process of angiogenesis. NDUFS8 (NADH:ubiquinone oxidoreductase core subunit S8) is a protein that plays a critical role in the function of mitochondrial Complex I. We aimed to investigate the potential involvement of NDUFS8 in angiogenesis. In human umbilical vein endothelial cells (HUVECs) and other endothelial cell types, we employed viral shRNA to silence NDUFS8 or employed the CRISPR/Cas9 method to knockout (KO) it, resulting in impaired mitochondrial functions in the endothelial cells, causing reduction in mitochondrial oxygen consumption and Complex I activity, decreased ATP production, mitochondrial depolarization, increased oxidative stress and reactive oxygen species (ROS) production, and enhanced lipid oxidation. Significantly, NDUFS8 silencing or KO hindered cell proliferation, migration, and capillary tube formation in cultured endothelial cells. In addition, there was a moderate increase in apoptosis within NDUFS8-depleted endothelial cells. Conversely, ectopic overexpression of NDUFS8 demonstrated a pro-angiogenic impact, enhancing cell proliferation, migration, and capillary tube formation in HUVECs and other endothelial cells. NDUFS8 is pivotal for Akt-mTOR cascade activation in endothelial cells. Depleting NDUFS8 inhibited Akt-mTOR activation, reversible with exogenous ATP in HUVECs. Conversely, NDUFS8 overexpression boosted Akt-mTOR activation. Furthermore, the inhibitory effects of NDUFS8 knockdown on cell proliferation, migration, and capillary tube formation were rescued by Akt re-activation via a constitutively-active Akt1. In vivo experiments using an endothelial-specific NDUFS8 shRNA adeno-associated virus (AAV), administered via intravitreous injection, revealed that endothelial knockdown of NDUFS8 inhibited retinal angiogenesis. ATP reduction, oxidative stress, and enhanced lipid oxidation were detected in mouse retinal tissues with endothelial knockdown of NDUFS8. Lastly, we observed an increase in NDUFS8 expression in retinal proliferative membrane tissues obtained from human patients with proliferative diabetic retinopathy. Our findings underscore the essential role of the mitochondrial protein NDUFS8 in regulating endothelial cell activation and angiogenesis.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

Lycium barbarum polysaccharide's protective effects against PM2.5-induced cellular senescence in HUVECs

Fine particulate matter (PM2.5) exposure is strongly associated with vascular endothelial senescence, a process implicated in cardiovascular diseases. While there is existing knowledge on the impact of Lycium barbarum polysaccharide (LBP) on vascular endothelial damage, the protective mechanism of LBP against PM2.5-induced vascular endothelial senescence remains unclear. In this study, we investigated the impact of PM2.5 exposure on vascular endothelial senescence and explored the intervention effects of LBP in human umbilical vein endothelial cells (HUVECs). We found that PM2.5 exposure dose-dependently reduced cell viability and proliferation in HUVECs while increasing the production of reactive oxygen species (ROS), malondialdehyde (MDA), and hydrogen peroxide (H2O2). Additionally, PM2.5 exposure inhibited the activity of superoxide dismutase (SOD). Notably, PM2.5 exposure induced autophagy impairments and cellular senescence. However, LBP mitigated PM2.5-induced cell damage. Further studies demonstrated that correcting autophagy impairment in HUVECs reduced the expression of the senescence markers P16 and P21 induced by PM2.5. This suggests the regulatory role of autophagy in cellular senescence and the potential of LBP in improving HUVECs senescence. These findings provide novel insights into the mechanisms underlying PM2.5-induced cardiovascular toxicity and highlight the potential of LBP as a therapeutic agent for improving vascular endothelial health.

A Dexamethasone-Loaded Polymeric Electrospun Construct as a Tubular Cardiovascular Implant.

