scholarly journals Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

Processes ◽  
2019 ◽  
Vol 7 (6) ◽  
pp. 356 ◽  
Author(s):  
Si Chen ◽  
P. I. Imoukhuede
Keyword(s):  
Systems Biology ◽  
Growth Factor ◽  
Plasma Membranes ◽  
Temporal Dynamics ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Dermal Fibroblasts ◽  
Steady Increase

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.

scholarly journals Pharmacologic Characterization of a Kinetic In Vitro Human Co-Culture Angiogenesis Model Using Clinically Relevant Compounds

2013 ◽  
Vol 18 (10) ◽  
pp. 1234-1245 ◽  
Author(s):  
Ashley Wolfe ◽  
Belinda O’Clair ◽  
Vincent E. Groppi ◽  
Dyke P. McEwen
Keyword(s):  
Growth Factor ◽  
Umbilical Vein ◽  
Dermal Fibroblasts ◽  
Tube Length ◽  
Endothelial Cell Proliferation ◽  
Discovery Research ◽  
Drug Discovery Research ◽  
Angiogenesis Model

Angiogenesis, the formation of new vessels from preexisting vessels, involves multiple cell types acting in concert to cause endothelial cell proliferation, migration, and differentiation into microvascular arrays. Under pathologic conditions, microenvironment changes result in altered blood vessel production. Historically, in vitro angiogenesis assays study individual aspects of the process and tend to be variable, difficult to quantify, and limited in clinical relevance. Here, we describe a kinetic, quantitative, co-culture angiogenesis model and demonstrate its relevance to in vivo pharmacology. Similar to in vivo angiogenesis, a co-culture of human umbilical vein endothelial cells with normal human dermal fibroblasts remains sensitive to multiple cytokines, resulting in a concentration-dependent stimulation of tube formation over time. Treatment with axitinib, a selective vascular endothelial growth factor (VEGF) antagonist, inhibited VEGF-mediated tube length and branch point formation and was selective for inhibiting VEGF over basic fibroblast growth factor (bFGF), similar to previous studies. Conversely, an FGFR-1 selective compound, PD-161570, was more potent at inhibiting bFGF-mediated angiogenesis. These results demonstrate the cytokine dynamics, selective pharmacology, and translational application of this model system. Finally, combining quantitative angiogenic biology with kinetic, live-content imaging highlights the importance of using validated in vitro models in drug discovery research.

Anti-Angiogenic Activity of Rotenoids from the Stems of Derris trifoliata

Planta Medica ◽  
2018 ◽  
Vol 84 (11) ◽  
pp. 779-785 ◽  
Author(s):  
Yanisa Mittraphab ◽  
Nattaya Ngamrojanavanich ◽  
Kuniyoshi Shimizu ◽  
Kiminori Matsubara ◽  
Khanitha Pudhom
Keyword(s):  
Endothelial Cells ◽  
Cancer Cells ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Ex Vivo Model ◽  
Cell Growth And Migration ◽  
Vitro Assay ◽  
Angiogenic Activity

The plants in the genus Derris have proven to be a rich source of rotenoids, of which cytotoxic effect against cancer cells seem to be pronounced. However, their effect on angiogenesis playing a crucial role in both cancer growth and metastasis has been seldom investigated. This study aimed at investigating the effect of the eight rotenoids (1–8) isolated from Derris trifoliata stems on three cancer cells and angiogenesis. Among them, 12a-hydroxyrotenone (2) exhibited potent inhibition on both cell growth and migration of HCT116 colon cancer cells. Further, anti-angiogenic assay in an ex vivo model was carried out to determine the effect of the isolated rotenoids on angiogenesis. Results revealed that 12a-hydroxyrotenone (2) displayed the most potent suppression of microvessel sprouting. The in vitro assay on human umbilical vein endothelial cells was performed to determine whether compound 2 elicits anti-angiogenic effect and its effect was found to occur via suppression of endothelial cells proliferation and tube formation, but not endothelial cells migration. This study provides the first evidence that compound 2 could potently inhibit HCT116 cancer migration and anti-angiogenic activity, demonstrating that 2 might be a potential agent or a lead compound for cancer therapy.

scholarly journals Long non-coding RNA H19 promotes corneal neovascularization by targeting microRNA-29c

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Baoqi Sun ◽  
Yiheng Ding ◽  
Xin Jin ◽  
Shuo Xu ◽  
Hong Zhang
Keyword(s):  
Growth Factor ◽  
Umbilical Vein ◽  
Corneal Neovascularization ◽  
Tube Formation ◽  
Vascular Endothelial ◽  
Non Coding Rna ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Growth Factor ◽  
Long Non Coding Rna

