The requirement of the mitochondrial protein NDUFS8 for angiogenesis.

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

Inhibiting MMP13 Attenuates Deep Vein Thrombosis in a Mouse Model by Reducing the Expression of Pdpn.

OBJECTIVE: Matrix metalloproteinase 13 (MMP13) is an extracellular matrix protease that affects the progression of atherosclerotic plaques and arterial thrombi by degrading collagens, modifying protein structures and regulating inflammatory responses, but its role in deep vein thrombosis (DVT) has not been determined. The purpose of this study was to investigate the potential effects of MMP13 and MMP13-related genes on the formation of DVT. METHODS: We altered the expression level of MMP13 in vivo and conducted a transcriptome study to examine the expression and relationship between MMP13 and MMP13-related genes in a mouse model of DVT. After screening genes possibly related to MMP13 in DVT mice, the expression levels of candidate genes in human umbilical vein endothelial cells (HUVECs) and the venous wall were evaluated. The effect of MMP13 on platelet aggregation in HUVECs was investigated in vitro. RESULTS: Among the differentially expressed genes, interleukin 1 beta, podoplanin (Pdpn), and factor VIII von Willebrand factor (F8VWF) were selected for analysis in mice. When MMP13 was inhibited, the expression level of PDPN decreased significantly in vitro. In HUVECs, overexpression of MMP13 led to an increase in the expression level of PDPN and induced platelet aggregation, while transfection of PDPN-siRNA weakened the ability of MMP13 to increase platelet aggregation. CONCLUSIONS: Inhibiting the expression of MMP13 could reduce the burden of DVT in mice. The mechanism involves downregulating the expression of Pdpn through MMP13, which could provide a novel gene target for DVT diagnosis and treatment.

DFMG decreases angiogenesis to uphold plaque stability by inhibiting the TLR4/VEGF pathway in mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 µmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

NLRC4-mediated pyroptosis was involved in coagulation disorders of acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a potentially lethal acute disease highly involved in coagulation disorders. Pyroptosis has been reported to exacerbate coagulation disorders, yet this implication has not been illustrated completely in AP. METHODS: RNA sequencing data of peripheral blood of AP patients were downloaded from the Gene Expression Omnibus database. Gene set variation analysis and single sample gene set enrichment analysis were used to calculate the enrichment score of coagulation-related signatures and pyroptosis. Spearman and Pearson correlation analysis was used for correlation analysis. Peripheral blood samples and related clinical parameters were collected from patients with AP and healthy individuals. A severe AP (SAP) model of mice was established using caerulein and lipopolysaccharide. Enzyme-linked immunosorbent assay, chemiluminescence immunoassay and immunohistochemical analysis were employed to detect the level of coagulation indicators and pyroptosis markers in serum and pancreas tissues. Additionally, we evaluated the effect of pyroptosis inhibition and NLRC4 silence on the function of human umbilical vein endothelial cells (HUVECs). RESULTS: Coagulation disorders were significantly positively correlated to the severity of AP, and they could be a predictor for AP severity. Further analyses indicated that six genes-DOCK9, GATA3, FCER1G, NLRC4, C1QB and C1QC-may be involved in coagulation disorders of AP. Among them, NLRC4 was positively related to pyroptosis that had a positive association with most coagulation-related signatures. Data from patients showed that NLRC4 and other pyroptosis markers, including IL-1ß, IL-18, caspase1 and GSDMD, were significant correlation to AP severity. In addition, NLRC4 was positively associated with coagulation indicators in AP patients. Data from mice showed that NLRC4 was increased in the pancreas tissues of SAP mice. Treatment with a pyroptosis inhibitor effectively alleviated SAP and coagulation disorders in mice. Finally, inhibiting pyroptosis or silencing NLRC4 could relieve endothelial dysfunction in HUVECs. CONCLUSIONS: NLRC4-mediated pyroptosis damages the function of endothelial cells and thereby exacerbates coagulation disorders of AP. Inhibiting pyroptosis could improve coagulation function and alleviate AP.

Epidermal Growth Factor-Like Repeats and Discoidin I-Like Domains 3 Deficiency Attenuates Dilated Cardiomyopathy by Inhibiting Ubiquitin Specific Peptidase 10 Dependent Smad4 Deubiquitination.