Cardiovascular tissue engineering is providing many solutions to cardiovascular diseases. The complex disease demands necessitating tissue-engineered constructs with enhanced functionality. In this study, we are presenting the production of a dexamethasone (DEX)-loaded electrospun tubular polymeric poly(l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) construct which contains iPSC-CMs (induced pluripotent stem cell cardiomyocytes), HUVSMCs (human umbilical vein smooth muscle cells), and HUVECs (human umbilical vein endothelial cells) embedded in fibrin gel. The electrospun tube diameter was calculated, as well as the DEX release for 50 days for 2 different DEX concentrations. Furthermore, we investigated the influence of the polymer composition and concentration on the function of the fibrin gels by imaging and quantification of CD31, alpha-smooth muscle actin (αSMA), collagen I (col I), sarcomeric alpha actinin (SAA), and Connexin 43 (Cx43). We evaluated the cytotoxicity and cell proliferation of HUVECs and HUVSMCs cultivated in PLA and PLGA polymeric sheets. The immunohistochemistry results showed efficient iPSC-CM marker expression, while the HUVEC toxicity was higher than the respective HUVSMC value. In total, our study emphasizes the combination of fibrin gel and electrospinning in a functionalized construct, which includes three cell types and provides useful insights of the DEX release and cytotoxicity in a tissue engineering perspective.

XB130 inhibits healing of diabetic skin ulcers through the PI3K/Akt signalling pathway.

Diabetic skin ulcers, a significant global healthcare burden, are mainly caused by the inhibition of cell proliferation and impaired angiogenesis. XB130 is an adaptor protein that regulates cell proliferation and migration. However, the role of XB130 in the development of diabetic skin ulcers remains unclear. To investigate whether XB130 can regulate the inhibition of proliferation and vascular damage induced by high glucose. Additionally, we aim to determine whether XB130 is involved in the healing process of diabetic skin ulcers, along with its molecular mechanisms. We conducted RNA-sequencing analysis to identify the key genes involved in diabetic skin ulcers. We investigated the effects of XB130 on wound healing using histological analyses. In addition, we used reverse transcription-quantitative polymerase chain reaction, Western blot, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, immunofluorescence, wound healing, and tubule formation experiments to investigate their effects on cellular processes in human umbilical vein endothelial cells (HUVECs) stimulated with high glucose. Finally, we performed functional analysis to elucidate the molecular mechanisms underlying diabetic skin ulcers. RNA-sequencing analysis showed that the expression of XB130 was up-regulated in the tissues of diabetic skin ulcers. Knockdown of XB130 promoted the healing of skin wounds in mice, leading to an accelerated wound healing process and shortened wound healing time. At the cellular level, knockdown of XB130 alleviated high glucose-induced inhibition of cell proliferation and angiogenic impairment in HUVECs. Inhibition of the PI3K/Akt pathway removed the proliferative effects and endothelial protection mediated by XB130. The findings of this study indicated that the expression of XB130 is up-regulated in high glucose-stimulated diabetic skin ulcers and HUVECs. Knockdown of XB130 promotes cell proliferation and angiogenesis via the PI3K/Akt signalling pathway, which accelerates the healing of diabetic skin ulcers.

Astragaloside IV Suppresses the Effects of Hepatocellular Carcinoma Cells on Proliferation, Angiogenesis, and Invasion in Human Umbilical Vein Endothelial Cells by Controlling Exosomes by Inhibiting Rab27a

