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Background BM-MSCs contribute to Helicobacter pylori (H. pylori)-induced gastric cancer, but their mechanism is still unclear. The aim of our study was to investigate the specific role and mechanism of BM-MSCs in H. pylori-induced gastric cancer. Main methods Mice received total bone marrow transplants and were then infected with H. pylori. BM-MSCs were extracted and transplanted into the gastric serosal layer of mice chronically infected with H. pylori. Hematoxylin and eosin staining, immunohistochemistry staining and immunofluorescence were performed to detect tumor growth and angiogenesis in mouse stomach tissues. Chicken chorioallantoic membrane assays, xenograft tumor models, and human umbilical vein endothelial cell tube formation assays were used for in vivo and in vitro angiogenesis studies. THBS4 was screened from RNA-seq analysis of gastric tissues of BM-MSCs transplanted into H. pylori-infected mice. Results BM-MSCs can migrate to the site of chronic mucosal injury and promote tumor angiogenesis associated with chronic H. pylori infection. Migration of BM-MSCs to the site of chronic mucosal injury induced the upregulation of THBS4, which was also evident in human gastric cancer and correlated with increased blood vessel formation and worse outcome. The THBS4/integrin α2 axis promoted angiogenesis by facilitating the PI3K/AKT pathway in endothelial cells. Conclusions Our results revealed a novel proangiogenic effect of BM-MSCs in the chronic H. pylori infection microenvironment, primarily mediated by the THBS4/integrin α2 axis, which activates the PI3K/AKT pathway in endothelial cells and eventually induces the formation of new tumor vessels.

Background. Hepatocellular carcinoma (HCC) is the main form of primary liver cancer and is one of the most prevalent and life-threatening malignancies globally. Hypoxia activates hypoxia-inducible factor-1α (HIF-1α), which is the key factor in promoting angiogenesis in HCC. Currently, there are few studies on the effects of HIF-1α-targeted gene therapy combined with traditional Chinese herbal extracts. Objective. We investigated the effects of HIF-1α RNA interference (RNAi) combined with asparagus polysaccharide (ASP) on HCC in vitro and in vivo. Methods. CCK-8, wound-healing, transwell, and human umbilical vein endothelial cell tube formation assays were performed to evaluate the proliferation, migration, invasion, and angiogenesis of HCC cells in vitro. In addition, western blotting, qPCR, and immunohistochemistry were performed to detect the expression of HIF-1α, vascular endothelial growth factor, AKT, p-AKT, ERK, p-ERK, and CD34 in HCC cells. Results. The combination of HIF-1α RNAi and ASP significantly inhibited the proliferation, migration, invasion, and angiogenesis of SK-Hep1 and Hep-3B cells compared with the use of HIF-1α RNAi or ASP alone. In addition, this combined treatment was shown to exert these effects by regulating the PI3K and MAPK signaling pathways. These results were observed both in vitro and in vivo. Conclusion. Our study indicates that HIF-1α RNAi combined with ASP inhibits angiogenesis in HCC via the PI3K and MAPK signaling pathways. Thus, we suggest that this combination may be an effective method for the comprehensive treatment of HCC, which may provide new ideas for the treatment of other malignant tumors.

For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Micro-computed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. STATEMENT OF SIGNIFICANCE: This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Tetrahydroxystilbene glucoside (TSG), an active ingredient of Polygonum multiflorum, has been known for certain anti-aging effects. In this study, the possible protective mechanism of TSG on human umbilical vein endothelial cell (HUVEC) senescence induced by angiotensin II (Ang II) was investigated. The results revealed that TSG pretreatment could reduce the percentage of senescence-associated β-galactosidase (SA-β-gal) positive cells and decrease the expression levels of the cellular senescence biomarker proteins p53 and PAI-1. At the same time, the expression of SIRT1 in senescent cells showed an upward trend due to TSG treatment. When inhibiting the expression of SIRT1 by EX527, our results showed that TSG reversed the effect of EX527 by promoting the expression level of SIRT1, reducing the expression of SA-β-gal positive cells and the expression level of p53 and PAI-1 proteins. The present study demonstrated that TSG could protect against HUVEC senescence induced by Ang II, potentially through modulation of SIRT1 activity.

BACKGROUND: Coronary artery diseases are the most important cause of premature death, and these diseases are predominantly related to atherosclerosis. Soluble lectin-like oxidized low-density lipoprotein receptor-1 and microRNAs are closely associated with atherosclerotic coronary heart diseases. AIMS: To investigate the relationship between the severity and risk of coronary artery disease and plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 and miR-98. STUDY DESIGN: Case-control study. METHODS: Angiographically documented patients with 38 single-vessel, 75 double-vessel, and 62 multi-vessel coronary artery disease; 62 healthy control participants; and 24-hour hypoxic (1% oxygen) human umbilical vein endothelial cells were included in this study. Circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 concentrations were determined through enzyme-linked immunosorbent assays, and miR-98 expressions were measured by quantitative real-time polymerase chain reaction. RESULTS: The expressions of plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 levels were progressively and significantly higher in patients with single-vessel, double-vessel, and multi-vessel coronary artery disease than in healthy controls (p

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3′,5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion: Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.

Wound healing is a complex physiological process in which fibrocytes serve a vital role. However, the mechanism underlying the recruitment of fibrocytes during wound healing remains largely unknown. The present study aimed to investigate whether endothelial cells are involved in the recruitment of fibrocytes in wound healing. To mimic the in vivo angiogenic process, a co‑culture system consisting of endothelial cells and fibrocytes was achieved using a permeable Transwell co‑culture system. The expression of chemokines produced by endothelial cells with or without co‑culture was then measured using a gene chip. Based on the dataset from chip analysis, chemokine ligand 15 (CCL15) produced by endothelial cells was identified, which likely serves a regulatory role in mediating the transmigration of fibrocytes. Overexpression of CCL15 in endothelial cells or chemokine receptor 1 (CCR1) in fibrocytes promoted the transmigration of fibrocytes, whilst silencing the expression of CCL15 in endothelial cells or that of CCR1 in fibrocytes attenuated the transmigration of fibrocytes. Results from the present study suggested that the CCL15‑CCR1 axis between endothelial cells and fibrocytes serves a vital role in mediating the recruitment of fibrocytes during wound healing.

Nitric oxide (NO) is a second messenger playing crucial roles in the signaling of a variety of cellular functions. Due to its pathophysiological significance, various NO modulators have been developed to explore NO pathways and some have been used as therapies. These modulators are often used directly to observe pharmacological effects in cell lines, but their actual effect on intracellular NO level is seldom analyzed. Herein, facilitated by a selective and sensitive fluorescence probe, we observed that some NO modulators displayed unexpected behaviors with both NO scavenger carboxy-PTIO and endothelial nitric oxide synthase (eNOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME) failing to decrease intracellular free NO level in EA. hy926 cells while NO donor diethylamine-NONOate (DEA·NONOate) and eNOS activator calcimycin (A23187) failing to increase free NO level in human umbilical vein endothelial cell line (HUV-EC-C), although the reagents were confirmed to work normally in the primary human umbilical vein endothelial cells (primary HUVECs) and RAW 264.7 macrophage cells. Further research suggested that these unusual behaviors might be attributed to the cellular microenvironments including both the NO synthase (NOS) level and the endogenous glutathione (GSH) level. Genetically manipulating eNOS level in both cells restores the expected response, while decreasing GSH level restores the ability of DEA·NONOate to increase NO level in HUV-EC-C. These results reveal that the cellular microenvironment has a profound impact on pharmacological effect. Our study suggests GSH as a reservoir for NO in live cells and highlights the value of chemical probes as valuable tools to reveal microenvironment-dependent pharmacological effects., Graphical abstract Image 1, Highlights • An imaging based method for evaluating the efficacy of NO modulators in live cells was developed. • Some NO modulators were observed yielding unexpected effects on cellular NO levels in some endothelial cell lines. • Further study unveiled intracellular microenvironments have profound effects on the efficacy of NO modulators in live cells. • This result highlights the pitfall of the specific cellular microenvironments on drug efficacy.

