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Journal of Taibah University Medical... Oct 2023Angiogenesis plays an important role in various physiological and pathological conditions and is essential for tumor growth and metastasis. The aim of this study was to...
OBJECTIVE
Angiogenesis plays an important role in various physiological and pathological conditions and is essential for tumor growth and metastasis. The aim of this study was to evaluate the effect of a combination of vandetanib and celecoxib on angiogenic tube formation and its effect on angiogenic genes (MMP-2 and MMP-9) using an model of human umbilical vein endothelial cells (HUVECs).
METHODS
HUVECs were cultured and verified by flow cytometry. HUVECs were then treated with vandetanib, celecoxib, and the combination of both drugs. Then, we investigated cell viability and cell apoptosis by MTT assays and flow cytometry. The process of angiogenesis was analyzed by tube formation assays, and the effect on angiogenic genes was determined by RT-qPCR.
RESULTS
HUVECs were positive for CD144 and negative for CD14. Vandetanib, celecoxib, and their combination inhibited HUVEC viability in a dose-dependent manner (p < 0.001). The rate of apoptosis was 13.1%, 9%, and 23.7% (p < 0.001) when treated with vandetanib, celecoxib, or the combination of both drugs, respectively. Vandetanib inhibited tube formation by 43.7%, celecoxib by 21%, and their combination by 77.3% (p < 0.001), respectively. RT-qPCR revealed that both vandetanib and celecoxib reduced the expression levels of MMP-2 and MMP-9, and their combination resulted in an even greater extent of reduction in expression levels (p < 0.001).
CONCLUSION
Celecoxib enhanced the effect of vandetanib in inhibiting angiogenesis and the combination of these two drugs led to even greater extents of inhibition than vandetanib alone.
PubMed: 36959917
DOI: 10.1016/j.jtumed.2023.02.016 -
Arsenic Sulfide Inhibits Hepatocellular Carcinoma Metastasis by Suppressing the HIF-1α/VEGF Pathway.Frontiers in Bioscience (Landmark... Jul 2023Metastasis is one of the principal reasons of cancer mortality from hepatocellular carcinoma (HCC). The goal of our investigation was to examine the mechanism by which...
BACKGROUND
Metastasis is one of the principal reasons of cancer mortality from hepatocellular carcinoma (HCC). The goal of our investigation was to examine the mechanism by which arsenic sulfide (As4S4) represses the metastasis of HCC.
METHODS
The cell counting kit-8 (CCK-8) assay was conducted to observe cell viability of HCC cell lines HepG2 and Hep3B following As4S4 treatment, and their metastasis was studied using the wound-healing and transwell assays. HCC-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) was assessed by tube formation assay. Enzyme-linked immunosorbent assay (ELISA), western blot, quantitative polymerase chain reaction and immunofluorescence staining were utilized to evaluate key molecules involved in metastasis, including hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), Vimentin, N-cadherin and E-cadherin.
RESULTS
As4S4 suppressed the proliferation, migration and invasion of HepG2 and Hep3B cell lines in a concentration-dependent pattern, and inhibited HCC cell-induced angiogenesis of HUVEC in the tube formation assay. Treatment with As4S4 also decreased the expression of crucial elements involved in the metastasis of HCC cells, including HIF-1α and VEGF, while reducing epithelial-mesenchymal transition, as shown by Western blot, ELISA and immunofluorescence staining.
CONCLUSIONS
Overall, results above indicate that As4S4 suppresses the metastasis of HCC cells via the HIF-1α/VEGF pathway.
Topics: Humans; Carcinoma, Hepatocellular; Vascular Endothelial Growth Factor A; Liver Neoplasms; Cell Line, Tumor; Vascular Endothelial Growth Factors; Human Umbilical Vein Endothelial Cells; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Pathologic
PubMed: 37525902
DOI: 10.31083/j.fbl2807152 -
Life Sciences Sep 2023To investigate the effect of connexin 43 (Cx43) on corneal neovascularization and its regulation of VEGFR2 on vascular endothelial cells.
