Matrigel Tube Formation Assay Research Articles

CAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Abstract Background and Aims Others and we have previously demonstrated that cAMP/PKA signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function. Methods The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated over expression of dominant negative (DN) and constitutive active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using cAMP analogue N6-Benzoyl-cAMP (5 mM) and PKI (1 µM), respectively. Pharmacologically, Rac1 was inhibited using NSC 23766 (10 µM) and RhoA was activated using Rho activator I (Cytoskeleton Inc.). In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. Results Pharmacologically, specific activation of PKA induced EC migration (Wound healing assay), tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. These pro-angiogenesis activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA-signalling resulted in an increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and a strong inhibition of RhoA/Rock pathway. Pharmacological activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, over expression of either dominant negative RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis. Conclusion We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/ROCK signalling may offer an important target to intervene therapeutic angiogenesis.

Effect of EMB-FUBINACA on brain endothelial cell angiogenesis: Expression analysis of angiogenic markers.

Angiogenesis is the process by which blood vessels are generated from preexisting ones. Synthetic cannabinoids represent new psychoactive substances that bind to the cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) and simulate similar effects of tetrahydrocannabinol, the primary component found in cannabis. In the present study, we used the synthetic cannabinoid EMB-FUBINACA to study its impact on brain angiogenesis. Human brain microvascular endothelial cells (HBMECs) were cultivated in DMEM media before being subjected to different concentrations of EMB-FUBINACA and the control. Cell viability and the migration rates of HBMECs were evaluated using the viability and wound healing assays, respectively. An in vitro Matrigel Tube Formation Assay was carried out to measure the angiogenic capacity of endothelial cells. Angiopoietin-1 (ANG-1), Angiopoietin-2 (ANG-2), and vascular endothelial growth factor (VEGF) mRNA expression were detected using Real-Time PCR. The released VEGF, ANG-1, and ANG-2 concentrations were detected using ELISA. Western blotting was performed to measure the levels of phosphorylated GSK-3β, VEGF, ANG-1, and ANG-2. EMB-FUBINACA stimulated endothelial cell proliferation, migration, and capillary tube-like formation and promoted the expression of proangiogenic factors on RNA and protein levels. This study points out that the synthetic cannabinoid EMB-FUBINACA is a potential candidate for further investigations to confirm its potential as an inducer of brain angiogenesis. This could encourage researchers to create a new therapeutic approach for angiogenesis-related diseases.

MiR-143-5p regulates the proangiogenic potential of human dental pulp stem cells by targeting HIF-1α/RORA under hypoxia: A laboratory investigation in pulp regeneration.

Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process. Human dental pulp stem cells were isolated, cultured and characterized invitro. Cobalt chloride (CoCl2) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured invitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration. Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The invivo experiments showed that hypoxia treatment (50 and 100 μM CoCl2) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis invitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration. CoCl2-induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA.

Fibroblast growth factor 23 inhibition attenuates steroid-induced osteonecrosis of the femoral head through pyroptosis

Steroid-induced osteonecrosis of the femoral head (SONFH) is the predominant cause of non-traumatic osteonecrosis of the femoral head (ONFH). Impaired blood supply and reduced osteogenic activity of the femoral head are the key pathogenic mechanisms of SONFH. Fibroblast growth factor 23 (FGF23) levels are not only a biomarker for early vascular lesions caused by abnormal mineral metabolism, but can also act directly on the peripheral vascular system, leading to vascular pathology. The aim of this study was to observe the role of FGF23 on bone microarchitecture and vascular endothelium, and to investigate activation of pyroptosis in SONFH. Lipopolysaccharide (LPS) combined with methylprednisolone (MPS) was applied for SONFH mouse models, and adenovirus was used to increase or decrease the level of FGF23. Micro-CT and histopathological staining were used to observe the structure of the femoral head, and immunohistochemical staining was used to observe the vascular density. The cells were further cultured in vitro and placed in a hypoxic environment for 12 h to simulate the microenvironment of vascular injury during SONFH. The effect of FGF23 on osteogenic differentiation was evaluated using alkaline phosphatase staining, alizarin red S staining and expression of bone formation-related proteins. Matrigel tube formation assay in vitro and immunofluorescence were used to detect the ability of FGF23 to affect endothelial cell angiogenesis. Steroids activated the pyroptosis signaling pathway, promoted the secretion of inflammatory factors in SONFH models, led to vascular endothelial dysfunction and damaged the femoral head structure. In addition, FGF23 inhibited the HUVECs angiogenesis and BMSCs osteogenic differentiation. FGF23 silencing attenuated steroid-induced osteonecrosis of the femoral head by inhibiting the pyroptosis signaling pathway, and promoting osteogenic differentiation of BMSCs and angiogenesis of HUVECs in vitro.

