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A moderate inflammatory response at the early stages of fracture healing is necessary for callus formation. Over-active and continuous inflammation, however, impairs fracture healing and leads to excessive tissue damage. Adequate fracture healing could be promoted through suppression of local over-active immune cells in the fracture site. In the present study, we achieved an enriched concentration of PD-L1 from exosomes (Exos) of a genetically engineered Human Umbilical Vein Endothelial Cell (HUVECs), and demonstrated that exosomes overexpressing PD-L1 specifically bind to PD-1 on the T cell surface, suppressing the activation of T cells. Furthermore, exosomal PD-L1 induced Mesenchymal Stem Cells (MSCs) towards osteogenic differentiation when pre-cultured with T cells. Moreover, embedding of Exos into an injectable hydrogel allowed Exos delivery to the surrounding microenvironment in a time-released manner. Additionally, exosomal PD-L1, embedded in a hydrogel, markedly promoted callus formation and fracture healing in a murine model at the early over-active inflammation phase. Importantly, our results suggested that activation of T cells in the peripheral lymphatic tissues was inhibited after local administration of PD-L1-enriched Exos to the fracture sites, while T cells in distant immune organs such as the spleen were not affected. In summary, this study provides the first example of using PD-L1-enriched Exos for bone fracture repair, and highlights the potential of Hydrogel@Exos systems for bone fracture therapy through immune inhibitory effects.

Background. Vascular endothelial cell injury is not only the initiating factor of cardiovascular and cerebrovascular diseases but also the essence of blood stasis. Levistilide A (LA), a natural component isolated from the traditional Chinese herb, Ligusticum chuanxiong Hort, has traditional effects on improving blood circulation and removing stasis. In this study, the effects and potential mechanisms of LA in the rat model of blood stasis and the mechanism in endothelial cell injury have been explored. Materials and Methods. In this experiment, the effects of LA on the model of acute blood stasis in rats were explored. The blood samples were collected for the measurement of coagulation and hemorheological indices, and the carotid arteries were also excised from rats for hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). In addition, the improvement effects of LA on the H2O2-induced human umbilical vein endothelial cell (HUVEC) injury model were evaluated. And the cell viability detection was conducted by the CCK8 assay, and the pathway-related protein expressions were detected by western blotting. Results. In vivo, compared with the model group, the treatment of LA (10 mg/kg) could reduce the FIB (fibrinogen) content ( P < 0.01 ), increase the INR (international normalized ratio) and PT (prothrombin time) ( P < 0.01 ), and reduce the plasma viscosity ( P < 0.05 ) and whole blood viscosities of low, medium, and high shear rates in the blood of blood stasis model rats ( P < 0.01 ). In vitro, the cell viability in the LA-pretreated group was higher than that of the model group ( P < 0.05 ). The expression levels of PI3K, AKT, and eNOs in the LA-pretreated group were increased ( P < 0.01 ) as compared to the model group. Conclusion. These findings demonstrated that LA has the ability to improve blood hypercoagulation and blood viscosity, and enhance the viability of cells. It is more likely that it exerts a protective effect on the endothelial cell through the PI3K-AKT-eNOs pathway. These results indicate LA will be a potential candidate to cure blood stasis with endothelial cell injury.

Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) co-cultured in vitro are widely used in adipose tissue engineering but exhibit various limitations. Chemokine (C-C motif) ligand 2 (CCL2) has been proved essential during adipogenesis and angiogenesis in vivo. We examined whether adipogenesis and angiogenesis could also be directly promoted by CCL2 in vitro. Cells were cultured with 0, 10, 50, and 100 ng/ml CCL2. The effects of CCL2 on adipogenesis of hADSCs, and lipid accumulation in the positive control group (hADSCs), blank control group (hADSCs + HUVECs), and experimental group (hADSCs + HUVECs + CCL2) in the hADSC and HUVEC direct co-culture system were evaluated by Oil Red O staining. Angiogenesis in the presence of CCL2 was evaluated by Matrigel tube formation assay. Angiogenic- and adipogenic-associated gene and protein expression in the co-culture system were measured by Quantitative Real-time Polymerase Chain Reaction and western blotting, respectively. All concentrations of CCL2 promoted hADSC adipogenic differentiation and HUVEC tube formation (P

Background and aims LncRNA plasmacytoma variant translocation 1 (PVT1) plays a regulatory role in some cardiovascular diseases, but its role in atherosclerosis (AS) remains barely explored. The study aimed to investigate the effects of PVT1 on high fat diet-induced AS and its potential mechanisms. Methods and results ApoE −/− mice were fed with high fat diet for 8 weeks to establish an AS model. Lentiviral vectors containing PVT1 short hairpin RNA (PVT1-shRNA) or NC-shRNA were administered by tail vein injection. Cell viability, apoptosis, inflammatory factor secretion, and cellular oxidative stress were measured to evaluate oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) injury. Dual-luciferase reporter gene and RNA immunoprecipitation assays were used to confirm the interaction between miR-153-3p and PVT1 or growth factor receptor binding protein 2 (GRB2). Atherosclerotic lesions, lipid deposition, and cell apoptosis in aorta were analyzed by H&E, Oil Red O, and TUNEL straining. PVT1 knockdown alleviated ox-LDL-induced inflammation, apoptosis and oxidative stress in HUVECs. PVT1 acted as a sponge of miR-153-3p, and GRB2 was confirmed as a target of miR-153-3p. MiR-153-3p overexpression attenuated the enhanced effects of PVT1 on ox-LDL-induced cell damage. GRB2 overexpression reversed the mitigating effects of miR-153-3p on ox-LDL-caused injury. Inhibiting PVT1 restrained the activation of ERK1/2 and p38 pathway via miR-153-3p/GRB2 axis. Additionally, silencing PVT1 in vivo reduced atherosclerotic plaques, lipid deposition, inflammation, oxidative stress, and apoptosis in AS mice. Conclusion PVT1 knockdown alleviated ox-LDL-induced vascular endothelial cell injury and atherosclerosis through miR-153-3p/GRB2 axis via ERK1/2 and p38 pathway.