PURPOSE
To investigate the effect of connexin 43 (Cx43) on corneal neovascularization and its regulation of VEGFR2 on vascular endothelial cells.
METHODS
In vivo, we used mouse corneal suture model to induce corneal neovascularization and discovered the function of gap26 in corneal neovascularization. In vitro, the effect of gap26 on HUVEC was observed by cell proliferation, tube formation and scratch experiments. WB and PCR detected the changes in angiogenic protein and mRNA expression. Knockdown of key mRNA in neovascularization using siRNA confirmed that Cx43 regulates neovascularization through the β-catenin-VE-cadherin-VEGFR2-Erk signaling pathway.
RESULTS
In vivo, gap26 can reduce mouse corneal neovascularization. In vitro, we show that Cx43 expression is increased in the presence of VEGFA stimulation, and when we use gap26 to inhibit Cx43 can reduce vascular endothelial cell proliferation, tube formation and migration. We found that the expression of pVEGFR2 and pErk increased in response to VEGFA, while they decreased after using gap26. And the expression of β-catenin and VE-cadherin decreased in response to VEGFA, while they increased after using gap26. Furthermore, we found that Cx43 regulates angiogenesis through the β-catenin-VE-cadherin-VEGFR2-Erk pathway.
CONCLUSIONS
Gap26 can downregulate VEGFR2 phosphorylation by stabilizing the expression of β-catenin and VE-cadherin on the cell membrane, thereby inhibiting VEGFA-induced HUVECs proliferation, migration and tube formation and inhibiting corneal neovascularization.
Topics: Animals; Humans; Mice; beta Catenin; Cadherins; Cell Movement; Cell Proliferation; Connexin 43; Corneal Neovascularization; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Signal Transduction; Vascular Endothelial Growth Factor A
PubMed: 37295713
DOI: 10.1016/j.lfs.2023.121836 -
Translational Research : the Journal of... Aug 2024Post-ischemic angiogenesis is critical for perfusion recovery and tissue repair. ELABELA (ELA) plays an essential role in embryonic heart development and vasculogenesis....
BACKGROUND
Post-ischemic angiogenesis is critical for perfusion recovery and tissue repair. ELABELA (ELA) plays an essential role in embryonic heart development and vasculogenesis. However, the mechanism of ELA on post-ischemic angiogenesis is poorly characterized.
METHODS
We first assessed ELA expression after hind limb ischemia (HLI) in mice. We then established a HLI model in tamoxifen-inducible endothelial-ELA-specific knockout mice (ELA) and assessed the rate of perfusion recovery, capillary density, and VEGFR2 pathway. Knockdown of ELA with lentivirus or siRNA and exogenous addition of ELA peptides were employed to analyze the effects of ELA on angiogenic capacity and VEGFR2 pathway in endothelial cells in vitro. The serum levels of ELA in healthy people and patients with type 2 diabetes mellitus (T2DM) and diabetic foot ulcer (DFU) were detected by a commercial ELISA kit.
RESULTS
In murine HLI models, ELA was significantly up-regulated in the ischemic hindlimb. Endothelial-specific deletion of ELA impaired perfusion recovery and angiogenesis. In physiologic conditions, no significant difference in VEGFR2 expression was found between ELA mice and ELA mice. After ischemia, the expression of VEGFR2, p-VEGFR2, and p-AKT was significantly lower in ELA mice than in ELA mice. In cellular experiments, the knockdown of ELA inhibited endothelial cell proliferation and tube formation, and the addition of ELA peptides promoted proliferation and tube formation. Mechanistically, ELA upregulated the expression of VEGFR2, p-VEGFR2, and p-AKT in endothelial cells under hypoxic conditions. In clinical investigations, DFU patients had significantly lower serum levels of ELA compared to T2DM patients.
CONCLUSION
Our results indicated that endothelial ELA is a positive regulator of post-ischemic angiogenesis via upregulating VEGFR2 expression. Targeting ELA may be a potential therapeutic option for peripheral arterial diseases.