BACKGROUND: Dilated cardiomyopathy (DCM) is the leading cause of heart failure with a poor prognosis. Recent studies suggest that endothelial to mesenchymal transition (EndMT) may be involved in the pathogenesis and cardiac remodeling during DCM development. EDIL3 (epidermal growth factor-like repeats and discoidin I-like domains 3) is an extracellular matrix glycoprotein that has been reported to promote EndMT in various diseases. However, the roles of EDIL3 in DCM still remain unclear. METHODS AND RESULTS: A mouse model of DCM and human umbilical vein endothelial cells were used to explore the roles and mechanisms of EDIL3 in DCM. The results indicated that EndMT and EDIL3 were activated in DCM mice. EDIL3 deficiency attenuated cardiac dysfunction and remodeling in DCM mice. EDIL3 knockdown alleviated EndMT by inhibiting USP10 (ubiquitin specific peptidase 10) dependent Smad4 deubiquitination in vivo and in vitro. Recombinant human EDIL3 promoted EndMT via reinforcing deubiquitination of Smad4 in human umbilical vein endothelial cells treated with IL-1ß (interleukin 1ß) and TGF-ß (transforming growth factor beta). Inhibiting USP10 abolished EndMT exacerbated by EDIL3. In addition, recombinant EDIL3 also aggravates doxorubicin-induced EndMT by promoting Smad4 deubiquitination in HUVECs. CONCLUSIONS: Taken together, these results indicate that EDIL3 deficiency attenuated EndMT by inhibiting USP10 dependent Smad4 deubiquitination in DCM mice.

Succinylated chitosan derivative restore HUVEC cells function damaged by TNF-α and high glucose in vitro and enhanced wound healing.

The inflammation of chronic wounds plays a key hindering role in the wound healing process. Slowing down the inflammatory response is significant for the repair of chronic wounds. Studies have revealed that succinate can inactivate gastrin D (GSDMD) and prevent cell pyroptosis. Chitosan has anti-inflammatory properties and is commonly used as wound healing material. Therefore, we used succinic anhydride to modify chitosan and found that N-succinylated chitosan (NSC) was more effective in inhibiting inflammation. The results showed that the stimulation of TNF-α and high glucose induces overexpression of capase-1 and TNF-α in human umbilical vein endothelial cells (HUVEC), and down-expression of CD31. However, the expression of capase-1 and TNF-α decreased, while the expression of CD31, VEGF and IL-10 was up-regulated significantly in dysfunctional HUVEC cells after treated by NSC. Moreover, NSC can speed wound healing, histological examination results showed that wounds treated with NSC exhibited faster epithelial tissue regeneration and thicker collagen deposition. Overall, this study results suggested that NSC has the function of restoring the physiological functions of dysfunctional HUVEC cells induced by high glucose and TNF-α, and can accelerate wound healing, indicating that NSC has good potential to be applied in inflammatory chronic wounds such as diabetic foot.

MiR-522-3p Attenuates Cardiac Recovery by Targeting FOXP1 to Suppress Angiogenesis.

Angiogenesis is crucial for blood supply reconstitution after myocardial infarction in patients with acute coronary syndrome (ACS). MicroRNAs are recognized as important epigenetic regulators of endothelial angiogenesis. The purpose of this study is to determine the roles of miR-522-3p in angiogenesis after myocardial infarction. The expression levels of miR-522-3p in rats' plasma and in the upper part of the ligation of the heart tissues at 28 days after myocardial infarction were significantly higher than those of the sham group. miR-522-3p mimics inhibited cell proliferations, migrations, and tube formations under hypoxic conditions in HUVECs (human umbilical vein endothelial cells), whereas miR-522-3p inhibitors did the opposite. Furthermore, studies have indicated that the inhibition of miR-522-3p by antagomir infusion promoted angiogenesis and accelerated the recovery of cardiac functions in rats with myocardial infarction.Data analysis and experimental results revealed that FOXP1 (Forkhead-box protein P1) was the target gene of miR-522-3p. Our study explored the mechanism of cardiac angiogenesis after myocardial infarction and provided a potential therapeutic approach for the treatment of ischemic heart disease in the future.