Exosomes might mediate the effects of remote ischemic post-conditioning (RIPostC) treatment on vital organs. The present study aimed to explore the role of RNA component of mitochondrial RNA-processing endoribonuclease (RMRP) in the effects of human umbilical vein endothelial cell (HUVEC)-derived exosomes on ischemic injuries in vitro and in vivo. HUVECs were subjected to oxygen-glucose deprivation (OGD) treatment and exosomes were collected OGD-treated human neural cells were incubated with HUVEC-derived exosomes. Changes in cell viability, apoptosis, and RMRP-mediated PI3K/Akt/mTOR pathway activity were detected. The role of RMRP inhibition in the anti-OGD effects of exosomes was further determined by upregulating RMRP expression in human neural cells. The potential RMRP inhibitory factors in exosomes were explored using microarray detection. The effects of exosomes were validated with MCAO mouse models. In OGD neurons incubated with the exosomes, cell viability was improved and cell apoptosis was suppressed. At molecular level, exosomes on downregulated RMRP, p-PI3K, p-Akt, and p-mTOR, while induced eNOS. After the overexpression of RMRP, the cell protective effects of exosomes were counteracted, which was associated with the re-activation of PI3K/Akt/mTOR pathway. Based on the detection of microarray, the induced levels of miR-206 and miR-1-3p by OGD in HVUECs contributed to the RMPR inhibition. Additionally, injection of exosomes restricted infarction area and suppressed RMRP in MCAO mice. Collectively, exosomes from OGD HUVECs could protect neurons against ischemia-induced injuries, and the effects were associated with the suppression of RMRP in neurons via distance transfer of miR-206 and miR-1-3p.

The underlying mechanism of the trans-sutural distraction osteogenesis (TSDO) technique as an effective treatment that improves the symptoms of midfacial hypoplasia syndromes is not clearly understood. Increasing findings in the orthopedics field indicate that macrophages are mechanically sensitive and their phenotypes can respond to mechanical cues. However, how macrophages respond to mechanical stretching and consequently influence osteoblast differentiation of suture-derived stem cells (SuSCs) remains unclear, particularly during the TSDO process. In the present study, we established a TSDO rat model to determine whether and how macrophages were polarized in response to stretching and consequently affected bone regeneration of the suture frontal edge. Notably, after performing immunofluorescence, RNA-sequencing, and micro-computed tomography, it was demonstrated that macrophages are first recruited by various chemokines factors and polarized to the M2 phenotype upon optimal stretching. The latter in turn regulates SuSC activity and facilitates bone regeneration in sutures. Moreover, when the activated M2 macrophages were suppressed by pharmacological manipulation, new bone microarchitecture could rarely be detected under mechanical stretching and the expansion of the sutures was clear. Additionally, macrophages achieved M2 polarization in response to the optimal mechanical stretching (10%, 0.5 Hz) and strongly facilitated SuSC osteogenic differentiation and human umbilical vein endothelial cell angiogenesis using an indirect co-culture system in vitro. Collectively, this study revealed the mechanical stimulation-immune response-bone regeneration axis and clarified at least in part how sutures achieve bone regeneration in response to mechanical force.

Techniques for harvest and delivery of cell sheets have been improving for decades. However, cell sheets with complicated patterns closely related to natural tissue architecture were hardly achieved. Here, we developed an efficient method to culture and harvest cell sheets with complex shape (noted as microtissues) using temperature-responsive hydrogel consisting of expandable polyethylene oxide polymer at low temperature. Firstly, a temperature-responsive hydrogel surface with honeycomb patterns (50 and 100 µm in width) were developed through microcontact printing of polydopamine (PD). The human dermal fibroblasts (HDFBs) and human umbilical vein endothelial cells (HUVECs) spontaneously formed honeycomb-shaped microtissues on the patterned hydrogel surface. The microtissues on the hydrogel were able to be harvested and directly delivered to the desired target through thermal expansion of the hydrogel at 4 °C with an efficiency close to 80% within 10 min which is faster than conventional method based on poly(N-isopropylacrylamide). The microtissues maintained their original honeycomb network and intact structures. Honeycomb-patterned cell sheets also were fabricated through serial seeding of various cell lines, including HDFBs, HUVECs, and human adipose-derived stem cells, in which cells were attached along the honeycomb pattern. The underlying honeycomb patterns in the cell sheets were successfully maintained for 3 days, even after delivery. In addition, patterned cell sheets were successfully delivered in vivo while maintaining an intact structure for 7 days. Together, our findings demonstrate that micropatterned temperature-responsive hydrogel is an efficient method of one-step culturing and delivery of complex microtissues and should prove useful in various tissue engineering applications. Statement of significance Scaffold-free cell delivery techniques, including cell sheet engineering, have been developed for decades. However, there is limited research regarding culture and delivery of microtissues with complex architecture mimicking natural tissue. Herein, we developed a micro-patterned hydrogel platform for the culture and delivery of honeycomb-shaped microtissues. Honeycomb patterns were chemically engineered on the temperature-responsive hydrogel through microcontact printing of polydopamine to selectively allow for human dermal fibroblast or human umbilical vein endothelial cell adhesion. They spontaneously formed honeycomb-shaped microtissues within 24 hr upon cell seeding and directly delivered to various target area including in vivo via thermal expansion of the hydrogel at 4 °C, suggesting that the micro-patterned hydrogel can be an efficient tool for culture and delivery of complex microtissue.

Herein, a novel series of dual histone deacetylase (HDAC) and vascular endothelial growth factor receptor (VEGFR) inhibitors were designed, synthesized and biologically evaluated based on previously reported pazopanib-based HDAC and VEGFR dual inhibitors. Most target compounds showed significant HDAC1, HDAC6 and VEGFR2 inhibition, which contributed to their potent antiproliferative activities against multiple cancer cell lines and significant antiangiogenic potencies in both human umbilical vein endothelial cell (HUVEC) tube formation assays and rat thoracic aorta ring assays. Further HDAC selectivity evaluations indicated that hydroxamic acids 5 and 9e possessed HDAC isoform selectivity profiles similar to that of the approved HDAC inhibitor suberoylanilide hydroxamic acid(SAHA), while hydrazide12 presented an HDAC isoform selectivity profilesimilar to that of the clinical HDAC inhibitor MS-275. The VEGFR inhibition profiles of 5, 9e and 12 were similar to that of the approved VEGFR inhibitor pazopanib. The intracellular target engagements of Compounds 5 and 12 were confirmed by western blot analysis. The metabolic stabilities of 5, 9e and 12 in mouse liver microsomes were inferior to that of pazopanib. These dual HDAC and VEGFR inhibitors provide lead compounds for further structural optimization to obtainpolypharmacological anticancer agents.

