Matrigel Tube Formation Research Articles

Aerobic Exercise-Mimetic Effects of Resveratrol on the Prevention of Vascular Endothelial Senescence

PURPOSE: Regular exercise has been shown to have anti-aging effects on the vascular system by improving endothelial function and diminishing oxidative stress. However, adherence to an exercise regimen can be difficult for some individuals, particularly those with limited mobility. This study investigated the effects of resveratrol as an exercise mimetic for preventing and improving oxidative stressinduced senescence in vascular endothelial cells.METHODS: Cultured human umbilical vein endothelial cells were used to investigate the reversal effects of laminar shear stress and the protective effects of resveratrol on oxidative stress-induced premature senescence. Senescence was estimated using SA-β-gal staining and western blot analysis, whereas oxidative stress levels were evaluated using MitoSOX staining. Matrigel tube formation and scratch assays were also performed to assess endothelial angiogenic and migration functions.RESULTS: The study showed that laminar shear stress could partly normalize premature endothelial senescence. Resveratrol pretreatment attenuated reactive oxygen species production and prevented senescence in H2O2-induced premature aging. Additionally, resveratrol contributed to maintaining endothelial cells’ angiogenic and migratory functions.CONCLUSIONS: This study demonstrated that laminar shear stress can mitigate the effects of oxidative stress-induced premature senescence. Resveratrol may be a potential substitute for exercise to prevent age-related damage to the endothelium by increasing resistance to oxidative stress and improving endothelial function.

EDIL3 and VEGF Synergistically Affect Angiogenesis in Endothelial Cells.

Angiogenesis is one of the histologically predominant characteristics of psoriasis. Vascular endothelial growth factor (VEGF) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) have critical effects on angiogenesis. Both these proteins are vital proangiogenic factors in tumor occurrence and progression; however, the relationship between EDIL3 and VEGF with psoriasis remains unclear. We aimed to elucidate the role of EDIL3 and VEGF and the involved mechanisms in psoriasis-associated angiogenesis. EDIL3 and VEGF expression in cutaneous tissue was determined by immunohistochemical assay. The effects of EDIL3 on VEGF, VEGFR2, and the growth, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were analyzed by Western blotting assay, cell counting kit-8 assay, Transwell assay, and Matrigel tube formation assay. EDIL3 and VEGF levels in psoriatic lesions significantly increased as compared to those in normal individuals and showed a positive relationship with the Psoriasis Area and Severity Index. The downregulation of EDIL3 decreased VEGF and VEGFR2 expression in HUVECs. Moreover, the decreased expression of EDIL3 and VEGF reduced the growth, invasion, and tube formation abilities of HUVECs, while EDIL3 resistance to VEGF and VEGFR2 was restored by using the EDIL3 recombinant protein. These results suggest that psoriasis is also characterized by EDIL3 and VEGF-mediated angiogenesis. Thus, EDIL3 and VEGF could serve as novel targets for treating psoriasis.

Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway

Endothelial impairment and dysfunction are closely related to the pathogenesis of steroid-associated osteonecrosis of the femoral head (SONFH). Recent studies have showed that hypoxia inducible factor-1α (HIF-1α) plays a crucial role in endothelial homeostasis maintenance. Dimethyloxalylglycine (DMOG) could suppress HIF-1 degradation and result in nucleus stabilization by repressing prolyl hydroxylase domain (PHD) enzymatic activity. Our results showed that methylprednisolone (MPS) remarkably undermined biological function of endothelial progenitor cells (EPC) by inhibiting colony formation, migration, angiogenesis, and stimulating senescence of EPCs, while DMOG treatment alleviated these effects by promoting HIF-1α signaling pathway, as evidenced by senescence-associated β-galactosidase (SA-β-Gal) staining, colony-forming unit, matrigel tube formation, and transwell assays. The levels of proteins related to angiogenesis were determined by ELISA and Western blotting. In addition, active HIF-1α bolstered the targeting and homing of endogenous EPCs to the injured endothelium in the femoral head. Histopathologically, our in vivo study showed that DMOG not only alleviated glucocorticoid-induced osteonecrosis but also promoted angiogenesis and osteogenesis in the femoral head as detected by microcomputed tomography (Micro-CT) analysis and histological staining of OCN, TRAP, and Factor Ⅷ. However, all of these effects were impaired by an HIF-1α inhibitor. These findings demonstrate that targeting HIF-1α in EPCs may constitute a novel therapeutic approach for the treatment of SONFH.

Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2

Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

Piezo1 (Piezo-Type Mechanosensitive Ion Channel Component 1)-Mediated Mechanosensation in Macrophages Impairs Perfusion Recovery After Hindlimb Ischemia in Mice.

Angiogenesis is a promising strategy for those with peripheral artery disease. Macrophage-centered inflammation is intended to govern the deficiency of the angiogenic response after hindlimb ischemia. However, little is known about the mechanism of macrophage activation beyond signals from cytokines and chemokines. We sought to identify a novel mechanical signal from the ischemic microenvironment that provokes macrophages and the subsequent inflammatory cascade and to investigate the potential role of Piezo-type mechanosensitive ion channels (Piezo) on macrophages during this process. Myeloid cell-specific Piezo1 (Piezo-type mechanosensitive ion channel component 1) knockout (Piezo1ΔMΦ) mice were generated by crossing Piezo1fl/fl (LysM-Cre-/-; Piezo1 flox/flox) mice with LysM-Cre transgenic mice to assess the roles of Piezo1 in macrophages after hindlimb ischemia. Furthermore, in vitro studies were carried out in bone marrow-derived macrophages to decipher the underlying mechanism. We found that tissue stiffness gradually increased after hindlimb ischemia, as indicated by Young's modulus. Compared to Piezo2, Piezo1 expression and activation were markedly upregulated in macrophages from ischemic tissues in concurrence with increased tissue stiffness. Piezo1ΔMΦ mice exhibited improved perfusion recovery by enhancing angiogenesis. Matrigel tube formation assays revealed that Piezo1 deletion promoted angiogenesis by enhancing FGF2 (fibroblast growth factor-2) paracrine signaling in macrophages. Conversely, activation of Piezo1 by increased stiffness or the agonist Yoda1 led to reduced FGF2 production in bone marrow-derived macrophages, which could be blocked by Piezo1 silencing. Mechanistically, Piezo1 mediated extracellular Ca2+ influx and activated Ca2+-dependent CaMKII (calcium/calmodulin-dependent protein kinase II)/ETS1 (ETS proto-oncogene 1) signaling, leading to transcriptional inactivation of FGF2. This study uncovers a crucial role of microenvironmental stiffness in exacerbating the macrophage-dependent deficient angiogenic response. Deletion of macrophage Piezo1 promotes perfusion recovery after hindlimb ischemia through CaMKII/ETS1-mediated transcriptional activation of FGF2. This provides a promising therapeutic strategy to enhance angiogenesis in ischemic diseases.

LncRNA FOXD3-AS1/miR-128-3p axis-mediated IGF2BP3 in glioma stimulates cancer angiogenesis and progression.

The aim of the study was to research the mechanism by which IGF2BP3 regulates glioma progression as well as its upstream regulatory axis. The researched mRNA was determined using differential expression analysis based on bioinformatics data, and its upstream miRNAs and lncRNAs were predicted. Interaction between genes we researched was identified by dual-luciferase method. The viability, migration, invasion and angiogenesis of glioma were measured with MTT, colony formation, Transwell and Matrigel tube formation experiments, respectively. The mRNA expression of each gene was tested with qRT-PCR. IGF2BP3 level was determined via western blot and immunohistochemistry. Subcellular fractionation of FOXD3-AS1 was tested with fluorescence in situ hybridization. In vivo tumorigenesis assay was conducted on nude mice. IGF2BP3 high level in glioma cells correlated with patient's prognosis. Downregulation of IGF2BP3 restrained proliferation, migration, invasion and angiogenesis in glioma cells both in vitro and in vivo. There was a binding relationship between IGF2BP3 and miR-128-3p. Besides, FOXD3-AS1 as a sponge of miR-128-3p was located mainly in cytoplasm. Additionally, FOXD3-AS1 facilitated IGF2BP3 level via sponging miR-128-3p to stimulate glioma angiogenesis. FOXD3-AS1 was a sponge of miR-128-3p through upregulating IGF2BP3 in glioma. Our findings shed light on diagnosis and treatment of glioma.