Angiogenesis plays important roles in pathological conditions such as cancer and inflammation as well as normal tissue development and homeostasis. Here, we investigated the effects and molecular mechanisms of α-viniferin, an oligostilbene isolated from Caragana sinica, on human umbilical vein endothelial cell responses in vitro and angiogenic sprouting in aortic rings ex vivo. α-viniferin treatment inhibited mitogen-induced HUVEC proliferation by retinoblastoma protein hypophosphorylation. In addition, α-viniferin suppressed mitogen-induced HUVEC adhesion, migration, invasion, and microvessel outgrowth. These anti-angiogenic activities of α-viniferin might be mediated through downregulation of cell cycle-related proteins, vascular endothelial growth factor receptor-2 (VEGFR-2), and matrix metalloproteinase-2. Furthermore, inactivation of VEGFR-2/p70 ribosomal S6 kinase signaling pathway was found to be involved in α-viniferin-mediated modulation of endothelial cell responses. Our results demonstrate the pharmacological functions and molecular mechanisms of α-viniferin in regulating angiogenesis, suggesting the therapeutic potential of α-viniferin to treat and prevent various angiogenesis-related diseases.

Purpose Dysfunction of endothelial cells plays a key role in the pathogenesis of diabetic atherosclerosis. High glucose (HG) has been found as a key factor in the progression of diabetic complications, including atherosclerosis. PI3K/Akt/eNOS signaling pathway has been shown to involve in HG-induced vascular injuries. Hydrogen sulfide (H2S) has been found to exhibit protective effects on HG-induced vascular injuries. Moreover, H2S activates PI3K/Akt/eNOS pathway in endothelial cells. Thus, the present study aimed to determine if H2S exerts protective effects against HG-induced injuries of human umbilical vein endothelial cells (HUVECs) via activating PI3K/Akt/eNOS signaling. Materials and methods The endothelial protective effects of H2S were evaluated and compared to the controlled groups. Cell viability, cell migration and tube formation were determined by in vitro functional assays; protein levels were evaluated by Western blot assay and ELISA; cell apoptosis was determined by Hoechst 33258 nuclear staining; Reactive oxygen species (ROS) production was evaluated by the ROS detection kit. Results HG treatment significantly inhibited PI3K/Akt/eNOS signaling in HUVECs, which was partially reversed by the H2S treatment. HG treatment inhibited cell viability of HUVECs, which were markedly prevented by H2S or PI3K agonist Y-P 740. HG treatment also induced HUVEC cell apoptosis by increasing the protein levels of cleaved caspase 3, Bax and Bcl-2, which were significantly attenuated by H2S or 740 Y-P. ROS production and gp91phox protein level were increased by HG treatment in HUVECs and this effect can be blocked by the treatment with H2S or Y-P 740. Moreover, HG treatment increased the protein levels of pro-inflammatory cytokines, caspase-1 and phosphorylated JNK, which was significantly attenuated by H2S or Y-P 740. Importantly, the cytoprotective effect of H2S against HG-induced injury was inhibited by LY294002 (an inhibitor of PI3K/Akt/eNOS signaling pathway). Conclusion The present study demonstrated that exogenous H2S protects endothelial cells against HG-induced injuries by activating PI3K/Akt/eNOS pathway. Based on the above findings, we proposed that reduced endogenous H2S levels and the subsequent PI3K/Akt/eNOS signaling impairment may be the important pathophysiological mechanism underlying hyperglycemia-induced vascular injuries.

Hydroxytyrosol (HT), a peculiar olive and olive oil phenolic antioxidant, plays a significant role in the endothelial and cardiovascular protection associated with olive oil consumption. However, studies examining the effects of HT on the whole-genome expression of endothelial cells, which are prominent targets for vasculo-protective effects of olive oil polyphenols, have been lacking. This study aims to comprehensively evaluate the genomic effects exerted by HT, at the transcriptional level, in endothelial cells under resting or proinflammatory conditions. Human umbilical vein endothelial cells (HUVECs) were treated with 10 µmol/L HT for 1 h and then stimulated with 5 ng/mL interleukin (IL)-1β for 3 h. Total RNA was extracted, and gene expression profile assessed with microarray analysis. Functional enrichment analysis and pathway analysis were performed by Ingenuity Pathways Analysis. Microarray data were validated by qRT-PCR. Fixing a significance threshold at 1.5-fold change, HT affected the expression of 708 and 599 genes, respectively, in HUVECs under resting and IL-1β-stimulated conditions, among these, 190 were common to both conditions. Unfolded protein response (UPR) and endoplasmic reticulum stress resulted from the two top canonical pathways common between HT and HT-IL-1β affected genes. IL-17F/A signaling was found in the top canonical pathways of HT modified genes under resting unstimulated conditions, whereas cardiac hypertrophy signaling was identified among the pathways affected by HT-IL-1β. The transcriptomic analysis allowed pinpointing immunological, inflammatory, proliferative, and metabolic-related pathways as the most affected by HT in endothelial cells. It also revealed previously unsuspected genes and related gene pathways affected by HT, thus broadening our knowledge of its biological properties. The unbiased identification of novel genes regulated by HT improves our understanding of mechanisms by which olive oil prevents or attenuates inflammatory diseases and identifies new genes to be enquired as potential contributors to the inter-individual variation in response to functional food consumption.

Background: Acute kidney injury (AKI) increases the risk of chronic kidney disease. Atorvastatin (ATV)-loaded lipid bilayer-coated mesoporous silica nanoparticles (L-AMSNs) were synthesized, and their physicochemical parameters were characterized. L-AMSNs exhibited excellent stability; it did not increase in size over time, indicating that the lipid membrane coating prohibited mesoporous silica nanoparticles (MSNs) coalescence. Results: The rate of drug release differed significantly between AMSNs and L-AMSNs at all tested time points. A remarkable improvement in hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cell (HUVEC) viability was observed after treatment with L-AMSNs; the malondialdehyde (MDA) level was significantly reduced compared to control cells. The extent of apoptosis was only 15% that of control H2O2-treated cells. L-AMSNs induced a remarkable decrease in the levels of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6), showing the therapeutic potential of nanocarrier-based ATV. L-AMSNs significantly increased the superoxide dismutase level and decreased the MDA level, indicating superior anti-inflammatory activity under conditions of oxidative stress. The L-AMSN showed a remarkable improvement in the outer stripe of outer medulla (OSOM) region and maintained the tubular structure of the kidney tissue. Besides, kidney injury score of L-AMSN is significantly lower compared to that of LPS-AKI and ATV indicating the excellent therapeutic efficacy of nanoparticulate system based L-AMSN. Conclusions: Nanoparticles system-based L-AMSNs maintained the tubular structure of kidney tissue, indicating excellent therapeutic efficacy. After clinical translation, L-AMSNs could serve as a promising treatment for AKI.