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.

Abstract 15406: Loss of Estrogen Receptor α Inhibits Angiogenic Function in Right Ventricular Endothelial Cells (RVECs)

Introduction: Right ventricular failure (RVF) is the leading cause of death in patients with pulmonary hypertension (PH). While the pathophysiology of RVF is complex and multifactorial, insufficient angiogenesis and impaired RV endothelial cell (RVEC) function have been identified as key contributors. 17β-estradiol (E2), the most abundant female sex steroid, exerts RV-protective effects including stimulation of angiogenesis. However, the mechanism underlying E2’s pro-angiogenic function are unknown. Hypothesis: Estrogen Receptor (ER) α is necessary for RV angiogenesis. Methods: To identify the role of ERα in RVEC function and RV angiogenesis, ERα loss-of-function mutant (ERα Mut ) rats were generated by CRISPR-Cas9. In vivo studies were performed in both male and female wild-type (WT) and ERα Mut rats. RV capillary density was assessed by immunofluorescence. RVECs were isolated from male or female WT and ERα Mut rats. Angiogenic function was evaluated by matrigel tube formation assay at 10h. RVEC proliferation (at 24h) was measured by Incucyte image-based analysis and Thymidine incorporation assay. RVEC migration (at 24h) was examined by transwell migration assay. Angiogenesis mediators in RVECs were quantified via Western Blot. P<0.05 was considered significant. Results: Female WT rats exhibited increased RV capillary density compared to males (p<0.001). However, loss of functioning ERα decreased RV capillary density (p<0.05). Loss of ERα impaired proliferation (24h) capability in female, but not male, RVECs (p<0.001). On the other hand, male, but not female, ERα Mut RVECs exhibited reduced cell migration in response to VEGF-A (p<0.01). Loss of ERα reduced RVEC tube formation in both sexes (p<0.05). At a molecular level, male ERα Mut RVECs expressed less VEGF-A, angiopoietin-2, and Id1 than WT (p<0.01) . Conclusions: ERα is necessary for RVEC angiogenic function. However, ERα effects on angiogenesis are sexually dimorphic. Male ERα Mut RVECs exhibit reduced cell migration potential and less angiogenesis mediators, whereas female ERα Mut RVECs exhibit decreased proliferation potential in vitro and reduced female RV capillary density in vivo . Targeting ERα may allow for sex-specific therapies aimed at enhancing RV adaptation in PH.

Site-specific periosteal cells with distinct osteogenic and angiogenic characteristics.

This study aimed to investigate the site-specific characteristics of rat mandible periosteal cells (MPCs) and tibia periosteal cells (TPCs) to assess the potential application of periosteal cells (PCs) in bone tissue engineering (BTE). MPCs and TPCs were isolated and characterized. The potential of proliferation, migration, osteogenesis and adipogenesis of MPCs and TPCs were evaluated by CCK-8, scratch assay, Transwell assay, alkaline phosphatase staining and activity, Alizarin Red S staining, RT‒qPCR, and Western blot (WB) assays, respectively. Then, these cells were cocultured with human umbilical vein endothelial cells (HUVECs) to investigate their angiogenic capacity, which was assessed by scratch assay, Transwell assay, Matrigel tube formation assay, RT‒qPCR, and WB assays. MPCs exhibited higher osteogenic potential, higher alkaline phosphatase activity, and more mineralized nodule formation, while TPCs showed a greater capability for proliferation, migration, and adipogenesis. MPCs showed higher expression of angiogenic factors, and the conditioned medium of MPCs accelerated the migration of HUVECs, while MPC- conditioned medium induced the formation of more tubular structure in HUVECs in vitro. These data suggest that compared to TPCs, MPCs exert more consequential proangiogenic effects on HUVECs. PCs possess skeletal site-specific differences in biological characteristics. MPCs exhibit more eminent osteogenic and angiogenic potentials, which highlights the potential application of MPCs for BTE. Autologous bone grafting as the main modality for maxillofacial bone defect repair has many limitations. Constituting an important cell type in bone repair and regeneration, MPCs show greater potential for application in BTE, which provides a promising treatment option for maxillofacial bone defect repair.

SAMD1 attenuates antiphospholipid syndrome-induced pregnancy complications.

This study was intended to investigate the effect of SAMD1 on antiphospholipid syndrome (APS)-induced pregnancy complications in mice. The mRNA and protein expression of SAMD1 in APS patients and healthy controls was detected by qRT-PCR and western blot. Anti-B2 GPI and ACA levels were tested by ELISA, 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. SAMD1 expression was low in serum of APS patients, which was correlated with the history of thrombosis and the number of adverse pregnancies. Anti-B2 GPI 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. SAMD1 may reduce APS-induced embryo loss by regulating cellular senescence, proliferation, migration, and angiogenesis.