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.

Diabetic foot ulcers (DFU) are a vascular complication of diabetes mellitus (DM). It has been confirmed that irisin is closely related to DM. However, the effect of irisin on DFU is obscure and needs further study. After human umbilical vein endothelial cell lines (HUVECs) were treated with different concentrations’ irisin, normal glucose, high glucose (HG), HG plus irisin-high (H) or sh-Notch1, cell biological behaviors, LDH, and VEGFA were detected by cell function experiments. Apoptosis- and Notch pathway-related protein levels were evaluated by Western blot. Irisin has no cytotoxicity, and irisin-H elevated cell viability and inhibited apoptosis and LDH level in HG-induced HUVECs. Meanwhile, irisin-H restored HG-repressed migration and angiogenesis in HUVECs. Irisin-H inhibited apoptosis-related protein levels and promoted VEGFA and Notch pathway-related protein levels in HG-treated HUVECs. Additionally, sh-Notch1 reversed the protective effect of irisin-H in HG-treated HUVECs. Irisin restores HG-induced cell injury and angiogenesis in HUVECs by activating Notch pathway via Notch1.

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.

OBJECTIVES To evaluate the effect of zoledronic acid (ZA) on human umbilical vein endothelial cells (HUVECs) attached to different surfaces. MATERIALS AND METHODS A total of three groups were evaluated in this study: sandblasting and acid etching (SLA) + HUVECs; mechanically polished (MP) + HUVECs; and plastic cell culture plates + HUVECs. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, surface roughness and water contact angle were tested for titanium surface characterisation. ZA was added at different concentrations (0, 1, 10, 50 and 100 μM). Cell adhesion, proliferation, viability, apoptosis and gene expression were evaluated. RESULTS Mechanically polished and SLA surfaces showed negative effects on cell adhesion and proliferation and promoted cell apoptosis with 100 μM ZA (p

A novel umami peptide, IPIPATKT, showed excellent dual dipeptidyl peptidase-IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory activities, the IC50 values were 64 and 265 μM, respectively. Molecular docking displayed that IPIPATKT was docked into the S1 and S2 pockets of ACE, and it was close to the active site pocket of DPP-IV. The insulin-resistant-HepG2 (IR-HepG2) cell model and human umbilical vein endothelial cell (HUVEC) model showed that the peptide significantly increased the content of glucose, the activities of hexokinase, pyruvate kinase, and the concentration of nitric oxide (p < 0.01), while it reduced the content of endothelin-1 (ET-1). IPIPATKT exhibited a hypotensive effect (-23.5 ± 2.2 mmHg) and attenuated the increase in glucose levels in vivo, as demonstrated using spontaneous hypertensive rats (SHRs) and C57BL/6N mice. We reported the in vivo activities of the umami peptide with dual hypertensive and hypoglycemic effects for the first time.

Objective It is unclear whether nucleophosmin (NPM) participates in cardiovascular disease. The present study aimed to investigate the role and underlying mechanisms of NPM in atherosclerosis. Methods Levels and location of NPM in human carotid atherosclerotic plaques and healthy controls were detected by real-time polymerase chain reaction, immunoblots, and immunofluorescence. Atherosclerotic prone ApoE–/– mice were fed with a Western diet for 16 weeks as an in vivo model. Human primary umbilical vein endothelial cells (HUVECs) were cultured as an in vitro model. Results Compared with controls, we found that NPM levels in human carotid atherosclerotic plaques were more than twice as high as in normal arteries, which mainly localized in endothelial cells. In vivo, adenovirus-containing NPM small hairpin RNA attenuated atherosclerotic lesion and promoted plaque stabilization in ApoE–/– mice fed a Western diet by reducing vascular inflammation, maintaining endothelial function, and decreasing macrophage infiltration. Furthermore, NPM knockdown decreased nuclear factor-κB (NF-κB) p65 phosphorylation. In cultured HUVECs, palmitic acid increased the protein levels of NPM and induced the expression of inflammatory cytokines and monocyte adhesion, whereas NPM knockdown attenuated this effect. In HUVECs, NPM protein physically interacted with NF-κB p65 subunit and promoted its nuclear transposition. NPM also increased the transcriptional activity of NF-κB p65 promoter and enhance its binding to target genes, including interleukin-1β, interleukin-6, intercellular adhesion molecule-1, and E-selectin. Conclusions These data provide novel evidence that NPM promotes atherosclerosis by inducing vascular inflammation and endothelial dysfunction through the NF-κB signaling pathway and suggest that NPM may be a promising target for atherosclerosis prevention and treatment.

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.

Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.

Insufficient vascularization of grafts often leads to delayed tissue ingrowth and impaired tissue function in tissue engineering. The surface topography of grafts plays critical roles in angiogenesis. In the present study, we prepared silk fibroin (SF)-based microtopography films with the number of convex dots ranging from 37 to 4835/mm2. The convex dot-featured topography surfaces were characterized by scanning electron microscopy, a Profilm3D optical profilometer, atomic force microscopy, and a contact angle goniometer. The effect of microtopographic films on the proliferation, adhesion, and expression of angiogenic factors of human umbilical vein endothelial cells (HUVECs) was investigated. Our results demonstrated that the SF film surface with 2899 convex dots/mm2 significantly enhanced adhesion, viability, and levels of vascular endothelial growth factors and basic fibroblast growth factors of HUVECs and significantly downregulated the level of α-SMA in human aortic smooth muscle cells, indicating that the microtopographic films could promote angiogenesis. Furthermore, in vitro results showed that HUVEC proliferation was positively correlated with yes-associated protein (YAP) activation, suggesting that the enhanced angiogenesis was mediated via the YAP pathway. Finally, mice subcutaneous embedding model results indicated that the SF film surface with 2899 convex dots/mm2 could significantly enhance angiogenesis in vivo. Altogether, our results showed that the SF film surface with 2899 convex dots/mm2 promoted the angiogenesis of HUVECs and offered a novel angiogenesis-promoting strategy of implant surface design for tissue engineering.