Hepatocellular carcinoma (HCC) is the third most common cancer worldwide, and invasion and metastasis are the leading causes of death. Astragaloside IV (AS-IV), a kind of traditional Chinese medicine, has been widely used in cancer therapy. Cell Counting Kit-8, tube formation, and Transwell analyses were performed to determine cell proliferation, invasion, and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the treatment of Hep3B or SK-Hep1 cells-derived exosomes or Hep3B or SK-Hep1 cells with or without AS-IV treatment. Then, Rab27a overexpression in Hep3B cells was selected to determine the function of AS-IV on the regulation of cell proliferation, invasion, and angiogenesis of HUVECs. Ultimately, the injection of Hep3B cell-derived exosomes or treatment with AS-IV on nude mice was used to comprehensively investigate AS-IV functions in vivo. Hep3B or SK-Hep1 cells promoted proliferation, invasion, and angiogenesis of HUVECs, which were inhibited by AS-IV therapy. The concentrations of Hep3B and SK-Hep1 cells-derived exosomes were markedly reduced after AS-IV therapy. Meanwhile, Rab27a expression was significantly decreased in SK-Hep1 or Hep3B cells after AS-IV therapy, and it inhibited the effects of AS-IV on Hep3B-induced proliferation, invasion, and angiogenesis of HUVECs. Tumor growth was also suppressed by AS-IV, which was accompanied by the decreased expression levels of Ki67 and CD34. Overall, this study demonstrated that AS-IV inhibits the effects of HCC cell-derived exosomes on proliferation, invasion, and angiogenesis of HUVECs via inhibiting Rab27a expression. Practical Applications. To our knowledge, this is the first study to explore the efficacy of AS-IV against HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs to reveal the underlying mechanism. In this study, CCK-8, Transwell, and tube formation assay detection show that AS-IV has an inhibitory effect on HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs by inhibiting Rab27a, which helps in the development of novel nutraceutical/functional food against HCC progression and thus could improve the quality of life in patients with HCC.

Exosomes from hypoxia-conditioned apical papilla stem cells accelerate angiogenesis in vitro through Notch/JAG1/VEGF signaling

Dental pulp angiogenesis is a committed step in pulp regeneration therapy, and exosomes provide a new cell-free choice for tissue regeneration. This study revealed the underlying regulatory mechanism of exosomes from stem cells of the apical papilla (SCAPs) under hypoxic state on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Exosomes extracted from normoxia or hypoxia-pretreated SCAPs were co-cultured with HUVECs, and hypoxia pretreatment increased the release of exosomes and the internalization of exosomes by HUVECs. Compared to normoxic SCAPs-derived exosomes, exosomes from hypoxic SCAPs were found to promote cell proliferation and migration in HUVECs, as it was respectively determined by Cell Counting Kit-8, RT-qPCR and Transwell assay. Besides, hypoxia-educated SCAPs-exosomes especially enhanced the angiogenesis abilities of HUVECs in vitro, which were confirmed by tube formation assay and RT-qPCR detection of angiogenesis-related molecular markers. Interestingly, we found that the hypoxia inducible factor-1α (HIF-1α)/Notch1 signaling pathway was activated in hypoxic SCAPs, and protein jagged-1 (JAG1) was delivered by hypoxic SCAPs-derived exosomes to increase vascular endothelial growth factor (VEGF) production in HUVECs. Moreover, exogenous interference of JAG1 expression in HUVECs partially neutralized the activities of hypoxic SCAPs-exosomes in promoting cell proliferation, migration and tube formation of HUVECs. In summary, this study elucidates that exosomes from hypoxic SCAPs shows high potential to promote angiogenesis in vitro through the HIF-1α/JAG1/VEGF signaling cascade, which may provide a new perspective for the development of vascular reconstruction measures during dental regeneration engineering.

MicroRNA-29 differentially mediates preeclampsia-dysregulated cellular responses to cytokines in female and male fetal endothelial cells.