AbstractLong non-coding RNA (lncRNA) H19 has been implicated in tumor angiogenesis. However, whether H19 regulates the progression of corneal neovascularization (CNV) is unclear. The present study aimed to determine the function of H19 in CNV and its possible molecular mechanism. Here, we found that the H19 levels were remarkably increased in vascularized corneas and basic fibroblast growth factor (bFGF)-treated human umbilical vein endothelial cells (HUVECs). In vitro, H19 up-regulation promoted proliferation, migration, tube formation and vascular endothelial growth factor A (VEGFA) expression in HUVECs, and it was found to down-regulate microRNA-29c (miR-29c) expression. Bioinformatics analysis revealed that H19 mediated the above effects by binding directly to miR-29c. In addition, miR-29c expression was markedly reduced in vascularized corneas and its expression also decreased in bFGF-treated HUVECs in vitro. MiR-29c targeted the 3′ untranslated region (3′-UTR) of VEGFA and decreased its expression. These data suggest that H19 can enhance CNV progression by inhibiting miR-29c, which negatively regulates VEGFA. This novel regulatory axis may serve as a potential therapeutic target for CNV.

scholarly journals ErMiao San Inhibits Angiogenesis in Rheumatoid Arthritis by Suppressing JAK/STAT Signaling Pathways

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Lianhua He ◽  
Qingxia Qin ◽  
Juan He ◽  
Han Wang ◽  
Yiping Hu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Antiangiogenic Effect ◽  
Necrosis Factor Alpha ◽  
Umbilical Vein Endothelial Cells ◽  
Sprout Formation

ErMiao San (EMS) is composed of the Cortex Phellodendri chinensis and Atractylodes lancea, and it has the function of eliminating heat and excreting dampness in terms of traditional Chinese medicine to damp heat syndrome. Previous reports indicate that EMS possesses anti-inflammatory activity; however, its action on angiogenesis of rheumatoid arthritis (RA) has not been clarified. The present study aims to determine the antiangiogenic activity of EMS in collagen-induced arthritis (CIA) mice and in various angiogenesis models. Our data showed that EMS (5 g/kg) markedly reduced the immature blood vessels in synovial membrane tissues of inflamed joints from CIA mice. It also inhibited vascular endothelial growth factor (VEGF)-induced microvessel sprout formation ex vivo. Meanwhile, EMS suppressed VEGF-induced migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, EMS significantly reduced the expression of angiogenic activators including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in synovium of CIA mice. More interestingly, EMS blocked the autophosphorylation of VEGF-induced JAK1, STAT1, and STAT6 in CIA mice and VEGF-induced HUVECs. These findings suggest for the first time that EMS possesses the antiangiogenic effect in RA in vivo, ex vivo, and in vitro by interrupting the targeting of JAK/STAT activation.

scholarly journals Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

2021 ◽  
Vol 10 (2) ◽  
pp. 183
Author(s):  
Nadia Meyer ◽  
Lars Brodowski ◽  
Katja Richter ◽  
Constantin S. von Kaisenberg ◽  
Bianca Schröder-Heurich ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Protein Expression ◽  
Tube Formation ◽  
In Vitro Assays ◽  
Heme Oxygenase 1 ◽  
Expression Levels ◽  
Functional Capacities

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction.

scholarly journals SiRNA Directed Against Annexin II Receptor Inhibits Angiogenesis via Suppressing MMP2 and MMP9 Expression

2015 ◽  
Vol 35 (3) ◽  
pp. 875-884 ◽  
Author(s):  
Hongyuan Song ◽  
Dongyan Pan ◽  
Weifeng Sun ◽  
Cao Gu ◽  
Yuelu Zhang ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Annexin Ii ◽  
Mmp9 Expression ◽  
Cell Arrest ◽  
Umbilical Vein Endothelial Cells

Background/Aims: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism. Methods/Results: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9. Conclusion: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

2012 ◽  
Vol 123 (3) ◽  
pp. 147-159 ◽  
Author(s):  
Ting-Hsing Chao ◽  
Shih-Ya Tseng ◽  
Yi-Heng Li ◽  
Ping-Yen Liu ◽  
Chung-Lung Cho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
No Production ◽  
Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

scholarly journals Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 356 ◽  
Author(s):  
Alessia Lo Curto ◽  
Simona Taverna ◽  
Maria Assunta Costa ◽  
Rosa Passantino ◽  
Giuseppa Augello ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fabry Disease ◽  
Healthy Subjects ◽  
Aging Process ◽  
Replicative Senescence ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Quantitative Polymerase Chain Reaction ◽  
Premature Aging

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) “young” and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

Hydroxysafflor yellow A promotes angiogenesis in rat brain microvascular endothelial cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA signaling pathway

2021 ◽  
Vol 18 ◽  
Author(s):  
Juxuan Ruan ◽  
Lei Wang ◽  
Jiheng Dai ◽  
Jing Li ◽  
Ning Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Ischemia Reperfusion ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Hydroxysafflor Yellow A ◽  
Microvascular Endothelial

Objective: Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. Methods: The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. Results: HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB were increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. Conclusion: SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

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