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 Circular RNAs (circRNAs) are aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC). In the current study, we investigated how circRNA_000684 affected the progression of PDAC, and how it regulated kruppel-like factor 5 (KLF5) and microRNA (miR)-145. Methods Differentially expressed circRNAs, miRs and genes related to PDAC as well as their targeting relationship were predicted using bioinformatics analyses. Binding relationships among circRNA_000684, miR-145 and KLF5 were verified using dual-luciferase reporter gene assay, RIP and RNA pull-down assay, respectively. The effects of circRNA_000684, miR-145, KLF5 on the malignant phenotypes of PDAC cells and human umbilical vein endothelial cell (HUVEC) angiogenesis were assessed using loss- and gain-of function experiments by CCK-8 assay, scratch test, Transwell and tube formation assays. RT-qPCR and Western blot analysis were used to determine MCM2, MMP2 and MMP9 and VEGFA expression. In addition, the roles of circRNA_000684, miR-145, and KLF5 in tumor growth were validated through in vivo experiments. Results Expression of CircRNA_000684 and KLF5 was upregulated, whereas miR-145 expression was downregulated in PDAC tissues and cells. CircRNA_000684 repression or miR-145 elevation inhibited the proliferation, invasion and migration of PDAC cells and HUVEC angiogenesis, as evidenced by lower levels of MCM2, MMP2 and MMP9 and VEGFA. CircRNA_000684 negatively regulated miR-145 expression, while miR-145 negatively regulated KLF5. In-vivo, circRNA_000684 elevation or miR-145 repression promoted tumor growth. Conclusion Taken together, the present study provided evidence clarifying that circRNA_000684 could downregulate miR-145 expression and elevate KLF5 to promote the progression of PDAC.

Introduction: For the regeneration of large volume tissue defects, the interaction between angiogenesis and osteogenesis is a crucial prerequisite. The surgically induced angiogenesis by means of a...

Objective Aims of this study were to (1) compare anti-proliferative activity between aqueous and ethanol Kaempferia parviflora (KP) extracts in both cancer (Human urinary bladder cancer cell, T24) and normal cell lines (Human umbilical vein endothelial cell, HUVEC). (2) confirm selective cytotoxicity of ethanol KP extract to normal and different cancer cell lines (3) investigate its cellular mechanism through p53 and SIRT1 gene expression. Methods Phytochemical difference between aqueous and ethanol extract was determined by thin layer chromatography (TLC). Screening for cytotoxic activity in human cell lines was performed by cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent. P53 and SIRT1 gene expression were quantified using RT-PCR. Results Results from the cell viability assay were shown as follows: (1) ethanol extract possessed higher toxicity to cancerous cells than aqueous extract (2) ethanol extract exhibited higher cytotoxicity to cancerous cells than normal cells (3) ethanol extract also showed cytotoxicity, with different levels, to three prostate cancer cell lines varying in aggressiveness. (4) ethanol KP extract induced cell death in T24 via p53 gene expression and prolonged cell survival in HUVEC through SIRT1 gene expression. Conclusion These findings implied that ethanol KP extract might possibly be an alternative for cancer adjuvant therapy through the mechanism of selective p53 and SIRT1 gene expression.

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.

Bacterial nanocellulose (BNC) is a promising material for small-caliber artificial blood vessels, although promoting its anticoagulant properties with more rapid endothelialization would improve long-term patency. Silk fibroin nanoparticles (SFNP) were introduced into the luminal wall surface of BNC conduits both with and without heparin (Hep) through pressurization followed by fixation. Hep was introduced in two ways: (1) embedded within SF nanoparticles to form SF-HepNPs for construction of the BNC-SF-HepNP conduit and (2) chemically grafted onto BNC and BNC-SFNP to form BNC-Hep and BNC-SFNP-Hep conduits. Fourier transform infrared spectroscopy confirmed the formation of SF-HepNPs, although they did not incorporate into the fibrillar network due to their large size. Hep was successfully grafted onto BNC and BNC-SFNP, verified by toluidine blue staining. The hemocompatibility and cytocompatibility of the five samples (BNC, BNC-SFNP, BNC-SF-HepNP, BNC-Hep, and BNC-SFNP-Hep conduits) were compared in vitro. The heparinized BNC-Hep and BNC-SFNP-Hep conduits improved the anticoagulant properties, and BNC-SFNP-Hep promoted human umbilical vein endothelial cell proliferation but also controlled excessive human arterial smooth muscle cell proliferation, assisting rapid endothelialization and improving lumen patency. No significant inflammatory reaction or material degradation was observed after subcutaneous implantation for 4 weeks. Autogenous tissues were observed around the conduits, and cells infiltrated into the edges of all samples, the BNC-SFNP conduit causing the deepest infiltration, providing an appropriate microenvironment for angiogenesis when used in small-caliber blood vessel applications. Few inflammatory cells were found around the BNC-Hep and BNC-SFNP-Hep conduits. Thus, the anticoagulant properties of the BNC-SFNP-Hep conduit and its stimulation of endothelialization suggest that it has great potential in clinical applications as a small-caliber artificial blood vessel.