AIM Arteriogenesis constitutes the most efficient endogenous rescue mechanism in cases of cerebral ischaemia. The aim of this work was to investigate whether angiotensin-converting enzyme inhibitors (ACEi) stimulates, and angiotensin II receptor type 1 blockers (ARB) inhibits cerebral collateral growth by applying a three-vessel occlusion (3-VO) model in rat. METHODS Cerebral collateral growth was measured post 3-VO (1) by assessing blood flow using the cerebrovascular reserve capacity (CVRC) technique, and (2) by assessing vessel diameters in the posterior cerebral artery (PCA) via the evaluation of latex angiographies. A stimulatory effect on arteriogenesis was investigated for ACEi administration ± bradykinin receptor 1 (B1R) and 2 (B2R) blockers, and an inhibitory effect was analysed for ARB administration. Results were validated by immunohistochemical analysis and mechanistic data were collected by human umbilical vein endothelial cell (HUVEC) viability or scratch assay and monocyte (THP-1) migration assay. RESULTS An inhibitory effect of ARB on arteriogenesis could not be demonstrated. However, collateral growth measurements demonstrated a significantly increased CVRC and PCA diameters in the ACEi group. ACEi stimulates cell viability and migration, which could be partially reduced by additional administration of bradykinin receptor 1 inhibitor (B1Ri). ACEi inhibits the degradation of pro-arteriogenic bradykinin derivatives, but combined ACEi + B1Ri + B1Ri (BRB) treatment did not reverse the stimulatory effect. Yet, co-administration of ACEi + BRB enhances arteriogenesis and cell migration. CONCLUSION We demonstrate a potent stimulatory effect of ACEi on cerebral arteriogenesis in rats, presumable via B1R. However, results imply a pleiotropic and compensatory effect of ACEi on bradykinin receptor-stimulated arteriogenesis.

According to the Centers for Disease Control and Prevention, tooth caries is a common problem affecting 9 out of every 10 adults worldwide. Dentin regeneration has since become one of the pressing issues in dentistry with tissue engineering emerging as a potential solution for enhancing dentin regeneration. In this study, we fabricated cell blocks with human dental pulp stem cells (hDPSCs)-laden alginate/fish gelatin hydrogels (Alg/FGel) at the center of the cell block and human umbilical vascular endothelial cells (HUVEC)-laden Si ion-infused fish gelatin methacrylate (FGelMa) at the periphery of the cell block. 1H NMR and FTIR results showed the successful fabrication of Alg/FGel and FGelMa. In addition, Si ions in the FGelMa were noted to be bonded via covalent bonds and the increased number of covalent bonds led to an increase in mechanical properties and improved degradation of FGelMa. The Si-containing FGelMa was able to release Si ions, which subsequently significantly not only enhanced the expressions of angiogenic-related protein, but also secreted some cytokines to regulate odontogenesis. Further immunofluorescence results indicated that the cell blocks allowed interactions between the HUVEC and hDPSCs, and taken together, were able to enhance odontogenic-related markers’ expression, such as alkaline phosphatase (ALP), dentin matrix phosphoprotein-1 (DMP-1), and osteocalcin (OC). Subsequent Alizarin Red S stain confirmed the benefits of our cell block and demonstrated that such a novel combination and modification of biomaterials can serve as a platform for future clinical applications and use in dentin regeneration.

Background Hypertension, as one of the most common chronic diseases, is a major public health issue. Previous studies have shown that there are miRNAs differentially expressed in hypertensive patients. In addition, hypertension is closely related to endothelial dysfunction, and miRNAs have been identified as important molecular mediators for endothelial function. Therefore, it is necessary to identify specific miRNAs related to hypertension and explore their molecular mechanism in the progression of hypertension. Methods We investigated the association of circulating levels of miR-3656 with hypertension. Furthermore, in-vitro studies were performed to investigate its possible mechanisms for hypertension in that the direct target genes of miR-3656 were confirmed using dual-luciferase reporter assay; moreover, the effects of miR-3656 on proliferation, migration, apoptosis, and microvascular rarefaction of HUVECs were investigated using MTS kit, wound-healing assay, FITC Annexin V apoptosis detection kit, and tube formation assay, correspondingly. Results Circulating miR-3656 was upregulated in patients with hypertension. MiR-3656 suppressed the proliferation, migration, and angiogenesis of HUVECs, but promoted the apoptosis of HUVECs. In addition, eNOS and ADAMTS13 were direct target genes of miR-3656, and overexpression of eNOS and ADAMTS13 abolished the effect of miR-3656 on HUVECs. Conclusion MiR-3656 is a potential biomarker for hypertension. MiR-3656 is involved in endothelial cellular injury implicated in hypertension by targeting eNOS and ADAMTS13.

Background Antiangiogenic therapy has increasingly become an important strategy for the treatment of colorectal cancer. Recent studies have shown that the tumour microenvironment (TME) promotes tumour angiogenesis. Bufalin is an active antitumour compound whose efficacy has been indicated by previous studies. However, there are very few studies on the antiangiogenic effects of bufalin. Methods Herein, human umbilical vein endothelial cell (HUVEC) tube formation, migration and adhesion tests were used to assess angiogenesis in vitro. Western blotting and quantitative PCR were used to detect relevant protein levels and mRNA expression levels. A subcutaneous xenograft tumour model and a hepatic metastasis model were established in mice to investigate the influence of bufalin on angiogenesis mediated by the TME in vivo. Results We found that angiogenesis mediated by cells in the TME was significantly inhibited in the presence of bufalin. The results demonstrated that the proangiogenic genes in HUVECs, such as VEGF, PDGFA, E-selectin and P-selectin, were downregulated by bufalin and that this downregulation was mediated by inhibition of the STAT3 pathway. Overexpression of STAT3 reversed the inhibitory effects of bufalin on angiogenesis. Furthermore, there was little reduction in angiogenesis when bufalin directly acted on the cells in the tumour microenvironment. Conclusion Our findings demonstrate that bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway in vascular endothelial cells, revealing that bufalin may be used as a new antiangiogenic adjuvant therapy medicine to treat colorectal cancer.

Long noncoding RNAs (lncRNAs) have been reported to be associated with the progression of coronary artery disease (CAD). In our previous study, the levels of lncRNA uc003pxg.1 were upregulated in patients with CAD compared with those in control subjects. However, the role and underlying mechanism of the effects of uc003pxg.1 in CAD remain unknown. Therefore, the aim of the present study was to investigate the expression pattern and biological function of uc003pxg.1 in CAD. First, uc003pxg.1 expression levels were assessed in peripheral blood mononuclear cells isolated from patients with CAD by reverse transcription-quantitative (RT-q)PCR. The results demonstrated that the levels of uc003pxg.1 were significantly upregulated (~4.6-fold) in samples from 80 patients with CAD compared with those in 80 healthy subjects. Subsequently, the present study demonstrated that small interfering RNA-mediated uc003pxg.1 knockdown inhibited human umbilical vein endothelial cell (HUVEC) proliferation and migration, which was analyzed using the Cell Counting Kit-8, cell cycle, EdU and Transwell assays. Additionally, the results of RT-qPCR and western blot analyses revealed that uc003pxg.1 regulated the mRNA and protein levels of cyclin D1 and cyclin-dependent kinase. Through high-throughput sequencing and dual-luciferase reporter assays, the present study demonstrated that microRNA (miR)-25-5p was a downstream target of uc003pxg.1. Further experiments verified that uc003pxg.1 regulated HUVEC proliferation and migration via miR-25-5p. The results of the present study may enhance the current understanding of the role of lncRNA uc003pxg.1 in CAD.