Preeclampsia (PE) differentially impairs female and male fetal endothelial cell function, which is associated with an increased risk of adult-onset cardiovascular disorders in children born to mothers with PE. However, the underlying mechanisms are poorly defined. We hypothesize that dysregulation of microRNA-29a-3p and 29c-3p (miR-29a/c-3p) in PE disturbs gene expression and cellular responses to cytokines in fetal endothelial cells in a fetal sex-dependent manner. RT-qPCR analysis of miR-29a/c-3p was performed on female and male unpassaged (P0) human umbilical vein endothelial cells (HUVECs) from normotensive (NT) pregnancies and PE. Bioinformatic analysis of an RNA-seq dataset was performed to identify PE-dysregulated miR-29a/c-3p target genes in female and male P0-HUVECs. Gain- and loss-of-function assays were conducted to determine the effects of miR-29a/c-3p on endothelial monolayer integrity and proliferation in response to transforming growth factor-β1 (TGFβ1) and tumour necrosis factor-α (TNFα) in NT and PE HUVECs at passage 1. We observed that PE downregulated miR-29a/c-3p in male and female P0-HUVECs. PE dysregulated significantly more miR-29a/c-3p target genes in female vs. male P0-HUVECs. Many of these PE-differentially dysregulated miR-29a/c-3p target genes are associated with critical cardiovascular diseases and endothelial function. We further demonstrated that miR-29a/c-3p knockdown specifically recovered the PE-abolished TGFβ1-induced strengthening of endothelial monolayer integrity in female HUVECs, while miR-29a/c-3p overexpression specifically enhanced the TNFα-promoted cell proliferation in male PE HUVECs. In conclusion, PE downregulates miR-29a/c-3p expression and differentially dysregulates miR-29a/c-3p target genes associated with cardiovascular diseases and endothelial function in female and male fetal endothelial cells, possibly contributing to the fetal sex-specific endothelial dysfunction observed in PE. KEY POINTS: Preeclampsia differentially impairs female and male fetal endothelial cell function in responses to cytokines. Pro-inflammatory cytokines are elevated in maternal circulation during pregnancy in preeclampsia. MicroRNAs are critical regulators of endothelial cell function during pregnancy. We have previously reported that preeclampsia downregulated microRNA-29a-3p and 29c-3p (miR-29a/c-3p) in primary fetal endothelial cells. However, it is unknown if PE differentially dysregulates the expression of miR-29a/c-3p in female and male fetal endothelial cells. We show that preeclampsia downregulates miR-29a/c-3p in male and female HUVECs and preeclampsia dysregulates cardiovascular disease- and endothelial function-associated miR-29a/c-3p target genes in HUVECs in a fetal sex-specific manner. MiR-29a/c-3p differentially mediate cell responses to cytokines in female and male fetal endothelial cells from preeclampsia. We have revealed fetal sex-specific dysregulation of miR-29a/c-3p target genes in fetal endothelial cells from preeclampsia. This differential dysregulation may contribute to fetal sex-specific endothelial dysfunction in offspring born to preeclamptic mothers.

Downregulation of VEGFR2 signaling by cedrol abrogates VEGF‑driven angiogenesis and proliferation of glioblastoma cells through AKT/P70S6K and MAPK/ERK1/2 pathways.

Cedrol is a sesquiterpene alcohol isolated from Cedrus atlantica, which has been traditionally used in aromatherapy and has anticancer, antibacterial and antihyperalgesic effects. One characteristic of glioblastoma (GB) is the overexpression of vascular endothelial growth factor (VEGF), which induces a high degree of angiogenesis. Although previous studies have reported that cedrol inhibits GB growth by inducing DNA damage, cell cycle arrest and apoptosis, its role in angiogenesis remains unclear. The aim of the present study was to investigate the effects of cedrol on VEGF-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 0-112 µM cedrol and 20 ng/ml VEGF for 0-24 h, and then anti-angiogenic activation of cedrol was determined by MTT assay, wound healing assay, Boyden chamber assay, tube formation assay, semi-quantitative reverse transcription-PCR and western blotting. These results demonstrated that cedrol treatment inhibited VEGF-induced cell proliferation, migration and invasion in HUVECs. Furthermore, cedrol prevented VEGF and DBTRG-05MG GB cells from inducing capillary-like tube formation in HUVECs and decreased the number of branch points formed. Moreover, cedrol downregulated the phosphorylation of VEGF receptor 2 (VEGFR2) and the expression levels of its downstream mediators AKT, ERK, VCAM-1, ICAM-1 and MMP-9 in HUVECs and DBTRG-05MG cells. Taken together, these results demonstrated that cedrol exerts anti-angiogenic effects by blocking VEGFR2 signaling, and thus could be developed into health products or therapeutic agents for the prevention or treatment of cancer and angiogenesis-related diseases in the future.