Long-term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction.

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long-term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET-1, ICAM-1, VCAM-1, and E-selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM-1. Expression of PDGF-BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT-PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM-1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF-BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.

Abstract P3156: Activated Fibroblast-Derived Exosomal Mir-216a-5p Promotes Endothelial Dysfunction By Targeting PGM5 In Diabetic Heart

Background: Diabetic patients are more susceptible to heart failure (HF). High blood glucose level in diabetic patients eventually triggers the body's inflammatory response and causes cardiac fibroblast activation, endothelial cell dysfunction (ECD), and, ultimately, HF. Previously, we have shown that activated fibroblast-mediated ECD leads to HF. However, the molecular mechanism of fibroblast-induced ECD in diabetics is not yet well defined. Therefore, we hypothesized that “myofibroblast in the diabetic heart secretes exosomes loaded with antiangiogenic/profibrotic factors, which impair EC function.” Methods: Exosomes were isolated from diabetic mice plasma and fibroblast conditioned media by ultracentrifugation and characterized by nanosight & electron microscopy. We cultured mouse primary heart endothelial cells in EC growth media. ECs were treated with exosomes derived from fibroblasts (treated with either 25mM glucose, 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Pathway-based miRNA array was screened with exosome-derived miR, and targets were predicted bioinformatically for selected miR. Results: Glu-AngII cotreatment significantly activated fibroblasts as shown by qPCR (Col1α, and FN expression) and western blot (pSmad2/Smad2, p-p38/p38). Interestingly, exosomes derived from activated fibroblasts significantly induced ECD (eNOS, VEGF, CD31 genes, and proteins expression). We further checked the effect of diabetic mice plasma exosomes on ECs function. We found significantly impaired endothelial function, as shown by Matrigel tube formation and Boyden chamber migration assays. Micro RNA array and qPCR data showed that miR-216a-5p was highly upregulated in exosomes derived from FBs cotreated with Glu-AngII. Pathway-based analysis revealed that PGM5 could be a potential target in miR-216-induced ECD. Interestingly, miR-216a-5p inhibition significantly rescued diabetes-induced PGM5 inhibition and improved endothelial function Conclusions: Taken together, this study demonstrates that fibroblast in the diabetic heart releases miR-216a-5p through exosomes, which promote endothelial dysfunction via the miR-216a-5p/PGM5 axis.

Bradykinin-pretreated Human cardiac-specific c-kit+ Cells Enhance Exosomal miR-3059-5p and Promote Angiogenesis Against Hindlimb Ischemia in mice.

Protection of cardiac function following myocardial infarction was largely enhanced by bradykinin-pretreated cardiac-specific c-kit+ (BK-c-kit+) cells, even without significant engraftment, indicating that paracrine actions of BK-c-kit+ cells play a pivotal role in angiogenesis. Nevertheless, the active components of the paracrine actions of BK-c-kit+ cells and the underlying mechanisms remain unknown. This study aimed to define the active components of exosomes from BK-c-kit+ cells and elucidate their underlying protective mechanisms. Matrigel tube formation assay, cell cycle, and mobility in human umbilical vein endothelial cells (HUVECs) and hindlimb ischemia (HLI) in mice were applied to determine the angiogenic effect of condition medium (CM) and exosomes. Proteome profiler, microRNA sponge, Due-luciferase assay, microRNA-sequencing, qRT-PCR, and Western blot were used to determine the underlying mechanism of the angiogenic effect of exosomes from BK-c-kit+. As a result, BK-c-kit+ CM and exosomes promoted tube formation in HUVECs and the repair of HLI in mice. Angiogenesis-related proteomic profiling and microRNA sequencing revealed highly enriched miR-3059-5p as a key angiogenic component of BK-c-kit+ exosomes. Meanwhile, loss- and gain-of-function experiments revealed that the promotion of angiogenesis by miR-3059-5p was mainly through suppression of TNFSF15-inhibited effects on vascular tube formation, cell proliferation and cell migration. Moreover, enhanced angiogenesis of miR-3059-5p-inhibited TNFSF15 has been associated with Akt/Erk1/2/Smad2/3-modulated signaling pathway. Our results demonstrated a novel finding that BK-c-kit+ cells enrich exosomal miR-3059-5p to suppress TNFSF15 and promote angiogenesis against hindlimb ischemia in mice.

Girdin regulates both migration and angiogenesis in pancreatic cancer cell lines.