Dual CXCR4/IL-10 Gene-Edited Human Amniotic Mesenchymal Stem Cells Exhibit Robust Therapeutic Properties in Chronic Wound Healing

Although stem cells have attracted attention as a novel therapeutic solution for tissue regeneration, their minimal efficacy remains controversial. In the present study, we aimed to investigate the enhanced therapeutic property of CXCR4/IL-10 dual angiogenic/anti-inflammatory gene knock-in amniotic mesenchymal stem cells (AMM) in a wound-healing model. Dual CXCR4 and IL-10 genes were inserted into the AMM genome using transcription-activator-like effector nuclease (TALEN). Matrigel tube formation and anti-inflammatory effects were assessed in vitro, and efficacy was tested in vivo in a diabetic wound-healing model. CXCR4/IL-10-expressing amniotic MSCs (AMM/CI) strongly expressed CXCR4 and IL-10 genes and robustly promoted tube formation and anti-inflammatory potential. AMM/CI transplantation resulted in accelerated wound healing, as well as high engraftment and re-epithelialization potential. Transplanted AMM/CI also exhibited high angiogenic and decreased pro-inflammatory gene expression in the wound tissue, indicating direct therapeutic effects on wound healing. Taken together, these data indicate that dual angiogenic/anti-inflammatory gene knock-in may be a novel approach to enhance the therapeutic effects of stem cells, and transplantation of AMM/CI can be an alternative therapeutic option in chronic wound healing.

TGF-β1 promotes human breast cancer angiogenesis and malignant behavior by regulating endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) is an important process of angiogenesis, which plays a significant role in in tumor invasion and metastasis, while its regulatory mechanisms in breast cancer remain to be fully elucidated. We previously demonstrated that tumor-associated macrophages (TAMs) can induce EndMT in endothelial cells by secreting CCL18 through the activation of the TGF-β and Notch signaling pathways in breast cancer. This study was designed to study the role of EndMT in breast cancer angiogenesis and progression in order to explore the underlying mechanism. Immunohistochemistry (IHC) was used to evaluate the expression of microvascular density (MVD) and EndMT markers in breast cancer. TGF-β1 was used to induce EndMT models of differentiated-endothelial breast cancer stem-like cells (BCSLCs). In vitro cell migration, proliferation and matrigel tube-formation assays, as well as in vivo nude mouse tumor-bearing model and nude mouse dorsal skinfold window chamber (DSWC) model, were utilized to investigate the effects in order to explore the mechanism of EndMT induced by TGF-β1 on breast cancer progression. In this study, we demonstrated that the EndMT markers were positively associated with MVD indicating unfavorable prognosis of invasive ductal carcinoma (IDC) patients. Functionally, TGF-β1 promoted migration, proliferation and angiogenesis of differentiated-endothelial BCSLCs by inducing EndMT in vitro and promoted tumor growth and angiogenesis in vivo. Mechanically, we revealed TGF-β1 induced EndMT by activation of TGF-β and Notch signaling pathways with increase of p-Smad2/3 and Notch1 expression. Moreover, we found Snail and Slug were key factors of TGF-β and Notch signaling pathways. Our findings elucidated the mechanism of TGF-β1 in the promotion of angiogenesis and progression by EndMT in breast cancer.

ROBO4 Inhibits Irradiation-Induced Microvascular Permeability, Endothelial to Mesenchymal Transition, and Improves Hematopoietic Reconstitution