Enhanced external counterpulsation improves dysfunction of forearm muscle caused by radial artery occlusion.

This study aimed to investigate the therapeutic effect of enhanced external counterpulsation (EECP) on radial artery occlusion (RAO) through the oscillatory shear (OS) and pulsatile shear (PS) models of human umbilical vein endothelial cells (HUVECs) and RAO dog models. We used high-throughput sequencing data GSE92506 in GEO database to conduct time-series analysis of functional molecules on OS intervened HUVECs, and then compared the different molecules and their functions between PS and OS. Additionally, we studied the effect of EECP on the radial artery hemodynamics in Labrador dogs through multi-channel physiological monitor. Finally, we studied the therapeutic effect of EECP on RAO at the histological level through Hematoxylin-Eosin staining, Masson staining, ATPase staining and immunofluorescence in nine Labrador dogs. With the extension of OS intervention, the cell cycle decreased, blood vessel endothelial cell proliferation and angiogenesis responses of HUVECs were down-regulated. By contrast, the inflammation and oxidative stress responses and the related pathways of anaerobic metabolism of HUVECs were up-regulated. Additionally, we found that compared with OS, PS can significantly up-regulate muscle synthesis, angiogenesis, and NO production related molecules. Meanwhile, PS can significantly down-regulate inflammation and oxidative stress related molecules. The invasive arterial pressure monitoring showed that 30Kpa EECP treatment could significantly increase the radial artery peak pressure (p = 0.030, 95%CI, 7.236-82.524). Masson staining showed that RAO significantly increased muscle interstitial fibrosis (p = 0.002, 95%CI, 0.748-2.128), and EECP treatment can reduce this change (p = 0.011, 95%CI, -1.676 to -0.296). ATPase staining showed that RAO significantly increased the area of type II muscle fibers (p = 0.004, 95%CI, 7.181-25.326), and EECP treatment could reduce this change (p = 0.001, 95%CI, -29.213 to -11.069). In addition, immunofluorescence showed that EECP increased angiogenesis in muscle tissue (p = 0.035, 95%CI, 0.024-0.528). EECP improves interstitial fibrosis and hypoxia, and increases angiogenesis of muscle tissue around radial artery induced by RAO.

Honokiol inhibits interleukin-induced angiogenesis in the NSCLC microenvironment through the NF-κB signaling pathway

Tumor angiogenesis, which may be affected by microenvironmental inflammation and promotes tumor development and metastasis, is one of the key reasons contributing to increased mortality. The goal of this study is to investigate how lignin analogs, specifically honokiol (HNK), block angiogenesis induced by the inflammatory milieu of lung cancer. The human lung cancer cell lines A549 and H460 were treated with HNK. Interleukin-1 was employed to mimic an inflammatory tumor microenvironment. Findings demonstrated that HNK drastically decreased the cell viability of A549 and H460 cells. In A549 and H460 cells, HNK also reduced the production of vascular endothelial growth factor (VEGF), the most important marker of tumor angiogenesis. Signal pathway studies revealed that HNK blocked the NF-κB signaling pathway. This effect, in turn, prevented the expression of VEGF by inhibiting the NF-κB signaling pathway. Human umbilical vein endothelial cells (HUVECs) from A549-conditioned medium cultures were subjected to HNK treatment, which decreased tubulogenesis, horizontal and vertical migration, and cell proliferation in HUVECs. Overall, HNK inhibited the NF–κB pathway. This effect resulted in the downregulation of VEGF, thus reducing the viability and angiogenesis of human lung cancer cell lines. In A549 cell xenografts, HNK decreased VEGF expression, tumor angiogenesis, and tumor development. Our research shows that HNK is a potential antiangiogenic molecule for the treatment of lung cancer.

UCMSCs-derived exosomal circHIPK3 promotes ulcer wound angiogenesis of diabetes mellitus via miR-20b-5p/Nrf2/VEGFA axis.

Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU. Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions. CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions. UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.

Icariside II induces rapid phosphorylation of endothelial nitric oxide synthase via multiple signaling pathways.

Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer’s disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.

Polyethylene glycol crosslinked decellularized single liver lobe scaffolds with vascular endothelial growth factor promotes angiogenesis in vivo

BackgroundImproving the mechanical properties and angiogenesis of acellular scaffolds before transplantation is an important challenge facing the development of acellular liver grafts. The present study aimed to evaluate the cytotoxicity and angiogenesis of polyethylene glycol (PEG) crosslinked decellularized single liver lobe scaffolds (DLSs), and establish its suitability as a graft for long-term liver tissue engineering. MethodsUsing mercaptoacrylate produced by the Michael addition reaction, DLSs were first modified using N-succinimidyl S-acetylthioacetate (SATA), followed by cross-linking with PEG as well as vascular endothelial growth factor (VEGF). The optimal concentration of agents and time of the individual steps were identified in this procedure through biomechanical testing and morphological analysis. Subsequently, human umbilical vein endothelial cells (HUVECs) were seeded on the PEG crosslinked scaffolds to detect the proliferation and viability of cells. The scaffolds were then transplanted into the subcutaneous tissue of Sprague-Dawley rats to evaluate angiogenesis. In addition, the average number of blood vessels was evaluated in the grafts with or without PEG at days 7, 14, and 21 after implantation. ResultsThe PEG crosslinked DLS maintained their three-dimensional structure and were more translucent after decellularization than native DLS, which presented a denser and more porous network structure. The results for Young's modulus proved that the mechanical properties of 0.5 PEG crosslinked DLS were the best and close to that of native livers. The PEG-VEGF-DLS could better promote cell proliferation and differentiation of HUVECs compared with the groups without PEG cross-linking. Importantly, the average density of blood vessels was higher in the PEG-VEGF-DLS than that in other groups at days 7, 14, and 21 after implantation in vivo. ConclusionsThe PEG crosslinked DLS with VEGF could improve the biomechanical properties of native DLS, and most importantly, their lack of cytotoxicity provides a new route to promote the proliferation of cells in vitro and angiogenesis in vivo in liver tissue engineering.

Caspase‐4/11 is critical for angiogenesis by repressing Notch1 signalling via inhibiting γ‐secretase activity

Notch1 activation mediated by γ-secretase is critical for angiogenesis. GeneCards database predicted that Caspase-4 (CASP4, with murine ortholog CASP11) interacts with presenilin-1, the catalytic core of γ-secretase. Therefore, we investigated the role of CASP4/11 in angiogenesis. In vivo, we studied the role of Casp11 in several angiogenesis mouse models using Casp11 wild-type and knockout mice. In vitro, we detected the effects of CASP4 on endothelial functions and Notch signalling by depleting or overexpressing CASP4 in human umbilical vein endothelial cells (HUVECs). The functional domain responsible for the binding of CASP4 and presenilin-1 was detected by mutagenesis and co-immunoprecipitation. Casp11 deficiency impaired adult angiogenesis in ischaemic hindlimbs, melanoma xenografts and Matrigel plugs, but not the developmental angiogenesis of retina. Bone marrow transplantation revealed that the pro-angiogenic effect depended on CASP11 derived from non-haematopoietic cells. CASP4 expression was induced by inflammatory factors and CASP4 knockdown decreased cell viability, proliferation, migration and tube formation in HUVECs. Mechanistically, CASP4/11 deficiency increased Notch1 activation in vivo and in vitro, while CASP4 overexpression repressed Notch1 signalling in HUVECs. Moreover, CASP4 knockdown increased γ-secretase activity. The γ-Secretase inhibitor DAPT restored the effects of CASP4 siRNA on Notch1 activation and angiogenesis in HUVECs. Notably, the catalytic activity of CASP4/11 was dispensable. CASP4 directly interacted with presenilin-1 through the caspase recruitment domain (CARD). These findings reveal a critical role of CASP4/11 in adult angiogenesis and make this molecule a promising therapeutic target for angiogenesis-related diseases in the future.

