The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells.

Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.

Eriocitrin Inhibits Angiogenesis by Targeting VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways.

Eriocitrin, a flavanone found in peppermint and citrus fruits, is known to possess many physiological activities. However, the anti-angiogenic effects of eriocitrin are yet to be fully elucidated. Therefore, the objective of this research was to explore the anti-angiogenic effects of eriocitrin both in vitro and in vivo as well as its underlying mechanism. Anti-angiogenic effects of eriocitrin were evaluated utilizing in vitro models of angiogenesis, including inhibition of tube formation, and induction of apoptosis in human umbilical vein endothelial cells (HUVECs). A chorioallantoic membrane (CAM) assay in chick embryos was also performed to evaluate the in vivo effects of eriocitrin on angiogenesis. Results showed significant eriocitrin effects on proliferation, tube formation, migration, and apoptosis in HUVECs. Furthermore, in vivo analysis revealed that eriocitrin significantly suppressed the formation of new blood vessels. In particular, it regulated MAPK/ERK signaling pathway and VEGFR2, inhibited the downstream PI3K/AKT/mTOR signaling pathway, and activated apoptosis signals such as caspase cascades. In HUVECs, the expression of matrix metalloproteinases (MMP-2 and MMP-9) exhibited an inhibitory effect on angiogenesis through the suppression of the signaling pathway. Therefore, eriocitrin presents potential for development into an antiangiogenic therapeutic agent.

Galectin-1 overexpression induces normal fibroblasts translate into cancer-associated fibroblasts and attenuates the sensitivity of anlotinib in lung cancer.

We aimed to investigate galectin-1 overexpression induces normal fibroblasts (NFs) translates into cancer-associated fibroblasts (CAFs). Galectin-1 overexpression was conducted in Human embryonic lung fibroblasts (HFL1) cell. The motilities of H1299 and A549 cells were measured. Human umbilical vein endothelial cell (HUVEC) proliferation and tube formation ability were assessed. Tumor volume and tumor weight was recorded. Cells motilities were increased, while apoptosis rates were decreased after CMs co-cultured. B-cell lymphoma-2 (Bcl-2) expression level was increased, while Bcl2-associatedX (Bax) and cleaved-caspase3 decreased. CMs treatment enhanced HUVEC proliferation and tube formation. Tumor volume and weight in CMs treated mice were increased, and the sensitivity of anlotinib in co-cultured cells was decreased. Our results revealed that galectin-1 overexpression induced NFs translated into CAFs.

Lysophosphatidic acid (LPA) receptor-mediated signaling regulates hypoxia-induced biological functions of lymphatic endothelial cells.

The tumor microenvironment is an extremely complex composed of cancer cells and various non-cancer cells, including lymphatic endothelial cells. Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) activate a variety of malignant properties in human malignancies. In the present study, we examined the roles of LPA receptor-mediated signaling in biological responses of lymphatic endothelial SVEC4-10 cells induced by hypoxia. Lpar1, Lpar2 and Lpar3 expressions were decreased in SVEC4-10 cells cultured at hypoxic conditions (1 % O2). LPA had no impact on the cell growth activity of SVEC4-10 cells in 21 % O2 culture conditions. Conversely, the cell growth activity of SVEC4-10 cells in 1 % O2 culture conditions was reduced by LPA. The cell motile activity of SVEC4-10 cells was elevated by 1 % O2 culture conditions. GRI-977143 (LPA2 agonist) and (2S)-OMPT (LPA3 agonist) stimulated SVEC4-10 cell motility as well as AM966 (LPA1 antagonist). In tube formation assay, the tube formation of SVEC4-10 cells in 1 % O2 culture conditions was markedly increased, in comparison with 21 % O2. GRI-977143 and (2S)-OMPT elevated the tube formation of SVEC4-10 cells. Furthermore, the tube formation of SVEC4-10 cells was increased by AM966. These results suggest that LPA receptor-mediated signaling contributes to the modulation of hypoxic-induced biological functions of lymphatic endothelial cells.

Tube Formation Capability and Chemotaxis of Skin Pericytes.

BACKGROUND: Pericytes (PCs), the critical components of vessels, are implicated in wound repair. This study aimed to explore the roles of PCs in wound healing and angiogenesis. METHODS: Skin PCs and human dermal microvascular endothelial cells (HDMECs) were isolated from patients' upper eyelid skin. Immunofluorescence staining was used to characterize the morphology of PCs. Tube formation and transwell chemotaxis assays were performed to explore PC's tube-forming capability and chemotaxis. Finally, we investigated the effects of PCs and endothelial cells on wound repair using skin wound of a rat model. RESULTS: Skin PCs exhibited a double-protrusion structure and characteristic antigen expression of neural/glial antigen 2 (NG2)+/platelet-derived growth factor receptor-ß (PDGFR-ß)+/alpha-smooth muscle actin (α-SMA)+/CD31-. Skin PCs could directly form lumen-like structures in a two dimensional (2D) culture environment, and mild hypoxia and starvation promoted the lumen-like structure formation. Furthermore, skin PCs quickly formed more stable lumen-like structures than HDMECs in matrigel, and they recruited HDMECs in a three dimensional (3D) culture environment. Transwell chemotaxis assay showed that PCs and HDMECs were chemotactic to each other. PCs could develop lumen-like structures in the skin wounds of rat models. The number of PCs mounted in wounded skin was compared to normal skin. The ratio of PCs to endothelial cells gradually increased after skin injury and reached its maximum on the 3rd day. CONCLUSIONS: Skin PCs have an excellent tube-forming capability and chemotaxis to endothelial cells. PCs might promote wound repair by recruiting endothelial cells.