Background: Hematopoietic Stem Cells Transplantation involves irradiation preconditioning which causes bone marrow endothelial cells dysfunction. While much emphasis is on the reconstitution of an adequate number of HSCs to repopulate the hematopoietic system after the death of malignant cells, EC preservation and normal functioning are indispensable to overcome the preconditioning damages. ROBO4 is an endothelial-specific receptor that is important in maintaining the vasculature. The purpose of this study is to ascertain ROBO4's role in regulating irradiation-induced damage of the endothelium and its subsequent effect on hematopoietic reconstruction. Method: Microvascular endothelial cells were treated with γ-radiation to establish an endothelial cell injury model. The expression of ROBO4 in the microvascular endothelial cells was manipulated employing lentiviral mediated RNAi (shRNA) and gene overexpression technology before irradiation treatment. The permeability of endothelial cells and its mesenchymal transition (Endo-MT) was measured using qPCR, immunocytochemistry, and immunoblotting to analyze the effect on the expression and distribution of junctional molecules (CD144, Connexin 43, and Occludin, etc.), endothelial cell-specific molecules (PECAM-1, VEGFR2 and TIE2, etc.), and specific mesenchymal cell markers (Fibronectin, Vimentin, S100A4, and ACTA2, etc.). Using Transwell and wound healing migration, Matrigel tube formation, and Transwell and FITC-Dextran permeability assays, we determined the changes in endothelial functions after ROBO4 gene manipulation and irradiation. In addition, a co-culture model of endothelial cells and hematopoietic stem cells isolated from C57BL/6 mice was constructed, and the effects of irradiated endothelial cells on the proliferation, differentiation and cell cycle of hematopoietic stem cells were analyzed using flow cytometry. Results: Ionizing radiation upregulates ROBO4 expression but disrupts endothelial junctional molecules. ROBO4 deletion enhances the irradiation-induced degradation of endothelial junctions hence increasing the permeability of the endothelium and also facilitating endothelial to mesenchymal transition (Endo-MT) by activating both the canonical (Smads) and non-canonical (PI3K/AKT/mTOR) pathways. Endothelial ROBO4 overexpression substantially reduces microvascular permeability and mitigates Endo-MT following irradiation. Even though ROBO4 overexpression does not dramatically affect endothelial functions, tube formation and migration potentials of endothelial cells significantly increase upon the knockdown of ROBO4 and irradiation. We also found that EC ROBO4 silencing inhibits the proliferation of HSPC and promotes HSC quiescence. On the other hand, ROBO4 overexpression lessened the inhibitory effects of the injured endothelium on hematopoietic stem cell expansion. Lastly, we saw that ROBO4 overexpression inhibits erythrocytosis and granulocytosis after irradiation compared to the control groups suggesting that EC ROBO4 may play a regulatory role in HSC differentiation. Conclusion: ROBO4 maintains microvascular integrity after radiation preconditioning treatment by regulating permeability and Endo-MT, protects endothelial function, and reduces the inhibitory effect of injured endothelium on hematopoietic reconstitution.

Synthesis, in vitro and in silico studies on novel 3-aryloxymethyl-5-[(2-oxo-2-arylethyl)sulfanyl]-1,2,4-triazoles and their oxime derivatives as potent inhibitors of mPGES-1

Human microsomal prostaglandin E synthase (mPGES)-1 is a glutathione-dependent membrane-bound enzyme which is involved in the terminal stage of prostaglandin E2 (PGE2) synthesis. It has been well reported as a key target for the discovery of new anti-inflammatory and anti-cancer drugs. Specific inhibitors of mPGES-1 are anticipated to selectively restrain the generation of PGE2 induced by the inflammatory stimuli, without obstructing of the regular biosynthesis of other homeostatic prostanoids. Therefore, the design of mPGES-1 inhibitors can represent a better choice to take control of PGE2 associated diseases, compared with conventional non-steroidal anti-inflammatory drugs and cyclooxygenase (COX) inhibitors, which are known for their serious side effects. Although there is an intensive effort for the identification of mPGES-1 inhibitors, none of the unveiled molecules so far have reached the clinical market. Therefore, the development of novel mPGES-1 inhibitors with proper drug-like properties is still an unmet medical need. As a continuation of our research for the identification of new chemotypes which might inhibit this enzyme, we now report the design and synthesis of 3-aryloxymethyl-5-[(2-oxo-2-arylethyl)sulfanyl]-1,2,4-triazoles and their oxime derivatives as inhibitors of human mPGES-1. All synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR (for compounds 12, 14, 15, 26, 27), HMBC (for compounds 6, 7, 8, 16, 19, 23, 28), and MS data. Twenty-four target compounds 7–30 were screened for their mPGES-1/COX-2 inhibitory activities as well as their cytotoxicity. Of these compounds, 20 and 24 showed potent mPGES-1 inhibition by IC50 values of 0.224±0.070 µM and 1.08±0.35 µM, respectively. These two compounds have also been observed to inhibit angiogenesis in matrigel tube formation assay with no toxicity toward HUVEC cells. In silico studies were also held to understand inhibition mechanisms of the most active compounds using molecular docking, molecular dynamics calculations and ADMET predictions.