Type 2 diabetes mellitus (T2DM) is a growing burden in low-and middle-income countries. Changing lifestyles and lack of physical activity are some of the reasons contributing to this epidemic increase. Co-morbidities associated with T2DM are largely due to the complications which arise as a consequence of endothelial dysfunction. Platelet derived growth factor-alpha (PDGFRA) is a protein responsible for cell proliferation, angiogenesis, migration and invasion. Increased levels of PDGFRA have been reported in T2DM. This study assessed the epigenetic regulation of PDGFRA through microRNAs (miR-181a/b-5p).Using a bioinformatics-based approach, we assessed the binding of miR-181a/b-5p to PDGFRA. Experimentally, this binding was confirmed using a dual luciferase reporter assay. Further, we overexpressed miR-181a/b-5p in Human umbilical vein endothelial cells (HUVECs) and the influence of over-expression on cell proliferation, migration and angiogenesis was assessed using in-vitro approaches. The influence of miR-181a/b-5p over expression on cellular apoptosis was ascertained using a TUNEL assay with concomitant changes being observed in the levels of Bcl-2 and cleaved Caspase-3.In HUVECs, PDGFRA is a direct target for miR-181a/b-5p. Over expression of miR-181a/b-5p decreased cellular proliferation, migration, invasion, and tube formation-a surrogate marker for angiogenesis. miR-181a/b-5p may be used as a therapeutic intervention to restrict uncontrolled levels of PDGFRA and thereby rescue the phenotypes of increased cell proliferation, migration, invasion and tube formation. miR-181a/b negatively regulates PDGFRA levels. Significance of the study: T2DM and its associated complications emerge from endothelial dysfunction. The associated phenotypes are regulated by a number of proteins, one such member being, PDGFRA. PDGFRA is in turn regulated by miR-181a/b-5p. Complementation with miR-181a/b-5p resulted in reversion of phenotypes. Thus, miR-181a/b-5p-mediated suppression of PDGFRA may be used as a therapeutic intervention in the management of type 2 diabetes.

BACKGROUND Ticagrelor is the first oral anti-platelet agent which has direct anti-platelet aggregation effect by combining with ADP P2Y12 receptors in platelets. It has been approved to reduce the incidence of thrombus cardiovascular events in acute coronary syndrome patients. However, the effects of ticagrelor on endothelial apoptosis have not been investigated. MATERIAL AND METHODS Oxidized low-density lipoprotein (ox-LDL) was used to establish a human umbilical vein endothelial cell (HUVEC) apoptosis model. To investigate the effects of ticagrelor on endothelial apoptosis, the HUVECs were treated with different dose of ticagrelor. Apoptosis rates of HUVECs was evaluated by flow cytometry, and the expression levels of Akt, p-Akt, Bcl-2, Bax, caspase-3, endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) concentration were assessed. RESULTS After treatment with 50 ug/mL ox-LDL or 100 ug/mL ox-LDL, we found that the late apoptosis and necrosis rate and the expression levels of Bax and caspase-3 were significantly increased in HUVECs, whereas the expression levels of Akt, p-Akt, Bcl-2, eNOS, and NO were significantly decreased. Ticagrelor restored the apoptosis rate of ox-LDL-induced HUVECs in a dose-dependent manner. In addition, compared with ox-LDL group, ticagrelor treatment significantly increased the expression levels of Akt, p-Akt, Bcl-2, eNOS, and NO concentration, and significantly decreased the expression levels of Bax and caspase-3. CONCLUSIONS We found that ox-LDL induced significant apoptosis and necrosis in our model, which was dose-dependently improved by ticagrelor. These changes might be explained by alterations in apoptosis and antioxidant pathways.

The biological effects of magnetic fields are a research hotspot in the field of biomedical engineering. In this study, we further investigated the effects of a rotating magnetic field (RMF; 0.2 T, 4 Hz) on the growth of human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. The results showed that RMF exposure prolonged the lifespan of C. elegans and slowed the aging of HUVECs. RMF treatment of HUVECs showed that activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) was associated with decreased mitochondrial membrane potential (MMP) due to increased intracellular Ca2+ concentrations induced by endoplasmic reticulum stress in anti-aging mechanisms. RMF also promoted the health status of C. elegans by improving activity, reducing age-related pigment accumulation, delaying Aβ-induced paralysis and increasing resistance to heat and oxidative stress. The prolonged lifespan of C. elegans was associated with decreased levels of daf-16 which related to the insulin/insulin-like growth factor signaling pathway (IIS) activity and reactive oxygen species (ROS), whereas the heat shock transcription factor-1 (hsf-1) pathway was not involved. Moreover, the level of autophagy was increased after RMF treatment. These findings expand our understanding of the potential mechanisms by which RMF treatment prolongs lifespan.

Atherosclerosis (AS) is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in the occurrence and development of AS. In our study, we attempted to evaluate the role of phosphatase and tensin homolog (PTEN) in endothelial cell apoptosis under oxidized low-density lipoprotein (ox-LDL) stimulation and identify the associated mechanisms. The results of our study demonstrated that ox-LDL induced human umbilical vein endothelial cell (HUVEC) death via mitochondrial apoptosis, as evidenced by reduced mitochondrial potential, increased mitochondria permeability transition pore opening, cellular calcium overload, and caspase-9/-3 activation. In addition, ox-LDL also suppressed cellular energy production via downregulating the mitochondrial respiratory complex. Moreover, ox-LDL impaired HUVECs migration. Western blot analysis showed that PTEN expression was upregulated after exposure to ox-LDL and knockdown of PTEN could attenuate ox-LDL-mediated endothelial cell damage. Furthermore, we found that ox-LDL impaired mitophagy activity, whereas PTEN deletion could improve mitophagic flux and this effect relied on the activity of the AMP-activated protein kinase (AMPK)-cAMP-response element-binding protein (CREB)-Mitofusin-2 (Mfn2) axis. When the AMPK-CREB-Mfn2 pathway was inhibited, PTEN deletion-associated HUVECs protection was significantly reduced, suggesting that the AMPK-CREB-Mfn2-mitophagy axis is required for PTEN deletion-mediated endothelial cell survival under ox-LDL. Taken together, our results indicate that ox-LDL-induced endothelial cell damage is associated with PTEN overexpression, and inhibition of PTEN could promote endothelial survival via activating the AMPK-CREB-Mfn2-mitophagy signaling pathway.

Chronic critical limb ischemia (CLI) represents a clinical end stage of peripheral arterial disease. Many CLI patients are ineligible for conventional revascularization therapies; thus, it is urgent to explore an alternative strategy to rescue the ischemic limb. Recent stem cell studies have greatly developed the field of therapeutic angiogenesis, which aims to significantly improve the limb blood supply. In our study, bone marrow mesenchymal stem cells (BMMSCs) served as the control to evaluate the function of umbilical cord mesenchymal stem cells (UCMSCs) in enhancing angiogenesis. We compared gene expression between BMMSCs and UCMSCs, and a bioinformatics analysis indicated that both UCMSCs and BMMSCs could stimulate angiogenesis and angiogenesis-related factors were upregulated in UCMSCs. In vitro assays indicated that both BMMSCs and UCMSCs promoted human umbilical vein endothelial cell proliferation, migration, and tube formation, and the effects of UCMSCs were more obvious. Consistent with in vitro results, both UCMSCs and BMMSCs improved the limb blood supply in a mouse model of hind limb ischemia, in which UCMSCs promoted angiogenesis more significantly. Finally, we found that activation of ERK and PI3K-Akt pathways might be the mechanism by which UCMSCs promote angiogenesis. These results indicate that UCMSCs play an important role in therapeutic angiogenesis to improve limb blood perfusion.