Topics: Animals; Vascular Endothelial Growth Factor Receptor-2; Ischemia; Humans; Up-Regulation; Mice, Knockout; Mice; Neovascularization, Physiologic; Hindlimb; Male; Diabetes Mellitus, Type 2; Mice, Inbred C57BL; Diabetic Foot; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Angiogenesis
PubMed: 38548174
DOI: 10.1016/j.trsl.2024.03.011 -
Journal of Cell Science Apr 2024Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The...
Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.
Topics: Animals; Humans; Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cell Movement; Feedback, Physiological; Focal Adhesions; GTPase-Activating Proteins; Human Umbilical Vein Endothelial Cells; Mechanotransduction, Cellular; Neovascularization, Physiologic; Tumor Suppressor Proteins; YAP-Signaling Proteins
PubMed: 38563084
DOI: 10.1242/jcs.261687 -
Cell Death & Disease Nov 2023Glioblastoma multiforme (GBM) is a highly vascularized malignant cancer of the central nervous system, and the presence of vasculogenic mimicry (VM) severely limits the...
Glioblastoma multiforme (GBM) is a highly vascularized malignant cancer of the central nervous system, and the presence of vasculogenic mimicry (VM) severely limits the effectiveness of anti-vascular therapy. In this study, we identified downregulated circHECTD1, which acted as a key VM-suppressed factor in GBM. circHECTD1 elevation significantly inhibited cell proliferation, migration, invasion and tube-like structure formation in GBM. RIP assay was used to demonstrate that the flanking intron sequence of circHECTD1 can be specifically bound by RBMS3, thereby inducing circHECTD1 formation to regulate VM formation in GBM. circHECTD1 was confirmed to possess a strong protein-encoding capacity and the encoded functional peptide 463aa was identified by LC-MS/MS. Both circHECTD1 and 463aa significantly inhibited GBM VM formation in vivo and in vitro. Analysis of the 463aa protein sequence revealed that it contained a ubiquitination-related domain and promoted NR2F1 degradation by regulating the ubiquitination of the NR2F1 at K396. ChIP assay verified that NR2F1 could directly bind to the promoter region of MMP2, MMP9 and VE-cadherin, transcriptionally promoting the expression of VM-related proteins, which in turn enhanced VM formation in GBM. In summary, we clarified a novel pathway for RBMS3-induced circHECTD1 encoding functional peptide 463aa to mediate the ubiquitination of NR2F1, which inhibited VM formation in GBM. This study aimed to reveal new mechanisms of GBM progression in order to provide novel approaches and strategies for the anti-vascular therapy of GBM. The schematic illustration showed the inhibitory effect of circHECTD1-463aa in the VM formation in GBM.
Topics: Humans; Glioblastoma; Cell Line, Tumor; Chromatography, Liquid; Tandem Mass Spectrometry; Peptides; Neovascularization, Pathologic; Trans-Activators; RNA-Binding Proteins
PubMed: 37968257
DOI: 10.1038/s41419-023-06269-y -
Science China. Life Sciences Feb 2024We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is...
We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is dysfunctional and impairs angiogenesis. Autophagy plays a critical role in angiogenesis, and HDL regulates autophagy. However, it is unclear whether nHDL and dHDL regulate angiogenesis by affecting autophagy. Endothelial cells (ECs) were treated with nHDL and dHDL with or without an autophagy inhibitor. Autophagy, endothelial nitric oxide synthase (eNOS) expression, miRNA expression, nitric oxide (NO) production, superoxide anion (O) generation, EC migration, and tube formation were evaluated. nHDL suppressed the expression of miR-181a-5p, which promotes autophagy and the expression of eNOS, resulting in NO production and the inhibition of O generation, and ultimately increasing in EC migration and tube formation. dHDL showed opposite effects compared to nHDL and ultimately inhibited EC migration and tube formation. We found that autophagy-related protein 5 (ATG5) was a direct target of miR-181a-5p. ATG5 silencing or miR-181a-5p mimic inhibited nHDL-induced autophagy, eNOS expression, NO production, EC migration, tube formation, and enhanced O generation, whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of dHDL. ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null (LDLr) mice when compared to C57BL/6 mice. ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr mice. Taken together, nHDL was able to stimulate autophagy by suppressing miR-181a-5p, subsequently increasing eNOS expression, which generated NO and promoted angiogenesis. In contrast, dHDL inhibited angiogenesis, at least partially, by increasing miR-181a-5p expression, which decreased autophagy and eNOS expression, resulting in a decrease in NO production and an increase in O generation. Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for dHDL-impaired angiogenesis.