Circ_0003423 Alleviates Oxidized Low-Density Lipoprotein-Induced Endothelial Cell Injury by Sponging miR-142-3p and Activating Sirtuin 3/Superoxide Dismutase 2 Pathway.

Atherosclerosis (AS) is a common cardiovascular disease with high morbidity and mortality globally. Circular RNAs (circRNAs) have been shown to regulate AS progression. However, the biological role of circ_0,003,423 in AS pathology and its associated mechanism is still unclear. Oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) were used as an AS cell model invitro. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assays were conducted to measure ribonucleic acid (RNA) and protein expression. Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. Cell apoptosis, migration, and angiogenesis were analyzed by flow cytometry, wound healing, and Matrigel tube formation assays. The oxidative stress factors were detected using reactive oxygen species activity (ROS) assay kit, malondialdehyde (MDA) assay kit, and superoxide dismutase (SOD) assay kit. The interaction between microRNA-142-3p (miR-142-3p) and circ_0,003,423 or sirtuin 3 (SIRT3) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Ox-LDL exposure dose-dependently reduced circ_0,003,423 expression in HUVECs. Ox-LDL suppressed the proliferation, migration, and angiogenesis and induced the apoptosis and oxidative stress of HUVECs, which were offset by circ_0,003,423 overexpression. miR-142-3p is a direct target of circ_0,003,423, and circ_0,003,423 overexpression-mediated protective effects in ox-LDL-induced HUVECs were largely reversed by the addition of miR-142-3p mimic. miR-142-3p directly interacted with the 3'UTR of SIRT3 and SIRT3 knockdown reversed anti-miR-142-3p-mediated protective effects in HUVECs upon ox-LDL exposure. Circ_0,003,423 positively regulated SIRT3 expression by sponging miR-142-3p in HUVECs. In addition, circ_0,003,423 could regulate the SIRT3/SOD2 signaling pathway in HUVECs. Circ_0,003,423 protected HUVECs from ox-LDL-induced dysfunction by sponging miR-142-3p and activating SIRT3/SOD2 signaling, which provided new potential targets for AS intervention and treatment.

SAMD1 attenuates antiphospholipid syndrome-induced vascular injury and pregnancy complications.

ObjectiveThis study was intended to investigate the effect of SAMD1 on antiphospholipid syndrome (APS)‐induced vascular injury in human umbilical vein endothelial cells (HUVECs) and pregnancy complications in mice.MethodsThe expression of SAMD1 in APS patients and healthy controls was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). Anti‐B2GPI and anticardiolipin antibody (ACA) levels were tested by enzyme‐linked immunosorbent assay, MMP‐9, iNOS, ICAM‐1, and MCP‐1 mRNA and protein levels determined by qRT‐PCR and Western blot, cellular senescence detected by β‐galactosidase staining, cell proliferation ability detected by CCK‐8 assay, cell viability detected by trypan blue staining, cell mobility detected by Transwell, and cell angiogenesis ability detected by matrigel tube formation assay. An APS pregnant mouse model was constructed, and the embryo absorption rate was calculated.ResultsSAMD1 expression was low in serum of APS patients, which was correlated with the history of thrombosis and the number of adverse pregnancies. Anti‐B2GPI and ACA levels were increased in APS. The expressions of MMP‐9, iNOS, ICAM‐1, and MCP‐1 were also significantly upregulated in HUVECs treated with APS serum. APS promoted HUVEC senescence and inhibited cell proliferation, migration, and angiogenesis. Overexpression of SAMD1 reversed the above results. Experiments on the APS pregnant mouse model confirmed that overexpression of SAMD1 reduced the rate of fetal loss.ConclusionSAMD1 may reduce APS‐induced vascular injury and embryo loss by regulating cellular senescence, proliferation, migration, and angiogenesis.