In a randomized trial, higher all-cause and cardiovascular mortality was observed in treatment with febuxostat than with allopurinol in patients with coexisting gout and serious cardiovascular conditions. In this study, we focus on an intervention of febuxostat or allopurinol as an anti-inflammatory treatment to reduce the transcription of nuclear factor-kappa B (NF-κB) and production of relevant inflammatory factors. We evaluated the effect of febuxostat on vascular cell adhesion protein 1 (VCAM-1) induction in cultured human umbilical vein endothelial cells (HUVECs). Cells were exposed to tumor necrosis factor (TNF)-α (10 ng/mL) treatment for 24 h. Febuxostat or allopurinol (0.1–100 μM) was added to the bath medium 15 min before TNF-α treatment. VCAM-1 levels in HUVECs increased after 24-h TNF-α treatment (n = 4). Febuxostat and allopurinol significantly suppressed VCAM-1 induced by treatment with TNF-α in a dose-dependent manner (p < 0.05, n = 4). Furthermore, these drugs suppressed the NF-κB protein levels in the nucleus 4 h after TNF-α treatment (n = 3 or 4). Our results suggest that TNF-α induces VCAM-1 production via NF-κB, which can be blocked by febuxostat or allopurinol. The effect of febuxostat treatment on cardiovascular events may be associated with protection against the infiltration of lymphocytes or monocytes through VCAM-1 induction in inflamed endothelial cells such as arterial sclerosis.

Objective To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). Design This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression in vitro; and exploring vessel formation induced by HMGA2 overexpression both in vitro and in vivo. Setting University research laboratory. Patients None. Interventions None. Main Outcome Measures The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and in vitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression in vitro and in vivo. Results Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth in vitro and formed xenografts with increased blood vessels. Conclusions HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

Our previous study confirmed the critical role of miR-125b and vascular endothelial growth factor (VEGF) in burn wound repair., The present study was aimed to identify the role of long noncoding RNAs (lncRNAs) related to the function of miR-125b and VEGF in burn wound repair and the underlying mechanism. First, we found that lncRNA PDK1-AS and VEGFA expression was significantly increased in heat-denatured dermal tissue samples and in human dermal microvascular endothelial cells (HDMECs) and human umbilical vein endothelial cells (HUVECs) after thermal injury. PDK1-AS knockdown significantly inhibited cell viability, cumulative tube length, cell migratory ability, and cell invasion of thermally injured HDMECs and HUVECs. PDK1-AS knockdown decreased VEGFA protein levels in HDMECs and HUVECs. While overexpression of PDK1-AS showed the opposite effects. Online tools prediction and luciferase assay confirmed that miR-125b-5p targeted PDK1-AS and VEGFA 3'-untranslated region. miR-125b-5p inhibition significantly increased VEGFA protein levels and enhanced viability, cumulative tube length, migratory ability, and invasion of HUVECs and HDMECs. Furthermore, the effects of PDK1-AS knockdown on VEGFA protein levels in the two cell lines were partially reversed by miR-125b-5p inhibition. Finally, in the tissue samples, PDK1-AS and VEGFA expression was increased, while miR-125b-5p expression was decreased in heat-denatured dermal tissues; the expression of miR-125b-5p had a negative correlation with PDK1-AS and VEGFA, respectively, and PDK1-AS and VEGFA were positively correlated with each other in tissue samples. In conclusion, PDK1-AS relieves miR-125b-5p-induced inhibition on VEGFA by acting as a endogenous RNA, therefore modulating HDMEC and HUVEC angiogenesis after thermal injury.

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.

Recent evidence suggests that metformin may prevent pre-eclampsia by reverting the angiogenic imbalance in maternal sera. In this study, we investigated effect of metformin on angiogenesis by quantifying tubule formation in a human-based in vitro test with co-culture of human adipose stromal cell (hASC) and human umbilical vein endothelial cell (HUVEC). A total of 20 pregnant women were recruited in the study. Serum samples were obtained from women with early- and late-onset pre-eclampsia and from women with pregnancies complicated by intrauterine growth restriction (IUGR) without pre-eclampsia (N = 5 in each of the three groups). Serum samples from women with healthy pregnancies served as controls (N = 5). The direct effect of metformin on angiogenesis was first assessed without maternal sera. Secondly, we investigated the impact of metformin on angiogenesis in the present of maternal sera. Metformin was used at 5, 50 and 600 µg/ml concentrations. Angiogenic and inflammatory biomarkers in maternal sera were analyzed by immunoassays. When the direct effect of metformin was studied, the two lowest concentrations of metformin did not affect tubule formation (angiogenesis), but the highest concentration inhibited angiogenesis. When metformin was supplemented at therapeutic concentrations of 5 and 50 µg/ml along with serum samples, there was no change in tubule formation in comparison to maternal sera alone. However, strong inhibitory effect on tubule formation was observed in all groups with the highest, non-therapeutic (600 µg/ml), concentration of metformin.

Nerve growth factor (NGF) is the protein responsible for the development and maintenance of sensory skin innervation. Given the role of appropriate innervation in skin healing, NGF has been indicated as a possible prohealing treatment in pathologic conditions characterized by nerve-ending loss, such as chronic ulcers in diabetes; however, its use as a therapeutic agent is limited by its hyperalgesic effect. We tested the effect of topical application of the nonalgogenic NGF derivative hNGFP61S/R100E in two models of skin ulcer induced in dbdb diabetic mice, investigating healing time, skin histology, reinnervation, and angiogenesis using morphologic and molecular approaches. We showed that the topical administration of CHF6467, a recombinant human NGF in which an amino acid substitution (R100E) abolished the hyperalgesic effect usually associated with NGF, accelerated skin repair in experimental wounds (full-excision and pressure-ulcer) induced in diabetic mice (dbdb). CHF6467-induced acceleration of wound healing was accompanied by increased re-epithelization, reinnervation, and revascularization as assessed by histology, immunohistochemistry, and image analysis. Bioinformatic analysis of differentially expressed genes and signaling pathways in the wound tissues showed that protein kinase B-mammalian target of rapamycin was the most regulated pathway. In spite of the transdermal absorption leading to measurable, dose-dependent increases in CHF6467 plasma levels, no systemic thermal or local mechanical hyperalgesia was observed in treated mice. When tested in vitro in human cell lines, CHF6467 stimulated keratinocyte and fibroblast proliferation and tube formation by endothelial cells. Collectively, these results support a possible use of CHF6467 as a prohealing agent in skin lesions in diabetes. SIGNIFICANCE STATEMENT: Topical application of CHF6467 accelerates reinnervation, neoangiogenesis, and wound healing in diabetic mice in both full-thickness skin-excision and pressure-ulcer models through the protein kinase B/mammalian target of rapamycin pathway and does not induce hyperalgesia.