Differential Proteomic Profiles of Coronary Serum Exosomes in Acute Myocardial Infarction Patients with or Without Diabetes Mellitus: ANGPTL6 Accelerates Regeneration of Endothelial Cells Treated with Rapamycin via MAPK Pathways.

Delayed re-endothelialization after coronary drug-eluting stent implantation is associated with an increased incidence of late in-stent thrombosis. Serum exosomes exhibit controversial effects on promoting endothelialization. This study aimed to compare the angiogenic effects of serum exosomes derived from patients with acute myocardial infarction (AMI) and AMI plus diabetes mellitus (DM) and to explore the underlying mechanisms. Serum exosomes derived from patients in the control (Con-Exos), AMI (AMI-Exos), and AMI plus DM (AMI+DM-Exos) groups were isolated and identified using standard assays. CCK-8, wound healing, and tube formation assays were performed to detect the angiogenic abilities of serum exosomes on rapamycin-conditioned human umbilical vein endothelial cells (HUVECs). Differential proteomic profiles between AMI-Exos and AMI+DM-Exos were analyzed by mass spectrometry. The effects and potential mechanisms of exosomal angiopoietin-like 6 (ANGPTL6) were investigated. Functional assays indicated that compared with Con-Exos, AMI-Exos enhanced, whereas AMI+DM-Exos inhibited the cell proliferation, migration, and tube formation of rapamycin-conditioned HUVECs. Subsequently, 28 differentially expressed proteins between AMI-Exos and AMI+DM-Exos were identified, which were correlated with material transportation, immunity, and inflammatory reaction. Moreover, ANGPTL6 was highly enriched in AMI-Exos. Overexpression and knockdown of ANGPTL6 enhanced and inhibited angiogenesis, respectively. Furthermore, the effect of ANGPTL6 on angiogenesis was mediated via the activation of ERK 1/2, JNK, and p38 pathways. The inhibition of ERK 1/2 signaling markedly attenuated the migration abilities of overexpressing ANGPTL6. Diabetes impairs the regenerative capacities of serum exosomes. Exosomal ANGPTL6 contributes to endothelial repair and is a novel therapeutic target for enhanced stent endothelization.

Regulatory Mechanism of circEIF4G2 Targeting miR-26a in Acute Myocardial Infarction.

Background Acute myocardial infarction (AMI) involves a series of complex cellular and molecular events, including circular RNAs (circRNAs), microRNAs (miRNAs) and other noncoding RNAs. Objective In this study, the regulation mechanism of circEIF4G2 acting on miR-26a on HUVECs (Human Umbilical Vein Endothelial Cells) proliferation, cell cycle and angiogenesis ability was mainly explored in the vascular endothelial growth factor induced (VEGF-induced) angiogenesis model. Methods VEGF induced HUVECs angiogenesis model was constructed, and the expression of circEIF4G2 and miR-26a in VEGF model was detected by qRT-PCR. When circEIF4G2 and miR-26a were knocked down or overexpressed in HUVECs, qRT-PCR was used to detect the expression of circEIF4G2 and miR-26a, CCK-8 was used to detect cell proliferation, flow cytometry was used to detect the cell cycle transition of HUVECs, and cell formation experiment was used to detect the ability of angiogenesis. MiRanda database and Targetscan predicted the binding site of circEIF4G2 and miR-26a, lucifase reporting assay and RNA pull down assay verified the interaction between circEIF4G2 and miR-26a. Results After HUVECs were treated with VEGF, circEIF4G2 was significantly upregulated. After circEIF4G2 was knocked down, the proliferation and angiogenesis of HUVECs cells were decreased, and the process of cell cycle G0/G1 phase was blocked. The overexpression of miR-26a reduced the proliferation and angiogenesis of HUVECs cells and blocked the cell cycle progression of G0/G1 phase. Double lucifase reporter gene assay verified that circEIF4G2 could directly interact with miR-26a through the binding site, and RNA Pull down assay further verified the interaction between circEIF4G2 and miR-26a. When circEIF4G2 and miR-26a were knocked down simultaneously in HUVECs, it was found that knocking down miR-26a could reverse the inhibition of circEIF4G2 on cell proliferation, cycle and angiogenesis. Conclusion In the VEGF model, circEIF4G2 was highly expressed and miR-26a was low expressed. MiR-26a regulates HUVECs proliferation, cycle and angiogenesis by targeting circEIF4G2.