PFKFB3-mediated glycometabolism reprogramming modulates endothelial differentiation and angiogenic capacity of placenta-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have a great potential ability for endothelial differentiation, contributing to an effective means of therapeutic angiogenesis. Placenta-derived mesenchymal stem cells (PMSCs) have gradually attracted attention, while the endothelial differentiation has not been fully evaluated in PMSCs. Metabolism homeostasis plays an important role in stem cell differentiation, but less is known about the glycometabolic reprogramming during the PMSCs endothelial differentiation. Hence, it is critical to investigate the potential role of glycometabolism reprogramming in mediating PMSCs endothelial differentiation.MethodsDil-Ac-LDL uptake assay, flow cytometry, and immunofluorescence were all to verify the endothelial differentiation in PMSCs. Seahorse XF Extracellular Flux Analyzers, Mito-tracker red staining, Mitochondrial membrane potential (MMP), lactate secretion assay, and transcriptome approach were to assess the variation of mitochondrial respiration and glycolysis during the PMSCs endothelial differentiation. Glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) was considered a potential modulator for endothelial differentiation in PMSCs by small interfering RNA. Furthermore, transwell, in vitro Matrigel tube formation, and in vivo Matrigel plug assays were performed to evaluate the effect of PFKFB3-induced glycolysis on angiogenic capacities in this process.ResultsPMSCs possessed the superior potential of endothelial differentiation, in which the glycometabolic preference for glycolysis was confirmed. Moreover, PFKFB3-induced glycometabolism reprogramming could modulate the endothelial differentiation and angiogenic abilities of PMSCs.ConclusionsOur results revealed that PFKFB3-mediated glycolysis is important for endothelial differentiation and angiogenesis in PMSCs. Our understanding of cellular glycometabolism and its regulatory effects on endothelial differentiation may propose and improve PMSCs as a putative strategy for clinical therapeutic angiogenesis.

LIPUS Promotes Endothelial Differentiation and Angiogenesis of Periodontal Ligament Stem Cells by Activating Piezo1.

Low-intensity pulsed ultrasound (LIPUS) promotes differentiation and regulates biological functions of various stem cells, but its effect on the endothelial differentiation of periodontal ligament stem cells (PDLSCs) is unclear. This study investigated the effect of LIPUS on endothelial differentiation and angiogenesis in PDLSCs and the role of the mechanically sensitive ion channel Piezo1 in this process. PDLSCs obtained from healthy people were used for endothelial induction, and 10 μg/ml lipopolysaccharide (LPS) was used to simulate the inflammatory state. The induced cells were treated with LIPUS (50 mW/cm2, 1.5 MHz) to study its effect on the endothelial differentiation of PDLSCs and the tube formation of differentiated cells. PCR, flow cytometry, immunofluorescence, and Matrigel tube formation assays were used to detect the differentiation and tube formation of PDLSCs. GsMTx4 was used to inhibit the expression of Piezo1, and the role of the Piezo1 pathway in the endothelial differentiation and microvascular formation of PDLSCs after LIPUS treatment was studied. The data showed that LIPUS increased endothelial differentiation and angiogenesis in PDLSCs under inflammatory or noninflammatory conditions. The use of an inhibitor weakened the effect of LIPUS. This study demonstrated that LIPUS can activate the expression of Piezo1 and promote the endothelial differentiation and microvascular formation of PDLSCs.

Human umbilical cord-derived mesenchymal stem cells not only ameliorate blood glucose but also protect vascular endothelium from diabetic damage through a paracrine mechanism mediated by MAPK/ERK signaling