Topics: Animals; Humans; Mice; Angiogenesis; Autophagy; Endothelial Cells; Mice, Inbred C57BL; MicroRNAs; Lipoproteins, HDL
PubMed: 37897614
DOI: 10.1007/s11427-022-2381-7 -
Journal of Cell Communication and... Dec 2023A trans-membrane receptor tyrosine kinase, cMET, belonging to the MET proto-oncogene family, is responsible for cancer metastasis and angiogenesis. But not much is known...
A trans-membrane receptor tyrosine kinase, cMET, belonging to the MET proto-oncogene family, is responsible for cancer metastasis and angiogenesis. But not much is known about the role of cMET in growth and progression of cancer stem cells (CSCs). Earlier studies have shown that Quinacrine (QC), a bioactive agent, has anti-CSCs activity. Here, the role of QC in deregulation of cMET-mediated metastasis and angiogenesis has been systematically evaluated in vitro in highly metastatic breast CSCs (mBCSCs), ex vivo in patient-derived breast cancer stem cells (PDBCSCs) and in vivo in xenograft mice model systems. Cell proliferation, migration, invasion and representative metastasis markers were upregulated in cMET-overexpressed cells and QC exposure inhibited these processes in both mBCSCs and PDBCSCs. Interestingly, metastasis was significantly inhibited by QC in cMET-overexpressed cells but comparatively lesser significant alteration of the process was noted in cMET-silenced cells. Increase in vascularization (in in ovo CAM assay), and cell-cell tube formation (in HUVECs), and enhanced MMP9 and MMP2 enzymatic activities (in gelatin zymography) were noted after cMET overexpression but these processes got reversed after cMET knockdown or QC treatment in cMET-overexpressed cells. QC inhibited angiogenesis significantly in cMET-overexpressed cells, but lesser significant change was observed in cMET-silenced cells. Reduction in tumor volume and decreased expression of metastatic and angiogenic markers were also noted in xenograft mice after QC treatment. Furthermore, QC inhibited cMET activity by dephosphorylation of its tyrosine residues (Y1234 and Y1356) and downregulation of its downstream cascade. Thus, QC inhibited the cMET-mediated metastasis and angiogenesis in in vitro, in ovo, in vivo and ex vivo model systems. Ligand (HGF) binding leads to receptor dimerization and phosphorylation of tyrosine kinase domain of cMET. This activates the cMET signaling cascade. The representative downstream metastasis and angiogenesis-related proteins get upregulated and induce the metastasis and angiogenesis process. But after the QC treatment, cMET get dephosphorylated and inactivated. As a result, the downstream signaling proteins of cMET along with the other representative metastatic and angiogenic factors get downregulated. These lead to inhibition of cMET-mediated metastasis and angiogenesis. (Created with BioRender.com).
PubMed: 37162635
DOI: 10.1007/s12079-023-00756-9 -
Molecular Cancer May 2024The hypoxic tumor microenvironment is a key factor that promotes metabolic reprogramming and vascular mimicry (VM) in ovarian cancer (OC) patients. ESM1, a secreted...
BACKGROUND
The hypoxic tumor microenvironment is a key factor that promotes metabolic reprogramming and vascular mimicry (VM) in ovarian cancer (OC) patients. ESM1, a secreted protein, plays an important role in promoting proliferation and angiogenesis in OC. However, the role of ESM1 in metabolic reprogramming and VM in the hypoxic microenvironment in OC patients has not been determined.