Identification of circular RNA hsa_circ_0034621 as a novel biomarker for carotid atherosclerosis and the potential function as a regulator of NLRP3 inflammasome.

BACKGROUND AND AIMS: NLRP3 inflammasome plays a key role in vascular inflammation and atherosclerosis. Circular RNAs (circRNAs) are involved in disease development by regulating gene expression, and have emerged as promising novel disease biomarkers. This study aimed to identify the NLRP3 inflammasome-associated circRNA biomarkers of carotid atherosclerosis. METHODS: Based on the differential expression profiles of circRNAs in patients with carotid artery plaque (CAP) and healthy controls, hsa_circ_0043621, hsa_circ_0051995, and hsa_circ_0123388 were screened and validated using real-time quantitative polymerase chain reaction (RT-qPCR). Potential circRNA-miRNA-mRNA interactions were explored using a luciferase assay. The biological roles of the validated circRNAs were investigated in human umbilical vein endothelial cells (HUVECs) using Western blotting, transwell, and CCK-8 assays. Clinical significance was assessed using receiver operating characteristic (ROC) curves and logistic regression analysis. RESULTS: The expression levels of all candidate circRNAs were significantly higher in patients with CAP than in controls (p<0.05), which was consistent with the results of the microarray analysis. Overexpression of hsa_circ_0043621 significantly increased the expression of NLRP3, induced migration of HUVECs, and inhibited cell proliferation. hsa_circ_0043621 demonstrated reasonable diagnostic accuracy for CAP detection and increased intima-media thickness (IMT). hsa_circ_0043621 upregulation was an independent predictor of an increased risk of CAP and increased IMT. CONCLUSIONS: hsa_circ_0043621 is a valuable circulating biomarker of carotid atherosclerosis and may contribute to its pathogenesis by regulating the NLRP3 inflammasome.

Steady Laminar Flow Decreases Endothelial Glycolytic Flux While Enhancing Proteoglycan Synthesis and Antioxidant Pathways.

Endothelial cells in steady laminar flow assume a healthy, quiescent phenotype, while endothelial cells in oscillating disturbed flow become dysfunctional. Since endothelial dysfunction leads to atherosclerosis and cardiovascular disease, it is important to understand the mechanisms by which endothelial cells change their function in varied flow environments. Endothelial metabolism has recently been proven a powerful tool to regulate vascular function. Endothelial cells generate most of their energy from glycolysis, and steady laminar flow may reduce endothelial glycolytic flux. We hypothesized that steady laminar but not oscillating disturbed flow would reduce glycolytic flux and alter glycolytic side branch pathways. In this study, we exposed human umbilical vein endothelial cells to static culture, steady laminar flow (20 dynes/cm2 shear stress), or oscillating disturbed flow (4 ± 6 dynes/cm2 shear stress) for 24 h using a cone-and-plate device. We then measured glucose and lactate uptake and secretion, respectively, and glycolytic metabolites. Finally, we explored changes in the expression and protein levels of endothelial glycolytic enzymes. Our data show that endothelial cells in steady laminar flow had decreased glucose uptake and 13C labeling of glycolytic metabolites while cells in oscillating disturbed flow did not. Steady laminar flow did not significantly change glycolytic enzyme gene or protein expression, suggesting that glycolysis may be altered through enzyme activity. Flow also modulated glycolytic side branch pathways involved in proteoglycan and glycosaminoglycan synthesis, as well as oxidative stress. These flow-induced changes in endothelial glucose metabolism may impact the atheroprone endothelial phenotype in oscillating disturbed flow.

Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment.

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

Beneficial Effects of Small-Molecule Oligopeptides Isolated from Panax Ginseng C. A. Meyer on Cellular Fates in Oxidative Stress-Induced Damaged Human Umbilical Vein Endothelial Cells and PC-12.