Sphingosine-1 phosphate receptor-1 (S1P1 ) activation maintains endothelial barrier integrity, whereas S1P1 desensitization induces peripheral blood lymphopenia. The latter is exploited in the approval and/or late-stage development of receptor-desensitizing agents targeting the S1P1 receptor in multiple sclerosis, such as siponimod, ozanimod, and ponesimod. SAR247799 is a recently described G protein-biased S1P1 agonist that activates S1P1 without desensitization and thus has endothelial-protective properties in patients without reducing lymphocytes. As SAR247799 demonstrated endothelial-protective effects at sub-lymphocyte-reducing doses, the possibility exists that other S1P1 modulators could also exhibit endothelial-protective properties at lower doses. To explore this possibility, we sought to quantitatively compare the biased properties of SAR247799 with the most advanced clinical molecules targeting S1P1 . In this study, we define the β-arrestin pathway component of the impedance profile following S1P1 activation in a human umbilical vein endothelial cell line (HUVEC) and report quantitative indices of the S1P1 activation-to-desensitization ratio of various clinical molecules. In a label-free impedance assay assessing endothelial barrier integrity and disruption, the mean estimates (95% confidence interval) of the activation-to-desensitization ratios of SAR247799, ponesimod, ozanimod, and siponimod were 114 (91.1-143), 7.66 (3.41-17.2), 6.35 (3.21-12.5), and 0.170 (0.0523-0.555), respectively. Thus, we show that SAR247799 is the most G protein-biased S1P1 agonist currently characterized. This rank order of bias among the most clinically advanced S1P1 modulators provides a new perspective on the relative potential of these clinical molecules for improving endothelial function in patients in relation to their lymphocyte-reducing (desensitization) properties.

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free. Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed. The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. Statement of Significance Cells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

Homocysteine (Hcy) is considered an independent risk factor for various cardiovascular diseases including atherosclerosis which is associated with lipid metabolism, inflammation, and oxidative stress. Results from our previous study suggested that Hcy-induced atherosclerosis could be reversed by Herpud1 knockout which inhibits vascular smooth muscle cell (VSMC) phenotype switching. Here, we aim to investigate more precise mechanisms behind the improvement in Hcy-induced atherosclerosis. Amyloid-β40 (Aβ40), a vital protein in Alzheimer disease (AD), has been regarded as an important component in the atherosclerosis program in recent years due to the biological similarity between AD and atherosclerosis. Thus, we determined to assess the value of Aβ40 in a Herpud1 knockout Hcy-induced atherosclerosis mouse model by measuring Aβ40 expression in tissue and biomarkers of lipid metabolism, inflammation, and oxidative stress in serum. Additionally, since endothelial dysfunction plays a prominent role in atherosclerosis, we tested human umbilical vein endothelial cell (HUVEC) function following Herpud1 silencing in vitro and evaluated JNK/AP1 signaling activation in our models because of its close relationship with Aβ40. As a result, our animal models showed that Herpud1 knockout reduced Aβ40 expression, inflammation, and oxidative stress levels other than lipid metabolism and alleviated atherosclerosis via JNK/AP1 signaling inhibition. Similarly, our cell experiments implied that Hcy-induced Aβ40 elevation and HUVEC dysfunction involving cell proliferation and apoptosis could be restored by Herpud1 silence through restraining JNK/AP1 pathway. Collectively, our study demonstrates that Herpud1 deficiency could reduce Aβ40 expression, thereby suppressing Hcy-induced atherosclerosis by blocking the JNK/AP1 pathway. This may provide novel potential targets for atherosclerosis prevention or treatment.

Elevated plasma homocysteine (Hcy) levels are associated with endothelial dysfunction. Previous studies have indicated the role of endoplasmic reticulum (ER) stress in Hcy‑induced endothelial injury. However, the mechanism of action remains unclear. In the current study, the role of human folate receptor α (hFRα) in Hcy‑induced ER stress was investigated, along with endothelial injury in human umbilical vein endothelial cells (HUVECs). The results showed that a moderate dose of Hcy induced morphological injury and reduced viability of HUVECs. Furthermore, moderate‑dose Hcy reduced hFRα expression in HUVECs. Notably, hFRα inhibition by RNA interference resulted in activation of the protein kinase RNA‑like endoplasmic reticulum kinase pathway and increased apoptosis of HUVECs. In conclusion, this study demonstrated the presence of hFRα in HUVECs, and indicated its role in the regulation of ER stress and cell injury by Hcy.

One of the earliest events in the development of atherosclerosis is endothelial activation, which is estimated in vitro at the functional level by quantifying monocyte adhesion. This involves the incubation of fluorescently labeled monocytes on top of cultured endothelial cells and quantifying the number of adhered monocytes. Currently, the quantification of adhered monocytes is done using microscopy or by lysing the cells and estimating the fluorescence. Here we present a novel flow cytometry-based method for the quantification of monocyte adhesion. This method could quantify the average number of monocytes adhered to a single endothelial cell after monocyte adhesion assay, and was also sensitive to the level of activation of endothelial cells. Flow cytometry-based quantification requires less time and effort compared with microscopy-based quantification.

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.

The protective effects of interleukin(IL)-35 against injury to human umbilical vein endothelial cells (HUVECs) induced by the serum of pre-eclampsia patients were analyzed.This cross-sectional stud...

Characteristic pathological changes in osteonecrosis of the femoral head (ONFH) include reduced osteogenic differentiation of bone mesenchymal stem cells (BMSCs), impaired osseous circulation and increased intramedullary adipocytes deposition. Osthole is a bioactive derivative from coumarin with a wide range of pharmacotherapeutic effects. The aim of this study was to unveil the potential protective role of osthole in alcohol‐induced ONFH. In vitro, ethanol (50 mmol/L) remarkably decreased the proliferation and osteogenic differentiation of BMSCs and impaired the proliferation and tube formation capacity of human umbilical vein endothelial cell (HUVECs), whereas it substantially promoted the adipogenic differentiation of BMSCs. However, osthole could reverse the effects of ethanol on osteogenesis via modulating Wnt/β‐catenin pathway, stimulate vasculogenesis and counteract adipogenesis. In vivo, the protective role of osthole was confirmed in the well‐constructed rat model of ethanol‐induced ONFH, demonstrated by a cascade of radiographical and pathological investigations including micro‐CT scanning, haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling, immunohistochemical staining and fluorochrome labelling. Taken together, for the first time, osthole was demonstrated to rescue the ethanol‐induced ONFH via promoting bone formation, driving vascularization and retarding adipogenesis.