Diabetes mellitus (DM) is a chronic, multifactorial metabolic disease whereby insulin deficiency or resistance results in hyperglycemia. A sustained high glucose environment results in inflammation and endothelial cell dysfunction. However, the underlying mechanisms are still not entirely clear. Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in diverse biological processes, including DM. Previous studies have found that hsa_circ_0068087 is increased in DM patients. In order to identify whether hsa_circ_0068087 plays a role in high glucose (HG)-induced inflammation and endothelial cell dysfunction Human Umbilical Vein Endothelial Cell (HUVECs), quantitative reverse transcription PCR (qRTPCR), tube formation assay, enzyme-linked immunosorbent assay (ELISA) and bifluorescein reporter experiments were employed in this study. The results showed that the expression of hsa_circ_0068087 was upregulated in HUVECs following increases in glucose. Knockdown of hsa_circ_0068087 suppressed HG-induced HUVEC dysfunction and inflammation by suppression of the TLR4/NF-κB/NLRP3 inflammasome signaling pathway. Downregulation of miR-197 reversed hsa_circ_0068087 silence-induced HUVEC dysfunction and inflammation in the HG condition. It was found that TLR4 was the target of miR-197 and that overexpression of TLR4 ameliorated miR-197-induced HUVEC dysfunction and inhibited inflammation in the HG condition. Bifluorescein report experiments confirmed that miR-197 is a potential target of hsa_circ_0068087 and that TLR4 is a potential miR-197 target. Taken together, these results suggest that downregulation of hsa_circ_0068087 ameliorates TLR4/NF-κB/NLRP3 inflammasome-mediated inflammation and endothelial cell dysfunction in the high glucose condition by sponging miR-197.

Platelet-derived microparticles (PMPs) are a type of microparticle budding from platelets undergoing activation or apoptosis in many autoimmune diseases, including antiphospholipid antibody syndrome (APS). PMPs may also contribute to recurrent miscarriage, although the exact mechanism is unclear. The aim of this study was to determine the potential biological mechanism by which abnormal PMP activation may affect recurrent miscarriage. PMPs were counted by fluorescence-activated cell sorting (FACS) and compared between the healthy control (HC) and recurrent miscarriage/APS groups. Different effects of PMPs isolated by FACS from patients with recurrent miscarriage/APS and HCs were explored. Capillary electrophoresis immunoquantification, RT-qPCR, Luminex xMAP and immunofluorescence staining were performed to investigate all these different effects of PMPs. We found that the difference in the counts of PMP was not significant. However the expression of the inflammatory cytokine tumour necrosis factor-α (TNF-α) and the adhesion molecules intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were increased by PMPs derived from the recurrent miscarriage/APS group. PMPs isolated from patients with recurrent miscarriage/APS also more potently stimulated monocyte recruitment, inhibited angiogenesis and promoted human umbilical vein endothelial cell (HUVEC) apoptosis, in comparison to PMPs from HCs matched for gestational week. Moreover, PMPs could be ternalized by HTR-8/SVneo cells and could increase apoptosis of these cells and decrease trophoblastic invasion and migration. To supplement our work, the limited sample size needs to be increased, and further in-vivo work is necessary. Findings from this study indicate that abnormal activation of PMPs contributes to recurrent miscarriage/APS progression and provides potential therapeutic targets.

Summary There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10–100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs., Highlights • Development of a functional 3D in vitro blood-brain barrier model • Evidence showing that perfusion stabilized barrier integrity for up to 21 days • Junctional analysis shows patterns supporting barrier formation in channels • Cells exhibit stable efflux transporter activity for 3 weeks in perfused channels, Faley et al. present a 3D in vitro model of the blood-brain barrier constructed using induced pluripotent stem cell-derived human brain microvascular endothelial cells cultured in 3D channels in gelatin hydrogels. The model displays robust tight junction protein expression, restricted paracellular permeability, and active efflux transporter activity, and represents an important step toward a representative human in vitro neurovascular model.

Objective Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand–receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. Methods In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. Results CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. Conclusion These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis., Highlights • Long-acting FKN-Fc reduced atherosclerosis in LDLR KO mice in a prevention model. • FKN-Fc administration accelerated diet-induced regression of established atherosclerosis. • Monocyte adhesion in vitro and in vivo is reduced in the presence of FKN-Fc.

Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from l-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2′,7′-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400 W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

Jiang Cao,1,* Li Li,2,* Xiao Han,1 Hai Cheng,1 Wei Chen,1 Kunming Qi,1 Chong Chen,1 Qingyun Wu,1 Mingshan Niu,1 Lingyu Zeng,1 Kailin Xu1 1Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou 221002, China; 2Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou 221002, China *These authors contributed equally to this work Background: miR-302 cluster has been reported as a tumor suppressor in many human cancers; yet, its function in chronic myeloid leukemia (CML) tumorigenesis remains largely unclear. The study was aimed to explore the functional roles of miR-302 cluster in CML progression. Materials and methods: Quantitative reverse transcriptase PCR and Western blot were performed to evaluate miR-302 cluster and vascular endothelial growth factor A (VEGFA) expression levels. Cell Counting Kit-8 assay, colony formation assay and human umbilical vein endothelial cell line capillary tube formation were used to determine the influence of miR-302 cluster on the growth and angiogenesis of K562 cells, respectively. Luciferase reporter assay was employed to confirm the direct target interaction between miR-302 cluster and VEGFA. Results: This study demonstrated that miR-302 cluster was frequently downregulated in CML samples and cell lines and high level of miR-302 cluster was significantly associated with good prognosis of CML patients. Compared with miRNA negative control, miR-302 cluster mimics obviously suppressed cell growth, colony formation and angiogenesis. Further studies revealed that VEGFA was a direct target gene of miR-302 cluster. Moreover, overexpression of VEGFA dramatically abated the inhibition of miR-302 cluster on cell growth and angiogenesis. Conclusion: The present study, for the first time, identified miR-302 cluster as a tumor suppressor, and overexpression of miR-302 cluster inhibited growth and angiogenesis in K562 cells. miR-302 cluster may be a potential therapeutic target in CML to develop the adjuvant antiangiogenic therapy based on VEGFA. Keywords: chronic myeloid leukemia, angiogenesis, miR-302, VEGFA

Transforming growth factor beta (TGFβ) signalling regulates extracellular matrix accumulation known to be essential for the pathogenesis of renal fibrosis; latent transforming growth factor beta binding protein 4 (LTBP4) is an important regulator of TGFβ activity. To date, the regulation of LTBP4 in renal fibrosis remains unknown. Herein, we report that LTBP4 is upregulated in patients with chronic kidney disease and fibrotic mice kidneys created by unilateral ureteral obstruction (UUO). Mice lacking the short LTBP4 isoform (Ltbp4S−/−) exhibited aggravated tubular interstitial fibrosis (TIF) after UUO, indicating that LTBP4 potentially protects against TIF. Transcriptomic analysis of human proximal tubule cells overexpressing LTBP4 revealed that LTBP4 influences angiogenic pathways; moreover, these cells preserved better mitochondrial respiratory functions and expressed higher vascular endothelial growth factor A (VEGFA) compared to wild-type cells under hypoxia. Results of the tube formation assay revealed that additional LTBP4 in human umbilical vein endothelial cell supernatant stimulates angiogenesis with upregulated vascular endothelial growth factor receptors (VEGFRs). In vivo, aberrant angiogenesis, abnormal mitochondrial morphology and enhanced oxidative stress were observed in Ltbp4S−/− mice after UUO. These results reveal novel molecular functions of LTBP4 stimulating angiogenesis and potentially impacting mitochondrial structure and function. Collectively, our findings indicate that LTBP4 protects against disease progression and may be of therapeutic use in renal fibrosis.