METHODS
Liquid chromatography coupled with tandem MS was used to analyze CAOV3 and OV90 cells. Interactions between ESM1, PKM2, UBA2, and SUMO1 were detected by GST pull-down, Co-IP, and molecular docking. The effects of the ESM1-PKM2 axis on cell glucose metabolism were analyzed based on an ECAR experiment. The biological effects of the signaling axis on OC cells were detected by tubule formation, transwell assay, RT‒PCR, Western blot, immunofluorescence, and in vivo xenograft tumor experiments.
RESULTS
Our findings demonstrated that hypoxia induces the upregulation of ESM1 expression through the transcription of HIF-1α. ESM1 serves as a crucial mediator of the interaction between PKM2 and UBA2, facilitating the SUMOylation of PKM2 and the subsequent formation of PKM2 dimers. This process promotes the Warburg effect and facilitates the nuclear translocation of PKM2, ultimately leading to the phosphorylation of STAT3. These molecular events contribute to the promotion of ovarian cancer glycolysis and vasculogenic mimicry. Furthermore, our study revealed that Shikonin effectively inhibits the molecular interaction between ESM1 and PKM2, consequently preventing the formation of PKM2 dimers and thereby inhibiting ovarian cancer glycolysis, fatty acid synthesis and vasculogenic mimicry.
CONCLUSION
Our findings demonstrated that hypoxia increases ESM1 expression through the transcriptional regulation of HIF-1α to induce dimerization via PKM2 SUMOylation, which promotes the OC Warburg effect and VM.
Topics: Female; Humans; Ovarian Neoplasms; Thyroid Hormone-Binding Proteins; Tumor Microenvironment; Animals; Thyroid Hormones; Mice; Membrane Proteins; Cell Line, Tumor; Fatty Acids; Neoplasm Proteins; Carrier Proteins; Warburg Effect, Oncologic; Hypoxia-Inducible Factor 1, alpha Subunit; Gene Expression Regulation, Neoplastic; Neovascularization, Pathologic; Xenograft Model Antitumor Assays; Cell Proliferation; Proteoglycans
PubMed: 38720298
DOI: 10.1186/s12943-024-02009-8 -
Advanced Science (Weinheim,... Feb 2024Chemotherapy is widely used to treat colorectal cancer (CRC). Despite its substantial benefits, the development of drug resistance and adverse effects remain...
Chemotherapy is widely used to treat colorectal cancer (CRC). Despite its substantial benefits, the development of drug resistance and adverse effects remain challenging. This study aimed to elucidate a novel role of glucagon in anti-cancer therapy. In a series of in vitro experiments, glucagon inhibited cell migration and tube formation in both endothelial and tumor cells. In vivo studies demonstrated decreased tumor blood vessels and fewer pseudo-vessels in mice treated with glucagon. The combination of glucagon and chemotherapy exhibited enhanced tumor inhibition. Mechanistic studies demonstrated that glucagon increased the permeability of blood vessels, leading to a pronounced disruption of vessel morphology. Signaling pathway analysis identified a VEGF/VEGFR-dependent mechanism whereby glucagon attenuated angiogenesis through its receptor. Clinical data analysis revealed a positive correlation between elevated glucagon expression and chemotherapy response. This is the first study to reveal a role for glucagon in inhibiting angiogenesis and vascular mimicry. Additionally, the delivery of glucagon-encapsulated PEGylated liposomes to tumor-bearing mice amplified the inhibition of angiogenesis and vascular mimicry, consequently reinforcing chemotherapy efficacy. Collectively, the findings demonstrate the role of glucagon in inhibiting tumor vessel network and suggest the potential utility of glucagon as a promising predictive marker for patients with CRC receiving chemotherapy.
Topics: Humans; Animals; Mice; Glucagon; Neovascularization, Pathologic; Colorectal Neoplasms; Signal Transduction; Cell Line, Tumor
PubMed: 38072640
DOI: 10.1002/advs.202307271