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Nerve growth factor (NGF) is recognized as a pleiotropic molecule, exerting a variety of biological effects on different cell types and pathophysiological conditions, and its role in tissue wound healing has been recently highlighted. However, the preferential cellular target of NGF is still elusive in the complex cellular and molecular cross talk that accompanies wound healing. Thus, to explore possible NGF cellular targets in skin wound healing, we investigated the in vitro NGF responsiveness of keratinocytes (cell line HEKa), fibroblasts (cell line BJ), and endothelial cells (cell line HUVEC), also in the presence of adverse microenvironmental conditions, e.g., hyperglycemia. The main results are summarized as follows: 1) NGF stimulates keratinocyte proliferation and HUVEC proliferation and angiogenesis in a dose-dependent manner although it has no effect on fibroblast proliferation; 2) NGF stimulates keratinocyte but not fibroblast migration in the wound healing assay; and 3) NGF completely reverts the proliferation impairment of keratinocytes and the angiogenesis impairment of HUVECs induced by high d-glucose concentration in the culture medium. These results contribute to better understanding possible targets for the therapeutic use of NGF in skin repair.

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.

Chlamydia pneumonia (C.pn) is a common respiratory pathogen that is involved in human cardiovascular diseases and promotes the development of atherosclerosis in hyperlipidemic animal models. C.pn reportedly up-regulated lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Recently, the anti-atherosclerotic activity of peroxisome proliferator-activated receptor γ (PPARγ) has been documented. In the present study, we investigated the effect of C.pn on LOX-1 expression in human umbilical vein endothelial cells (HUVECs) and identified the involvement of the PPARγ signaling pathway therein. The results showed that C.pn increased the expression of LOX-1 in HUVECs in a dose- and time-dependent manner. C.pn-induced up-regulation of LOX-1 was mediated by ERK1/2, whereas p38 MAPK and JNK had no effect on this process. C.pn induced apoptosis, inhibited cell proliferation, and decreased the expression PPARγ in HUVECs. Additionally, LOX-1 activity and cell injury caused by C.pn through activation of ERK1/2 was completely inhibited by rosiglitazone, a PPARγ agonist. In conclusion, we inferred that activation of PPARγ in HUVECs suppressed C.pn-induced LOX-1 expression and cell damage by inhibiting ERK1/2 signaling.

Cell-laden cardiac patches have recently been emerging to renew cellular sources for myocardial infarction (MI, commonly know as a heart attack) repair. However, the fabrication of cell-laden patches with porous structure remains challenging due to the limitations of currently available hydrogels and existing processing techniques. The present study utilized a bioprinting technique to fabricate hydrogel patches and characterize them in terms of printability, mechanical and biological properties. Cell-laden hydrogel (or bio-ink) was formulated from alginate dialdehyde (ADA) and gelatin (GEL) to improve the printability, degradability as well as bioactivity. Five groups of hydrogel compositions were designed to investigate the influence of the oxidation degree of ADA and hydrogels concentration on the properties of printed scaffolds. ADA–GEL hydrogels have generally shown favorable for living cells (EA.hy926 cells and hybrid human umbilical vein endothelial cell line). The hydrogel with an oxidation degree of 10% and a concentration ratio of 70/30 (or 10%ADA70–GEL30) demonstrated the best printability among the groups examined. Formulated hydrogels were also bioprinted with the living cells (EA.hy926), and the scaffolds printed were then subject to the cell culture for 7 days. Our results illustrate that the scaffolds bioprinted from 10%ADA70–GEL30 hydrogels had the best homogenous cell distribution and also the highest cell viability. Taken together, in the present study we synthesized a newly formulated bio-ink from ADA and GEL and for the fist time, used them to bioprint cardiac patches, which have the potential to be used in MI repair.

Preeclampsia is a multisystem disorder that contributes to morbidity and mortality worldwide. Recent studies show that elevated plasma and erythrocytic malondialdehyde (MDA), a marker of lipid peroxidation, has been documented in women with established preelampsia. This study aimed to observe antioxidant properties of black rice bran on preeclampsia. Level of MDA was measured with TBARS (thiobarbituric acid-reactive substances). Results showed that black rice bran has antioxidant properties by significantly decreasing MDA in preeclampsia-induced HUVEC. This makes black rice bran a promising agent which can be further used in preeclampsia treatment.

Cardiovascular disease (CVD) states are associated with endothelial dysfunction (ED) and increased production of ROS in endothelial cells. The present study aimed to explore the protective effects of antioxidant protein peroxiredoxin 6 (PRDX6) on angiotensin II (AngII)‑induced human umbilical vein endothelial cell (HUVEC) dysfunction. To investigate cell viability, levels of inflammatory molecules and proteins were assayed using the CCK-8 assay and evaluated by ELISA and Western blot. NO and ROS levels were determined by Griess assay and the fluorescent probe DCFH-DA. Cell migration capacity was assessed by Transwell assay. AngII decreased cell viability and PRDX6, upregulated the expression levels of TNF-α, IL-6, IL-1β, LDH and MDA, stimulated ROS production, and reduced NO synthase, the expressions of eNOS, MnSOD, ICAM-1, VCAM-1, and activated the MAPK family of signaling proteins. However, the stimulatory effects of AngII on HUVECs were remarkably suppressed by PRDX6. Furthermore, mercaptosuccinate (MS; PRDX6 inhibitor) had similar effects as AngII in aggravating HUVECs damage. Conversely, these adverse events caused by AngII and MS were obviously reversed by ML3404 and SP600125. The present study indicated that PRDX6 overexpression inactivated p38 MAPK and JNK pathway through decrease AngII-induced inflammation, oxidative stress and endothelial dysfunction leading to attenuation of endothelial cell damage.