Girdin, an actin‑binding protein, is reportedly involved in the invasion and angiogenesis of various cancers. It has been suggested that the flavonoid Scutellarin (SCU) inhibits Girdin signaling. In the present study, the function and therapeutic applications of Girdin in pancreatic cancer (PaCa) were investigated. Immunohistochemical staining of Girdin in resected PaCa specimens from the Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Science showed that high Girdin expression was associated with poor overall survival and relapse‑free survival, as well as with T factor, indicating invasion into the surrounding tissues. On the other hand, Girdin was highly expressed in almost all PaCa cell lines, and the migration ability of Girdin‑knockdown cell lines was decreased even under epidermal growth factor (EGF) stimulation. In addition, SCU suppressed PaCa cell migration by inhibiting the phosphorylation of Girdin. The expression and production of vascular endothelial growth factor A (VEGF‑A) was significantly decreased in Girdin‑knockdown cell lines. Furthermore, in Matrigel tube formation assays performed using culture supernatant, the lumen‑forming ability of vascular endothelial cells was also decreased in Girdin‑knockdown cell lines. However, SCU treatment did not significantly alter the expression or production of VEGF‑A. These results suggested that Girdin is involved in EGF signaling‑mediated migration of PaCa cells, that SCU inhibits PaCa invasion by suppressing Girdin activity, and that Girdin is also involved in angiogenesis via an activation pathway different from the action site of SCU. Girdin may be a prognostic biomarker, and the development of a novel molecular‑targeted drugs for Girdin may improve the prognosis of PaCa in the future.

Cryopreservation of human cerebral microvascular endothelial cells with glycerol

The cryopreservation of human cerebral microvascular endothelial cells (hCMEC) has facilitated their commercial availability for research studying the blood–brain barrier. The currently employed cryopreservation protocol uses 10% dimethyl sulfoxide (Me2SO) in cell medium, or 5% Me2SO in 95% fetal bovine serum (FBS) as cryoprotective agents (CPAs). However, Me2SO is toxic to cells and FBS is animal-derived and not chemically defined, so reducing the concentrations of these components is desirable. Recently, we showed that cryopreserving hCMEC in cell medium with 5% Me2SO and 6% hydroxyethyl starch (HES) results in over 90% post-thaw cell viability. This previous work was performed using an interrupted slow cooling (graded freezing) approach followed by SYTO13/GelRed staining to assay for membrane integrity. In this paper, we repeated graded freezing of hCMEC in cell medium containing 5% Me2SO and 6% HES, but this time using Calcein AM/propidium iodide staining to ensure that the stain is an equivalent alternative to SYTO13/GelRed for assessment of cell viability, and that results are comparable to those previously published. Next, using graded freezing experiments and Calcein AM/propidium iodide staining, we examined the effectiveness of non-toxic glycerol as a CPA at different concentrations, loading times, and cooling rates. The cryobiological response of hCMEC was used to develop a protocol that optimizes both the permeating and non-permeating capabilities of glycerol. HCMEC in cell medium loaded with 10% glycerol for 1 h at room temperature, ice nucleated at −5 °C and held for 3 min, and then cooled at −1 °C/min to −30 °C before plunging into liquid nitrogen had post-thaw viability of 87.7% ± 1.8%. Matrigel tube formation assay and immunocytochemical staining of junction protein ZO-1 were carried out on post-thaw hCMEC to ensure that the cryopreserved cells were viable and functional, in addition to being membrane-intact.

Platelet-Derived Growth Factor-BB Priming Enhances Vasculogenic Capacity of Bone Marrow-Derived Endothelial Precursor Like Cells.

Human endothelial progenitor cells (EPCs) were first identified in the peripheral blood and later in the cord blood and bone marrow (BM) with different vascularization capacity and different surface marker profiles. However, their identity and functional roles in neovascularization have not been clearly demonstrated in vivo and in vitro. Characterization of BM-EPC like cells were performed by fluorescence-activated cell sorting, immunofluorescence staining, enzyme-linked immunosorbent assay, Matrigel tube formation assay,andwestern blot analysis. BM-EPC like cells were identified by selective adhesion to fibronectin and collagen from BM mononuclear cells, which generate fast-growing colonies with spindle morphology, express surface markers of CD105, vWF, UEA-I lectin binding, secrete HGF, VEGF, TGF-beta1 but can be distinguished from circulating EPC and endothelial cells by no expression of surface markers such as CD31, CD309, CD45, and CD34. These BM-EPC like cells shared many cell surface markers of BM-mesenchymal stem cells (MSC) but also can be distinguished by their vasculogenic property and other unique surface markers. Furthermore, the vasculogenic capacity of BM-EPC like cells were enhanced by co-culture of BM-MSC or PDGF-BB priming. PDGF-BB stimulated cell migration, proliferation, and secretion of laminin β-1, which was proposed as one of the mechanisms involved in the better vascularization of BM-EPC like cells. PDGF-BB priming may be applied to improve the potency and function of BM-EPC like cells as vasculogenic cell therapy for the ischemic vascular repair.

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.

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.

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.