BackgroundEndothelial damage is an initial step of macro- and micro-vasculature dysfunctions in diabetic patients, accounting for a high incidence of diabetic vascular complications, such as atherosclerosis, nephropathy, retinopathy, and neuropathy. However, clinic lacks effective therapeutics targeting diabetic vascular complications. In field of regenerative medicine, mesenchymal stem cells, such as human umbilical cord-derived MSCs (hucMSCs), have great potential in treating tissue damage.MethodsTo determine whether hucMSCs infusion could repair diabetic vascular endothelial damage and how it works, this study conducted in vivo experiment on streptozotocin-induced diabetic rat model to test body weight, fasting blood glucose (FBG), serum ICAM-1 and VCAM-1 levels, histopathology and immunohistochemical staining of aorta segments. In vitro experiment was further conducted to determine the effects of hucMSCs on diabetic vascular endothelial damage, applying assays of resazurin staining, MTT cell viability, wound healing, transwell migration, and matrigel tube formation on human umbilical vein endothelial cells (HUVECs). RNA sequencing (RNAseq) and molecular experiment were conducted to clarify the mechanism of hucMSCs.ResultsThe in vivo data revealed that hucMSCs partially restore the alterations of body weight, FBG, serum ICAM-1 and VCAM-1 levels, histopathology of aorta and reversed the abnormal phosphorylation of ERK in diabetic rats. By using the conditioned medium of hucMSCs (MSC-CM), the in vitro data revealed that hucMSCs improved cell viability, wound healing, migration and angiogenesis of the high glucose-damaged HUVECs through a paracrine action mode, and the altered gene expressions of IL-6, TNF-α, ICAM-1, VCAM-1, BAX, P16, P53 and ET-1 were significantly restored by MSC-CM. RNAseq incorporated with real-time PCR and Western blot results clarified that high glucose activated MAPK/ERK signaling in HUVECs, while MSC-CM reversed the abnormal phosphorylation of ERK and overexpressions of MKNK2, ERBB3, MYC and DUSP5 in MAPK/ERK signaling pathway.ConclusionsHucMSCs not only ameliorated blood glucose but also protected vascular endothelium from diabetic damage, in which MAPK/ERK signaling mediated its molecular mechanism of paracrine action. Our findings provided novel knowledge of hucMSCs in the treatment of diabetes and suggested a prospective strategy for the clinical treatment of diabetic vascular complications.

Paradoxical role of β8 integrin on angiogenesis and vasculogenic mimicry in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive and highly vascularized brain tumor with poor prognosis. Endothelial cell-dependent angiogenesis and tumor cell-dependent Vasculogenic mimicry (VM) synergistically contribute to glioma vascularization and progression. However, the mechanism underlying GBM vascularization remains unclear. In this study, GBM stem cells (GSCs) were divided into high and low β8 integrin (ITGB8) subpopulations. Co-culture assays followed by Cell Counting Kit-8 (CCK-8), migration, Matrigel tube formation, and sprouting assays were conducted to assess the proliferative, migratory and angiogenic capacity of GBM cells and human brain microvascular endothelial cells (hBMECs). An intracranial glioma model was constructed to assess the effect of ITGB8 on tumor vascularization in vivo. Our results indicated that ITGB8 expression was elevated in GSCs and positively associated with stem cell markers in glioma tissues, and could be induced by hypoxia and p38 activation. ITGB8 in GSCs inhibited the angiogenesis of hBMECs in vitro, while it promoted the ability of network formation and expression of VM-related proteins. The orthotopic GBM model showed that ITGB8 contributed to decreased angiogenesis, meanwhile enhanced invasiveness and VM formation. Mechanistic studies indicated that ITGB8-TGFβ1 axis modulates VM and epithelial-mesenchymal transition (EMT) process via Smad2/3-RhoA signaling. Together, our findings demonstrated a differential role for ITGB8 in the regulation of angiogenesis and VM formation in GBM, and suggest that pharmacological inhibition of ITGB8 may represent a promising therapeutic strategy for treatment of GBM.

SDF-1-edited human amniotic mesenchymal stem cells stimulate angiogenesis in treating hindlimb ischaemia.