Background Maintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels. Methods We utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling. Results Western blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs. Conclusions This study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.

Silver nanoparticles (NPs) have attracted a considerable interest in the field of cancer research due to their potential utility in cancer therapy. In the present study, we developed bovine serum albumin (BSA)-coated silver NPs (BSA-Silver NPs) and characterized in vitro multimodal therapeutic activities of NPs for the treatment of skin cancer. BSA-Silver NPs were synthesized by a single-step reduction process, and the successful preparation was verified through a list of physical characterizations, including transmission electron microscopy (TEM), ultraviolet-visible (UV–VIS) light spectroscopy, dynamic light scattering (DLS), and Fourier transform infrared (FT-IR) spectroscopy. The synthesized BSA-Silver NPs showed marked cytocidal effects on B16F10 melanoma cells, which was likely caused by oxidative stress. BSA-Silver NPs also elicited significant anti-angiogenic effects on HUVEC (human umbilical vein endothelial cell) by inhibiting their proliferation, migration, and tube formation. Moreover, BSA-Silver NPs showed a considerable light-to-heat conversion ability, suggesting their utility as photothermal agents. Overall, our findings suggest that BSA-Silver NPs may be promising candidates for the multimodal therapy of skin cancer.

Deubiquitinases (DUBs) have important biological functions, but their roles in breast cancer metastasis are not completely clear. In this study, through screening a series of DUBs related to breast cancer distant metastasis-free survival (DMFS) in the Kaplan-Meier Plotter database, we identified ubiquitin-specific protease 12 (USP12) as a key deubiquitinating enzyme for breast cancer metastasis. We confirmed this via an orthotopic mouse lung metastasis model. We revealed that the DMFS of breast cancer patients with high USP12 was worse than that of others. Knockdown of USP12 decreased the lung metastasis ability of 4T1 cells, while USP12 overexpression increased the lung metastasis ability of these cells in vivo. Furthermore, our results showed that the supernatant from USP12-overexpressing breast cancer cells could promote angiogenesis according to human umbilical vein endothelial cell (HUVEC) migration and tube formation assays. Subsequently, we identified midkine (MDK) as one of its substrates. USP12 could directly interact with MDK, decrease its polyubiquitination and increase its protein stability in cells. Overexpression of MDK rescued the loss of angiogenesis ability mediated by knockdown of USP12 in breast cancer cells in vitro and in vivo. There was a strong positive relationship between USP12 and MDK protein expression in clinical breast cancer samples. Consistent with the pattern for USP12, high MDK expression predicted lower DMFS and overall survival (OS) in breast cancer. Collectively, our study identified that USP12 is responsible for deubiquitinating and stabilizing MDK and leads to metastasis by promoting angiogenesis. Therefore, the USP12–MDK axis could serve as a potential target for the therapeutic treatment of breast cancer metastasis.

Thin endometrium is a primary cause of failed embryo transfer, resulting in long-term infertility and negative family outcomes. While hormonal treatments have greatly improved fertility results for some women, these responses remain unsatisfactory due to damage and infection of the complex endometrial microenvironment. In this study, a multifunctional microenvironment-protected exosome-hydrogel is designed for facilitating endometrial regeneration and fertility restoration via in situ microinjection and endometrial regeneration. This exosome hydrogel is formulated via Ag+ -S dynamic coordination and fusion with adipose stem cell-derived exosomes (ADSC-exo), yielding an injectable preparation that is sufficient to mitigate infection risk while also possessing the antigenic contents and paracrine signaling activity of the ADSC source cells, enabling regeneration of the endometrial microenvironment. In vitro, this exosome-hydrogel exerts an outstanding neovascularization-promoting effect, increased human umbilical vein endothelial cell proliferation and tube formation for 1.87 and 2.2 folds. In vivo, microenvironment-protected exosome-hydrogel also reveals to promote neovascularization and tissue regeneration while suppressing local tissue fibrosis. Importantly, regenerated endometrial tissue is more receptive to give embryos and birth to a healthy newborn. This microenvironment-protected exosome-hydrogel system offers a convenient, safe, and noninvasive approach for repairing thin endometrium and fertility restoration.

Background Recent studies have suggested a crucial role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in ovarian cancer treatment. We, therefore, set out to explore the mechanism through which MSC-derived EVs delivered microRNA-424 (miR-424) to influence the development of ovarian cancer. Methods Bioinformatics analyses were first performed to screen ovarian cancer-related differentially expressed genes and to predict regulatory miRNAs. Then, dual-luciferase reporter gene assay was carried out to verify the relationship between miR-424 and MYB. Subsequently, the characterized MSCs and isolated EVs were co-cultured with ovarian cancer cells, followed by determination of the expression patterns of miR-424, MYB, vascular endothelial growth factor (VEGF), and VEGF receptor (VEGFR), respectively. In addition, the effects of EVs-delivered miR-424 on the proliferation, migration, invasion and tube formation of ovarian cancer cells were assessed using gain- and loss-of-function approaches. Lastly, tumor xenograft was induced in nude mice to illustrate the influence of EVs-loaded miR-424 on ovarian cancer in vivo. Results Our data exhibited that MYB was highly-expressed and miR-424 was poorly-expressed in ovarian cancer. More importantly, MYB was identified as a target gene of miR-424. Additionally, the transfer of miR-424 by MSC-derived EVs was found to repress the proliferation, migration, and invasion of ovarian cancer cells, with a reduction in the expressions of VEGF and VEGFR. Furthermore, MSC-derived EVs over-expressing miR-424 could inhibit the proliferation, migration, and tube formation of human umbilical vein endothelial cells, and also suppressed tumorigenesis and angiogenesis of ovarian tumors in vivo. Conclusion Collectively, our findings indicate that MSC-derived EVs transfer miR-424 to down-regulate MYB, which ultimately led to the inhibition of the tumorigenesis and angiogenesis of ovarian cancer. Hence, this study offers a potential prognostic marker and a therapeutic target for ovarian cancer.

Scope Pro‐inflammatory stimuli such as hyperglycemia and cytokines have been shown to negatively affect endothelial cell functions. The aim of this study is to assess the potential of quercetin and its human metabolites to overcome the deleterious effects of hyperglycemic or inflammatory conditions on the vascular endothelium by modulating endothelial cell metabolism. Methods and results A metabolomics approach enabled identification and quantification of 27 human umbilical vein endothelial cell (HUVEC) metabolites. Treatment of HUVECs with high‐glucose concentrations causes significant increases in lactate and glutamate concentrations. Quercetin inhibits glucose‐induced increases in lactate and adenosine 5′‐triphosphate (ATP) and also increased inosine concentrations. Tumor necrosis factor α‐treatment (TNFα) of HUVECs causes increases in asparagine and decreases in aspartate concentrations. Co‐treatment with quercetin reduces pyruvate concentrations compared to TNFα‐only treated controls. Subsequently, it was shown that quercetin and its HUVEC phase‐2 conjugates inhibit adenosine deaminase, xanthine oxidase and 5′nucleotidase (CD73) but not ectonucleoside triphosphate diphosphohydrolase‐1 (CD39) or purine nucleoside phosphorylase activities. Conclusion Quercetin was shown to alter the balance of HUVEC metabolites towards a less inflamed phenotype, both alone and in the presence of pro‐inflammatory stimuli. These changes are consistent with the inhibition of particular enzymes involved in purine metabolism by quercetin and its HUVEC metabolites., Pro‐inflammatory stimuli such as hyperglycemia and cytokines have been shown to negatively affect endothelial cell functions. In the present study, quercetin is shown to alter the balance of HUVEC metabolites towards a less inflamed phenotype, both alone and in the presence of pro‐inflammatory stimuli. These changes are consistent with inhibition of particular enzymes involved in purine metabolism by quercetin and its HUVEC metabolites.