Peptides derived from sea cucumber accelerate cells proliferation and migration for wound healing by promoting energy metabolism and upregulating the ERK/AKT pathway

Numerous bioactive peptides have high potential in accelerating skin repair. This study aimed to explore wound healing capacity of sea cucumber-derived tetrapeptides with amino acid sequence of Val-Thr-Pro-Tyr (VTPY) and Val-Leu-Leu-Tyr (VLLY) by human skin fibroblast (HSF) and human umbilical vein endothelial cells (HUVEC) in vitro. The results showed that VTPY and VLLY possessed excellent capacity to induce the proliferation and migration of HSF cells and HUVEC cells. Among them, VTPY possessed a higher cell migration-promotion capacity than VLLY. Meanwhile, VTPY and VLLY increased mitochondrial respiratory capacity of cells, leading to the significant increase in adenosine triphosphate (ATP) production. Additionally, VTPY and VLLY significantly increased the percentage of S phase in cell cycle of HUVEC via upregulation of extracellular regulated protein kinases (ERK) and protein kinase B (AKT) signaling pathway. Molecular docking further indicated the interaction of peptides with mitogen-activated protein kinase phosphatase (MKP) and PH domain leucine-rich repeat protein phosphatase (PHLPP) binding site in the ERK2 and AKT, respectively, leading to subsequent increased phosphorylation of ERK1/2 and AKT. Overall, peptides of VTPY and VLLY possessed outstanding capacity to induce the proliferation and migration of HSF cells and HUVEC cells in vitro and the mechanism was mainly related to improving mitochondrial respiratory capacity to produce more ATP for biological energy, blocking the binding of MKP to ERK2 and PHLPP to AKT and thus upregulating the ERK/AKT pathway.

MiR-29b-3p Inhibits the Inflammation Injury in Human Umbilical Vein Endothelial Cells by Regulating SEC23A.

This study aims to investigate the effects of miR-29b-3p on the inflammation injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) and explore the underlying mechanisms. The effects of different concentrations of LPS (0, 1, 5 and 10μg/mL) on inflammation injury in HUVECs are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses to determine the optimal stimulus concentration. After stimulating HUVECs with 10μg/mL LPS, the expression levels of miR-29b-3p are detected, and the effects of miR-29b-3p on inflammation injury are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses. Bioinformatic analysis, luciferase reporter assay and confirmatory experiments are applied to identify the target gene bound with miR-29b-3p. Rescue experiments have verified the roles of miR-29b-3p and the target gene in inflammation injury. We found that pro-inflammatory factor was increased, apoptosis was promoted, and cell proliferation was inhibited after the treatment of LPS in HUVECs. Overexpression of miR-29b-3p inhibited LPS-induced inflammatory response and apoptosis while promoting proliferation in HUVECs. Besides, bioinformatics analysis indicated that SEC23A was the target gene of miR-29b-3p and the confirmatory experiments showed that SEC23A was negatively correlated with miR-29b-3p and positively correlated with LPS concentration. Rescue experiments revealed that overexpression of SEC23A partially enhanced the inflammation injury effects in LPS-induced HUVECs with overexpression of miR-29b-3p. Hence, miR-29b-3p repressed inflammatory response, cell apoptosis and promoted cell proliferation in LPS-induced HUVECs by targeting SEC23A, providing a potential target for treating sepsis.