Although stem cells have extensively been studied as a novel vehicle for tissue repair, their sustained efficacy remains controversial. In this study, we aimed to investigate the angiogenic potency over time of stromal cell‐derived factor‐1 (SDF‐1) gene‐edited amniotic mesenchymal stem cells (AMM/S) in a hindlimb ischaemia model. An SDF‐1 transgene was inserted into the AMM cell genome via transcription activator‐like effector nuclease (TALEN) mediated knock‐in, and cell migration, Matrigel tube formation, and in vivo Matrigel plug assays were performed. AMM/S were also transplanted into hindlimb ischaemia model mice. Blood perfusion, therapeutic potential, histology, capillary density and in vivo angiogenic assays were performed. AMM/S exhibited high expression of the SDF‐1 gene, and robustly promoted migration, proliferation and microvascular formation. AMM/S transplantation significantly increased blood perfusion and limb loss prevention compared with AMM. AMM/S also significantly inhibited increased capillary density and expression of angiogenic factors in the ischaemic hindlimb. Our study demonstrated that AMM/S provides a significant therapeutic effect in ischaemic hindlimbs by enhancing angiogenesis.

Interleukin-33 protects mice against hindlimb ischemic injury by enhancing endothelial angiogenesis

Peripheral vascular disease usually leads to vascular injury and inflammatory reaction, and the main therapeutic measures are improving angiogenesis and restoring blood flow. Interleukin-33 (IL-33) is a pleiotropic cytokine implicated in immune responses and tissue repair. Here, we explore the effect of IL-33 in hindlimb ischemic injury and elucidate the potential mechanisms of action. The expression of IL-33 and its receptor ST2 were obviously elevated in ischemic hindlimb of mice underwent ligation surgery. Exogenous IL-33 apparently facilitated blood flow restoration in ischemic hindlimb, whereas ST2-deficient mice displayed severe defects in ischemic hindlimb repair. The activation of IL-33/ST2 signaling contributed to revascularization in ischemic hindlimb, which was related to modulation of proangiogenic function of endothelial cells. Further ex vivo and in vitro studies revealed that IL-33 clearly accelerated angiogenesis by Matrigel plug and tube formation assays. Mechanically, the angiogenic function of IL-33 is involved in regulation of Akt/eNOS pathway. All together, these findings imply that IL-33-mediated endothelial angiogenesis may represent a prospective effective therapy for hindlimb ischemic damage.

Establishment and biological effect evaluation of prevascularized porous β-tricalcium phosphate tissue engineered bone

To evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group ( n=6) and porous β-TCP scaffold as the control group ( n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. The isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel ( P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group ( P<0.05), except for area fraction at 4 weeks after implantation ( P>0.05). The prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.

Circ_ROBO2/miR-186-5p/TRIM14 axis regulates oxidized low-density lipoprotein-induced cardiac microvascular endothelial cell injury.

BackgroundCoronary artery disease (CAD) is one of the main risks of death, which is mainly caused by coronary arteries arteriosclerosis. Circular RNAs (circRNAs) have shown important regulatory roles in cardiovascular diseases. We amid to explore the role of circ_ROBO2 in CAD.MethodsCardiac microvascular endothelial cells (CMECs) stimulated by oxidized low-density lipoprotein (ox-LDL) were served as the cellular model of CAD. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay were performed to detect RNA levels and protein levels, respectively. Cell proliferation was assessed by 5-ethynyl-2′-deoxyuridine (EdU) assay and Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed for measuring cell apoptosis. Matrigel tube formation assay was used to evaluate angiogenesis ability. The intermolecular interaction was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA-pull down assays.ResultsThe expression of circ_ROBO2 was upregulated in CAD patients and ox-LDL-induced CMECs. Treatment of ox-LDL suppressed cell proliferation and angiogenic ability as well as promoted the apoptosis of CMECs partly by upregulating circ_ROBO2. MicroRNA-186-5p (miR-186-5p) was identified as a target of circ_ROBO2, and circ_ROBO2 knockdown attenuated ox-LDL-induced damage in CMECs by sponging miR-186-5p. Tripartite motif containing 14 (TRIM14) acted as a target of miR-186-5p, and TRIM14 overexpression alleviated miR-186-5p-mediated inhibitory effect on ox-LDL-induced injury in CMECs. Circ_ROBO2 positively regulated TRIM14 expression by sponging miR-186-5p.ConclusionCirc_ROBO2 played a promoting role in ox-LDL-induced CMECs injury by sponging miR-186-5p and regulating TRIM14, providing a promising treatment strategy for CAD.