Cell fate instability is a crucial characteristic of aging and appears to contribute to various age-related pathologies. Exploring the connection between bioactive substances and cell fate stability may offer valuable insights into longevity. Therefore, the objective of this study was to investigate the potential beneficial effects of ginseng oligopeptides (GOPs) isolated from Panax ginseng C. A. Meyer at the cellular level. Disruption of homeostasis of human umbilical vein endothelial cells (HUVECs) and PC-12 was achieved by culturing them in the growth medium supplemented with 200 µM of H2O2, and 25, 50, and 100 µg/mL GOPs for 4 h. Then, they were cultured in a H2O2-free growth medium containing different concentration of GOPs. We found that GOP administration retards the oxidative stress-induced cell instability in HUVECs by increasing cell viability, inhibiting the cell cycle arrest, enhancing telomerase (TE) activity, suppressing oxidative stress and an inflammatory attack, and protecting mitochondrial function. Furthermore, we hypothesized that GOPs may promote mitochondrial biosynthesis by upregulating PGC-1α expression. Similarly, GOPs positively regulated cell stability in PC-12; notably, the protective effect of GOPs on PC-12 mainly occurred through the inhibition of autophagic cell death of neuronal cells, while the protective effect on mitochondria was weak. In conclusion, it is evident that GOPs demonstrate potential beneficial effects in maintaining cell fate stability, thereby potentially contributing to an enhanced health span and overall well-being.

Antiseptic Functions of CGK012 against HMGB1-Mediated Septic Responses.

High mobility group box 1 (HMGB1), a protein with important functions, has been recognized as a potential therapeutic target for the treatment of sepsis. One possible mechanism for this is that inhibiting HMGB1 secretion can exert antiseptic effects, which can restore the integrity of the vascular barrier. (7S)-(+)-cyclopentyl carbamic acid 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester (CGK012) is a newly synthesized pyranocoumarin compound that could function as a novel small-molecule inhibitor of the Wnt/ß-catenin signaling pathway. However, no studies have yet determined the effects of CGK012 on sepsis. We investigated the potential of CGK012 to attenuate the excessive permeability induced by HMGB1 and enhance survival rates in a mouse model of sepsis with reduced HMGB1 levels following lipopolysaccharide (LPS) treatment. In both LPS-stimulated human endothelial cells and a mouse model exhibiting septic symptoms due to cecal ligation and puncture (CLP), we assessed proinflammatory protein levels and tissue damage biomarkers as indicators of reduced vascular permeability. CGK012 was applied after induction in human endothelial cells exposed to LPS and the CLP-induced mouse model of sepsis. CGK012 effectively mitigated excessive permeability and suppressed HMGB1 release, resulting in improved vascular stability, decreased mortality, and enhanced histological conditions in the mouse model of CLP-induced sepsis. In conclusion, our findings indicate that CGK012 treatment in mice with CLP-induced sepsis diminished HMGB1 release and increased the survival rate, suggesting its potential as a pharmaceutical intervention for sepsis.

[Protective effect of FAK inhibitor PF-562271 against human umbilical vein endothelial cell injury induced by aging platelets].

OBJECTIVE: To investigate the protective effect of PF-562271, a FAK inhibitor, against aging platelet-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were treated with vehicle, lipopolysaccharide (LPS), LPS+aging platelets, or LPS+aging platelets+PF-562271. The changes in protein expressions of FAK, pFAK and PECAM-1 in the treated cells were detected using Western blotting and immunofluorescence assay, and the level of reactive oxygen species (ROS) was detected with flow cytometry. The changes of barrier function of the cells were assessed with cell permeability test and transendothelial cell resistance test. RT-qPCR was used to analyze mRNA expressions of inflammatory factors, and pro-inflammatory cytokine levels in the culture supernatants was determined with enzyme-linked immunosorbent assay. Immunofluorescence assay was used to examine the effect of the ROS inhibitor vitamin C on PECAM-1 expression in the cells with different treatments. RESULTS: Treatment of HUVECs with LPS and aging platelets significantly increased cellular protein expressions of FAK, pFAK and PECAM-1, which were effectively lowered by addition of PF-562271 (P < 0.05). LPS and aged platelets obviously enhanced ROS production in the cells, which was inhibited by the addition of PF-562271 (P < 0.001). PF-562271 significantly alleviated the damage of endothelial cell barrier function of the cells caused by LPS and aging platelets (P < 0.01). The expressions of TNF-α, IL-6 and IL-8 in HUVECs increased significantly after exposure to LPS and aging platelets, and were obviously lowered after treatment with PF-562271 (P < 0.05). Treatment with vitamin C significantly decreased the expression of PECAM-1 protein in the cells (P < 0.01). CONCLUSION: The FAK inhibitor PF-562271 alleviates endothelial cell damage induced by LPS and aging platelets by lowering cellular oxidative stress levels and reducing inflammatory responses.