Background Deep vein thrombosis (DVT) is an early postoperative complication. Thrombosis formation, which is potentially life-threatening, seriously affects the rehabilitation of patients after surgery. We aimed to establish a C57 mouse model of DVT and to examine the changes in the expression of Kruppel-like factor 15 (KLF15) and endothelial nitric oxide synthase (eNOS) in venous wall tissues, and we also investigated the regulatory relationship of KLF15 and eNOS in the thrombin-induced human umbilical vein endothelial cell (HUVEC) injury cell model. Methods The DVT model was established using the inferior vena cava (IVC) stenosis method. The expression levels of KLF15 and eNOS were analyzed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In cell experiments, the expression of KLF15 and eNOS was analyzed in the model of thrombin-induced HUVEC injury with KLF15 siRNA. Results Compared to the control and sham-operated groups, KLF15 in the DVT group was upregulated, while eNOS was downregulated. The results of cell experiments revealed that KLF15 was downregulated in the thrombin+KLF15 siRNA group compared with the thrombin group. Meanwhile, eNOS was upregulated in the thrombin+KLF15 siRNA group compared with the thrombin group. These findings suggested that KLF15 regulated the expression of eNOS in the DVT model. Conclusions We successfully constructed a DVT mouse model. In the early stage of DVT formation, KLF15 regulated the expression and inhibited the antithrombotic effect of eNOS, resulting in thrombi formation.

Glioblastoma (GBM) is amongst the deadliest human cancers and is characterized by high levels of vascularisation. Angiogenesis is highly dynamic during brain development and almost quiescent in the adult brain, but is reactivated in vascular-dependent CNS pathologies such as brain tumors. Nucleolin (NCL) is a known regulator of cell proliferation and angiogenesis, but its roles on physiological and pathological brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit (NVU) in human fetal brains and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial- and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression in tumor- and blood vessel cells correlated with glioma malignancy in vivo. In culture, siRNA-mediated NCL knockdown reduced human umbilical vein endothelial cell (HUVEC) sprouting angiogenesis, proliferation and filopodia extension, and reduced glucose metabolism. Mechanistically, RNA sequencing of Nucleolin knockdown in HUVECs revealed a putative p53-TIGAR-HK2 regulation of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that positively regulates sprouting angiogenesis and endothelial metabolism. Our findings have important implications in therapeutic targeting of glioma.

Background As a large capillary network, the human placenta plays an important role throughout pregnancy. Placental vascular development is complex and delicate and involves many types of placental cells, such as trophoblasts, and mesenchymal stem cells. There has been no systematic, comparative study on the roles of these two groups of placental cells and the whole placental tissue in the placental angiogenesis. In this study, primary cytotrophoblasts (CTBs) from early pregnancy and primary human placenta-derived mesenchymal stem cells (hPDMSCs) from different stages of pregnancy were selected as the cell research objects, and full-term placental tissue was selected as the tissue research object to detect the effects of their conditioned medium (CM) on human umbilical vein endothelial cell (HUVEC) angiogenesis. Methods We successfully isolated primary hPDMSCs and CTBs, collected CM from these placental cells and sub-cultured placental tissue, and then evaluated the effects of the CM on a series of angiogenic processes in HUVECs in vitro. Furthermore, we measured the levels of angiogenic factors in the CM of placental cells or tissue by an angiogenesis antibody array. Results The results showed that not only placental cells but also sub-cultured placental tissue, to some extent, promoted HUVEC angiogenesis in vitro by promoting proliferation, adhesion, migration, invasion, and tube formation. We also found that primary placental cells in early pregnancy, whether CTBs or hPDMSCs, played more significant roles than those in full-term pregnancy. Placental cell-derived CM collected at 24 h or 48 h had the best effect, and sub-cultured placental tissue-derived CM collected at 7 days had the best effect among all the different time points. The semiquantitative angiogenesis antibody array showed that 18 of the 43 angiogenic factors had obvious spots in placental cell-derived CM or sub-cultured placental tissue-derived CM, and the levels of 5 factors (including CXCL-5, GRO, IL-6, IL-8, and MCP-1) were the highest in sub-cultured placental tissue-derived CM. Conclusions CM obtained from placental cells (primary CTBs or hPDMSCs) or sub-cultured placental tissue contained proangiogenic factors and promoted HUVEC angiogenesis in vitro. Therefore, our research is helpful to better understand placental angiogenesis regulation and provides theoretical support for the clinical application of placental components, especially sub-cultured placental tissue-derived CM, in vascular tissue engineering and clinical treatments.

Sonic hedgehog (Shh) pathway disruption causes craniofacial malformations including orofacial clefts (OFCs) of the lip and palate. In normal craniofacial morphogenesis, Shh signals to multipotent cranial neural crest cells (cNCCs) and was recently discovered to regulate the angiogenic transcriptome, including expression markers of perivascular cells and pericytes. The mural cells of microvasculature, pericytes in the brain and face differentiate from cNCCs, but their role in facial development is not known. Here, we examined microvascular morphogenesis in a mouse model of Shh pathway antagonist-induced cleft lip and the impact of cNCC-specific Shh pathway activation in a cNCC-endothelial cell co-culture system. During cleft pathogenesis in vivo, disrupted microvascular morphogenesis localized with attenuated tissue outgrowth in the medial nasal processes that form the upper lip. In vitro, we found that human umbilical vein endothelial cell (HUVEC) cord formation was not affected by direct Shh pathway perturbation. However, in a co-culture system in which cNCCs directly interact with endothelial cells, cNCC-autonomous Shh pathway activity significantly prolonged endothelial cord network stability. Taken together, these findings support the premise that Shh pathway activation in cNCCs promotes pericyte-like function and microvascular stability. In addition to suggesting a previously unrecognized role for Shh signaling in facial development, these studies also identify perivascular differentiation and microvascular morphogenesis as new focuses for understanding normal and abnormal craniofacial development.

Endometrial cancer (EC) is a common gynaecological cancer worldwide. Exosomes, secreted by living cells and detected in various body fluids, can exchange information between organs and compartments to affect cellular functions, such as proliferation, apoptosis, migration and angiogenesis. We hypothesise that plasma exosomal contents are altered during cancer progression and promote cancer growth and angiogenesis by delivering biomolecules to cancer and vascular endothelial cells. In this study, circulating exosomes derived from EC patients and age-matched healthy people were acquired by commercial kits. Cell counting kit-8, Transwell and Matrigel tube formation assays showed that circulating exosomes from EC patients promote EC cell growth and human umbilical vein endothelial cell (HUVEC) angiogenesis. Next, proteomic analysis and ELISA revealed that plasma exosomal lectin galactoside-binding soluble 3 binding protein (LGALS3BP) increased during EC progression. Moreover, to explore the function of exosomal LGALS3BP, we acquired exosomes containing high levels of LGALS3BP by overexpressing LGALS3BP in human embryonic kidney 293 cells, and we demonstrated that highly contained exosomal LGALS3BP contributed to EC cell proliferation and migration and HUVEC functions via the activation of the PI3K/AKT/VEGFA signalling pathway both in vitro and in vivo. Finally, high LGALS3BP expression was observed in human EC tissue, which indicated a poor prognosis. In addition, immunohistochemical analysis of human EC tissues revealed that LGALS3BP expression was correlated with VEGFA expression and blood vessel density. Hence, we proposed that plasma exosomes containing LGALS3BP contributed to EC growth and angiogenesis during EC progression, which also provided a novel perspective on EC diagnosis and prognosis.