Tissue-engineered heart valves are a promising alternative to current valve substitutes. As the main scaffold of tissue-engineered heart valves, the decellularized heart valve (DHV) has problems such as biomechanical property damage and rapid degradation. In this study, we applied a photo-crosslinking reaction induced by riboflavin and ultraviolet light A (UVA) in the DHV for improving its biomechanical properties and stability. The results showed that the biomechanical properties of the DHV significantly improved following riboflavin-UVA (R-UVA) crosslinking. Moreover, the R-UVA-crosslinked DHV (R-UV-DHV) showed better resistance to enzymatic degradation in vitro, with significantly higher thermal denaturation temperature compared to that of the untreated DHV, indicating that the stability of the R-UV-DHV improved. Histological staining and scanning electron microscopy showed that the leaflet ultrastructure was preserved better after R-UVA crosslinking compared to a glutaraldehyde-crosslinked DHV. In addition, we found that the R-UV-DHV exhibited excellent human umbilical vein endothelial cell adhesion and cells could readily grow on its surface. In an in vitro anti-calcification experiment, the R-UV-DHV demonstrated non-calcifying properties in a simulated body fluid. Furthermore, the R-UV-DHV showed characteristics of slow degradation, non-calcification, and reduced pro-inflammatory response through a rat subcutaneous implantation model. As a result, R-UVA can effectively crosslink the DHV and the R-UV-DHV possessed satisfactory biocompatibility. R-UVA crosslinking can be a new approach for improving the performance of the DHV to prepare a better scaffold for tissue-engineered valves.

Ferroptosis is a kind of cell death triggered by intracellular phospholipid peroxidation. Human umbilical vein blood endothelial progenitor cells-Exosomes (EPCs-Exos) affect ferroptosis. This study sought to explore the mechanism of EPCs-Exos in human umbilical vein endothelial cell (HUVEC) ferroptosis. EPCs-Exos were isolated and identified. HUVECs were treated with Erastin at IC50 concentration. Ferroptosis-related indexes and iron ion content were detected using kits. HUVEC migration and angiogenesis before/after ferroptosis inhibitor treatment were observed by cell scratch and angiogenesis assays. After Erastin induction, HUVECs were transfected with miR-30e-5p mimic, or treated with EPCs-Exos and EPCs-Exos transfected with miR-30e-5p inhibitor. miR-30e-5p expression was detected by RT-qPCR. The binding relationship between miR-30e-5p and specificity protein 1 (SP1) was verified by dual-luciferase assay. SP1 expression was detected by Western blot. HUVECs treated with Erastin and EPCs-Exos were transfected with pcDNA3.1-SP1. Protein levels of adenosine monophosphate-activated protein kinase (AMPK) and p-AMPK were detected by Western blot. EPCs-Exos inhibited Erastin-induced HUVEC ferroptosis and endothelial injury. Erastin inhibited miR-30e-5p and EPCs-Exo treatment recovered miR-30e-5p expression. miR-30e-5p was encapsulated in EPCs-Exos. After inhibiting miR-30e-5p in EPCs, the inhibitory effect of EPCs-Exos on HUVEC ferroptosis was attenuated. miR-30e-5p targeted SP1. Overexpression of SP1 partially reversed the effect of EPCs-Exos on improving HUVEC ferroptosis and increasing phosphorylation levels of AMPK. Collectively, EPCs-Exos inhibited Erastin-induced HUVEC ferroptosis by upregulating miR-30e-5p, inhibiting SP1, and activating the AMPK pathway.

Opioid overdose, dependence, and addiction are a major public health crisis. Patients with chronic kidney disease (CKD) are at high risk of opioid overdose, therefore novel methods that provide accurate prediction of renal clearance (CLr) and systemic disposition of opioids in CKD patients can facilitate the optimization of therapeutic regimens. The present study aimed to predict renal clearance and systemic disposition of morphine and its active metabolite morphine-6-glucuronide (M6G) in CKD patients using a vascularized human proximal tubule microphysiological system (VPT-MPS) coupled with a parent-metabolite full body physiologically-based pharmacokinetic (PBPK) model. The VPT-MPS, populated with a human umbilical vein endothelial cell (HUVEC) channel and an adjacent human primary proximal tubular epithelial cells (PTEC) channel, successfully demonstrated secretory transport of morphine and M6G from the HUVEC channel into the PTEC channel. The in vitro data generated by VPT-MPS were incorporated into a mechanistic kidney model and parent-metabolite full body PBPK model to predict CLr and systemic disposition of morphine and M6G, resulting in successful prediction of CLr and the plasma concentration–time profiles in both healthy subjects and CKD patients. A microphysiological system together with mathematical modeling successfully predicted renal clearance and systemic disposition of opioids in CKD patients and healthy subjects.

Polyetheretherketone (PEEK) is a biocompatible polymer, but its clinical application is largely limited due to its inert surface. To solve this problem, a multifunctional PEEK implant is urgently fabricated. In this work, a dual-metal-organic framework (Zn-Mg-MOF74) coating is bonded to PEEK using a mussel-inspired polydopamine interlayer to prepare the coating, and then, dexamethasone (DEX) is loaded on the coating surface. The PEEK surface with the multifunctional coating provides superior hydrophilicity and favorable stability and forms an alkaline microenvironment when Mg2+, Zn2+, 2,5-dihydroxyterephthalic acid, and DEX are released due to the coating degradation. In vitro results showed that the multifunctional coating has strong antibacterial ability against both Escherichia coli and Staphylococcus aureus; it also improves human umbilical vein endothelial cell angiogenic ability and enhances rat bone marrow mesenchymal stem cell osteogenic differentiation activity. Furthermore, the in vivo rat subcutaneous infection model, chicken chorioallantoic membrane model, and rat femoral drilling model verify that the PEEK implant coated with the multifunctional coating has strong antibacterial and angiogenic ability and promotes the formation of new bone around the implant with a stronger bone-implant interface. Our findings indicate that DEX loaded on the Zn-Mg-MOF74 coating-modified PEEK implant with bacteriostasis, angiogenesis, and osteogenesis properties has great clinical application potential as bone graft materials.

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.