Antiangiogenic properties of BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom by VEGF pathway in endothelial cells.

Angiogenesis is a process that is controlled by a delicate combination of proangiogenic and antiangiogenic molecules and can be disrupted in various illnesses, including cancer. Non-cancerous diseases can also have an abnormal or insufficient vascular growth, inflammation and hypoxia, which exacerbate angiogenesis. These conditions include atherosclerosis, psoriasis, endometriosis, asthma, obesity and AIDS. Based on that, the present work assessed the in vitro and ex vivo antiangiogenic properties stemming from BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom, via the VEGF pathway. BthMP at a concentration of 5 and 40 µg/mL showed no toxicity to endothelial cells (HUVEC) in the MTT assay and was not able to induce necrosis and colony proliferation. Interestingly, BthMP inhibited adhesion, migration and invasion of HUVECs in Matrigel and arrested in vitro angiogenesis by reducing the average number of nodules in toxin-treated cells by 9.6 and 17.32 at 5 and 40 µg/mL, respectively, and the number of tubules by 15.9 at 5 µg/mL and 21.6 at 40 µg/mL in a VEGF-dependent way, an essential proangiogenic property. Furthermore, BthMP inhibited the occurrence of the angiogenic process in an ex vivo aortic ring test by decreasing new vessel formation by 52% at 5 µg/mL and by 66% at 40 µg/mL and by increasing the expression of an antiangiogenic gene, SFLT-1, and decreasing the expression of the proangiogenic genes VEGFA and ANGPT-1. Finally, this toxin reduces the production of nitric oxide, a marker that promotes angiogenesis and VEGF modulation, and decreases the protein expression of VEGFA in the supernatant of the HUVEC culture by about 30 %. These results suggest that BthMP has a promising antiangiogenic property and proves to be a biotechnological mechanism for understanding the antiangiogenic responses induced by snake venom metalloproteinases, which could be applied to a variety of diseases that exhibit an imbalance of angiogenesis mechanisms.

Homocysteine metabolites inhibit autophagy by upregulating miR-21-5p, miR-155-5p, miR-216-5p, and miR-320c-3p in human vascular endothelial cells.

Nutritional and genetic deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis, which is a major cause of cardiovascular disease (CVD). Impaired autophagy causes the accumulation of damaged proteins and organelles and is associated with CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone and N-Hcy-protein. However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of Hcy-thiolactone, N-Hcy-protein, and Hcy on autophagy human umbilical vein endothelial cells. We found that treatments with Hcy-thiolactone, N-Hcy-protein, or Hcy significantly downregulated beclin 1 (BECN1), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 (LC3) mRNA and protein levels. We also found that these changes were mediated by upregulation by Hcy-thiolactone, N-Hcy-protein, and Hcy of autophagy-targeting microRNA (miR): miR-21, miR-155, miR-216, and miR-320c. The effects of these metabolites on levels of miR targeting autophagy as well as on the levels of BECN1, ATG5, ATG7, and LC3 mRNA and protein were abrogated by treatments with inhibitors of miR-21, miR-155, miR-216, and mir320c. Taken together, our findings show that Hcy metabolites can upregulate miR-21, miR-155, miR-216, and mir320c, which then downregulate autophagy in human endothelial cells, important for vascular homeostasis.

Flubendazole suppresses VEGF-induced angiogenesis in HUVECs and exerts antitumor effects in PC-3 cells.

Flubendazole, an FDA-approved anthelmintic, has been predicted to show strong VEGFR2 inhibitory activity in silico screening combined with in vitro experimental validation, and it has shown anti-cancer effects on some human cancer cell lines, but little is known about the anti-angiogenesis effects and anti-prostate cancer effects. In this study, we analyzed the binding modes and kinetic analysis of flubendazole with VEGFR2 and first demonstrated that flubendazole suppressed VEGF-stimulated cell proliferation, wound-healing migration, cell invasion and tube formation of HUVEC cells, and decreased the phosphorylation of extracellular signal-regulated kinase and serine/threonine kinase Akt, which are the downstream proteins of VEGFR2 that are important for cell growth. What's more, our results showed that flubendazole decreased PC-3 cell viability and proliferation ability, and suppressed PC-3 cell wound healing migration and invasion across a Matrigel-coated Transwell membrane in a concentration-dependent manner. The antiproliferative effects of flubendazole were due to induction of G2-M phase cell cycle arrest in PC-3 cells with decreasing expression of the Cyclin D1 and induction of cell apoptosis with the number of apoptotic cells increased after flubendazole treatment. These results indicated that flubendazole could exert anti-angiogenic and anticancer effects by inhibiting cell cycle and inducing cell apoptosis.