Tissue engineering is an emerging and promising concept to replace or cure failing organs, but its clinical translation currently encounters issues due to the inability to quickly produce inexpensive thick tissues, which are necessary for many applications. To circumvent this problem, we postulate that cells secrete the optimal cocktail required to promote angiogenesis when they are placed in physiological conditions where their oxygen supply is reduced. Thus, dermal fibroblasts were cultivated under hypoxia (2% O2) to condition their cell culture medium. The potential of this conditioned medium was tested for human umbilical vein endothelial cell proliferation and for their ability to form capillary-like networks into fibrin gels. The medium conditioned by dermal fibroblasts under hypoxic conditions (DF-Hx) induced a more significant proliferation of endothelial cells compared to medium conditioned by dermal fibroblasts under normoxic conditions (DF-Nx). In essence, doubling time for endothelial cells in DF-Hx was reduced by 10.4% compared to DF-Nx after 1 week of conditioning, and by 20.3% after 2 weeks. The DF-Hx allowed the formation of more extended and more structured capillary-like networks than DF-Nx or commercially available medium, paving the way to further refinements.

β2-Glycoprotein I (β2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to β2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of β2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and β2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to β2-GPI was detected on the surface of HUVECs, and colocalization of MBL with β2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because β2-GPI–mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the β2-GPI/MBL complex may contribute to amplify similar activities of anti–β2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine‐induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3‐kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF‐E2‐related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200‐800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl‐2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L‐NAME but enhanced by Cav‐1 knockdown, and MK‐2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p‐Akt, and eNOS and decreased Cav‐1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine‐induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.

Ubiquitin-proteasome pathways have a crucial role in tumor progression. PSMD4 (Rpn10, 26S proteasome non-ATPase subunit 4), which is a subunit of the regulatory particle, is a major ubiquitin (Ub) receptor of 26S proteasome. PSMD4 overexpression has been observed in colon carcinoma, hepatocellular carcinoma, and breast cancer. In this work, we elucidated the effect of hypoxia on PSMD4 gene expression in prostate cancer cells (PC3). Chemically mimicked hypoxia drastically upregulated PSMD4 gene expression at both mRNA and protein levels. Transient transfection experiments indicated that all promoter fragments were active in PC3 cells. Hypoxia increased transcriptional activity of all PSMD4 promoter constructs. EMSA analysis shows that HIF-1a transcription factor binds to the hypoxia response element (HRE) present within the -98/+52 region of PSMD4 promoter. We also used human umbilical vein endothelial cell (HUVEC) as a different cell model, in which increased PSMD4 expression was seen only at 24 h. The increased expression of the PSMD4 level in the PC3 cell line was not parallel to the expression in hypoxic HUVEC., Balikesir University Scientific Research Projects Units (BAP) 2014/109

Insufficient endothelialization of cardiovascular devices is a high-risk factor for implant failure. Presentation of extracellular matrix (ECM)-derived coatings is a well-known strategy to improve implant integration. However, the complexity of the system is challenging and strategies for applying multifunctionality are required. Here, we engineered mussel-derived surface-binding peptides equipped with integrin (c[RGDfK]) and proteoglycan binding sites (FHRRIKA) for enhanced endothelialization. Surface-binding properties of the platform containing l-3,4-dihydroxyphenylalanine (DOPA) residues were confirmed for hydrophilized polycaprolactone-co-lactide scaffolds as well as for glass and polystyrene. Further, heparin and the heparin-binding angiogenic factors VEGF, FGF-2 and CXCL12 were immobilized onto the peptide in a modular assembly. Presentation of bioactive peptides greatly enhanced human umbilical vein endothelial cell (HUVEC) adhesion and survival under static and fluidic conditions. In subsequent investigations, peptide-heparin-complexes loaded with CXCL12 or VEGF had an additional increasing effect on cell viability, differentiation and migration. Finally, hemocompatibility of the coatings was ensured. This study demonstrates that coatings combining adhesion peptides, glycosaminoglycans and modulators are a versatile tool to convey ECM-inspired multifunctionality to biomaterials and efficiently promote their integration.

The study aimed to investigate whether sulforaphane (SFN) protects against angiotensin II (Ang II)-mediated human umbilical vein endothelial cell (HUVEC) injury. Ang II treatment decreased HUVEC viability, increased cell apoptosis, decreased mitochondria membrane potential (MMP), impaired cytochrome c release, activated caspase 3/9, and induced reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate oxidase activity. Moreover, SFN treatment blunted Ang II-stimulated oxidative stress and mitochondria-related apoptosis in HUVECs. The ROS scavenger N-acetyl-l-cysteine reduced Ang II-induced oxidative stress and apoptosis, indicating that ROS generation is involved in the Ang II-induced mitochondria-mediated apoptotic pathway. SFN induced nuclear factor erythroid 2 (Nrf2) activation and expression in Ang II-stimulated HUVECs. Downregulation of Nrf2 expression by a target-specific siRNA revealed an Nrf2-dependent effect on the SFN-mediated attenuation of Ang II-induced apoptosis in HUVECs. Pretreatment with brusatol, an Nrf2-specific inhibitor, reversed the protective effects of SFN on Ang II-induced HUVEC injury. SFN treatment protected HUVECs from Ang II-induced damage by decreasing oxidative stress and ameliorating mitochondrial injury.

trans fatty acids (TFAs) have been reported to promote vascular diseases mainly by promoting apoptosis and inflammation of vascular endothelial cells. However, it has been reported in recent years that elaidic acid (9t18:1) and vaccenic acid (11t18:1) may have different effects on vascular health. This study investigated the effects of 9t18:1 and 11t18:1 on human umbilical vein endothelial cell (HUVEC) function and the possible mechanism of inflammation by analyzing the changes in the phospholipid composition and the relationship between phospholipase A2 (PLA2) and MAPK pathway. Here we found that the effect of 11t18:1 on cell viability, membrane damage and cellular inflammation was significantly lower than that of 9t18:1 (p < 0.05). And 9t18:1 and 11t18:1 had different effects on phospholipid composition. Both 9t18:1 and 11t18:1 significantly increased the protein expression of PLA2. Moreover, the MAPK pathway regulated the expression of PLA2, inflammatory cytokines and cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) in HUVECs induced by 9t18:1 and 11t18:1. In conclusion, 9t18:1 and 11t18:1 activated the MAPK pathway which regulated the expression of PLA2 to cause inflammation in HUVECs.

The CRISPR/Cas9 system is a versatile tool that enables targeted genome editing in various cell types, including hard-to-transfect endothelial cells. The required crRNA, tracrRNA, and Cas9 protein have mostly been introduced into endothelial cells by viral transduction or plasmid transfection so far. We here describe an effective lipofection-based delivery of pre-complexed crRNA:tracrRNA:Cas9 ribonucleoproteins into human umbilical vein endothelial cells (HUVEC) and immortalized HUVEC (CI-huVEC). Complete inactivation of either CCM1, CCM2, or CCM3 in endothelial cells mimics the situation in cavernous lesions of CCM patients and thus represents a suitable model for future studies.