Fibromodulin facilitates the osteogenic effect of Masquelet's induced membrane by inhibiting the TGF-ß/SMAD signaling pathway.

Masquelet's induced membrane (IM) technique is a promising treatment strategy for the repair of substantial bone defects. The formation of an IM around polymethylmethacrylate bone cement plays a crucial role in this technique. Several studies have indicated that IMs have bioactivity because they contain abundant blood vessels, a variety of cells, and biological factors. The bioactivity of an IM increases during the initial stages of formation, thereby facilitating bone regeneration and remodeling. Nevertheless, the precise mechanisms underlying the enhancement of IM bioactivity and the promotion of bone regeneration necessitate further investigation. In this study, we successfully developed a Masquelet IM model of critical femur defects in rats. By employing proteomics analysis and biological detection techniques, we identified fibromodulin (FMOD) as a pivotal factor contributing to angiogenesis and the enhanced bioactivity of the IM. A significant increase in angiogenesis and the expression of bioactive factors in the IM was also observed with the upregulation of FMOD expression. Furthermore, this effect is mediated through the inhibition of the transforming growth factor beta (TGF-ß)/SMAD signaling pathway. We also demonstrated that administering recombinant human FMOD enhanced osteogenesis in rat bone marrow mesenchymal stem cells and angiogenesis in human umbilical vein endothelial cells in vitro. Furthermore, the negative regulatory effect of the TGF-ß signaling pathway was verified. In conclusion, this study provides a novel theoretical basis for the application of IMs in bone-defect reconstruction and explores possible new mechanisms that may play an important role in promoting the bioactivity and osteogenic potential of IMs.

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

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

Peptide OM-LV20 promotes arteriogenesis induced by femoral artery ligature via the miR-29b-3p/VEGFA axis.

BACKGROUND AND AIMS: Therapeutic arteriogenesis is a promising direction for the treatment of ischemic disease caused by atherosclerosis. However, pharmacological or biological approaches to stimulate functional collateral vessels are not yet available. Identifying new drug targets to promote and explore the underlying mechanisms for therapeutic arteriogenesis is necessary. METHODS: Peptide OM-LV20 (20 ng/kg) was administered for 7 consecutive days on rat hindlimb ischemia model, collateral vessel growth was assessed by H&E staining, liquid latex perfusion, and specific immunofluorescence. In vitro, we detected the effect of OM-LV20 on human umbilical vein endothelial cells (HUVEC) proliferation and migration. After transfection, we performed quantitative real-time polymerase chain reaction, in situ-hybridization and dual luciferase reporters to assessed effective miRNAs and target genes. The proteins related to downstream signaling pathways were detected by Western blot. RESULTS: OM-LV20 significantly increased visible collateral vessels and endothelial nitric oxide synthase (eNOS), together with enhanced inflammation cytokine and monocytes/macrophage infiltration in collateral vessels. In vitro, we defined a novel microRNA (miR-29b-3p), and its inhibition enhanced proliferation and migration of HUVEC, as well as the expression of vascular endothelial growth factor A (VEGFA). OM-LV20 also promoted migration and proliferation of HUVEC, and VEGFA expression was mediated via inhibition of miR-29b-3p. Furthermore, OM-LV20 influenced the protein levels of VEGFR2 and phosphatidylinositol3-kinase (PI3K)/AKT and eNOS in vitro and invivo. CONCLUSIONS: Our data indicated that OM-LV20 enhanced arteriogenesis via the miR-29b-3p/VEGFA/VEGFR2-PI3K/AKT/eNOS axis, and highlighte the application potential of exogenous peptide molecular probes through miRNA, which could promote effective therapeutic arteriogenesis in ischemic conditions.