Coordination of cell cycle and morphogenesis during organ formation.

Organ formation requires precise regulation of cell cycle and morphogenetic events. Using the Drosophila embryonic salivary gland (SG) as a model, we uncover the role of the SP1/KLF transcription factor Huckebein (Hkb) in coordinating cell cycle regulation and morphogenesis. The hkb mutant SG exhibits defects in invagination positioning and organ size due to the abnormal death of SG cells. Normal SG development involves distal-to-proximal progression of endoreplication (endocycle), whereas hkb mutant SG cells undergo abnormal cell division, leading to cell death. Hkb represses the expression of key cell cycle and pro-apoptotic genes in the SG. Knockdown of cyclin E or cyclin-dependent kinase 1, or overexpression of fizzy-related rescues most of the morphogenetic defects observed in the hkb mutant SG. These results indicate that Hkb plays a critical role in controlling endoreplication by regulating the transcription of key cell cycle effectors to ensure proper organ formation.

Fibronectin mediates activin A-promoted human trophoblast migration and acquisition of endothelial-like phenotype.

BACKGROUND: During human early placentation, a proportion of extravillous trophoblasts (EVTs) migrate to the maternal decidua, differentiating into endovascular EVTs to remodel spiral arteries and ensure the establishment of blood circulation at the maternal-fetal interface. Inadequate EVT migration and endovascular differentiation are closely associated with adverse pregnancy outcomes such as miscarriage. Activin A and fibronectin are both secretory molecules abundantly expressed at the maternal-fetal interface. Activin A has been reported to regulate EVT biological functions. However, whether fibronectin mediates activin A-promoted EVT migration and acquisition of endothelial-like phenotype as well as the underlying molecular mechanisms remain unknown. Additionally, the role of fibronectin in pregnancy establishment and maintenance warrants further investigation. METHODS: Primary and immortalized (HTR8/SVneo) human EVTs were used as in vitro study models. Cultured human first-trimester chorionic villous explants were utilized for ex vivo validation. A local fibronectin knockdown model in ICR mouse uteri, achieved by nonviral in vivo transfection with small interfering RNA (siRNA) targeting fibronectin 1 (si-Fn1), was employed to explore the roles of fibronectin in the establishment and maintenance of early pregnancy. RESULTS: Our results showed that activin A treatment significantly induced fibronectin 1 (FN1) mRNA expression and fibronectin protein production, which is essential for human trophoblast migration and endothelial-like tube formation. Both basal and activin A-upregulated fibronectin expression were abolished by the TGF-ß type I receptor inhibitor SB431542 or siRNA-mediated knockdown of activin receptor-like kinase (ALK4) or SMAD4. Moreover, activin A-increased trophoblast migration and endothelial-like tube formation were attenuated following the depletion of fibronectin. Fibronectin knockdown via intrauterine siRNA administration reduced CD31 and cytokeratin 8 (CK8) expression at the maternal-fetal interface, resulting in a decrease in the number of implantation sites and embryos. CONCLUSIONS: Our study demonstrates that activin A promotes trophoblast cell migration and acquisition of endothelial-like phenotype via ALK4-SMAD2/3-SMAD4-mediated fibronectin upregulation. Furthermore, through a local fibronectin knockdown model in mouse uteri, we found that the absence of fibronectin at the maternal-fetal interface impedes endovascular migration of trophoblasts and decidual vascularization, thereby interfering with early embryo implantation and the maintenance of pregnancy. These findings provide novel insights into placental development during early pregnancy establishment and contribute to the advancement of therapeutic approaches for managing pregnancy complications related to trophoblast dysfunction.

A 1, 4-benzoquinone derivative isolated from Ardisia crispa (Thunb.) A. DC. root suppresses angiogenesis via its angiogenic signaling cascades.

The root hexane extract of Ardisia crispa (ACRH), which belongs to the Primulaceae family, has been reported to possess anti-inflammatory, chemopreventive, anti-arthritic, and antiangiogenic activities. In this study, we isolated a p-benzoquinone derivative, 2-methoxy-6-undecyl-1,4-benzoquinone (AC2), from ACRH and investigated its potential antiangiogenic activity in human umbilical vein endothelial cells (HUVECs) and zebrafish embryo models. Prior to this study, AC2 was characterized using 1H NMR spectroscopy and MS. AC2 significantly suppressed HUVEC proliferation in a time-independent manner, with an IC50 value of 1.35 ± 0.05, 1.15 ± 0.02, and 1.00 ± 0.01 µg/mL at 24, 48, and 72 h, respectively. AC2 also induced apoptosis in HUVECs and significantly suppressed their migration, invasion, and tube formation in a concentration-dependent manner. Additionally, AC2 significantly attenuated most of the analyzed protein markers, including pro-MMP-2, VEGF-C, VEGF-D, angiopoietin-2, endothelin-1, fibroblast growth factor (FGF)-1, FGF-2, follistatin, heparin-binding epidermal growth factor-like growth factor (HB-EGF), and hepatocyte growth factor (HGF) at all tested concentrations. Furthermore, AC2 significantly inhibited zebrafish embryo intersegmental vessels (ISVs), confirming its antiangiogenic role. In conclusion, AC2 exhibits a potential anti-angiogenic effect by suppressing several proangiogenic and growth factors. Further studies are needed to investigate their effects on other excessive angiogenic diseases.

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure.

BACKGROUND: 2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PURPOSE: In this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. STUDY DESIGN AND METHODS: First, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. RESULTS: The study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. CONCLUSIONS: This study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

A SIRT6 Inhibitor, Marine-Derived Pyrrole-Pyridinimidazole Derivative 8a, Suppresses Angiogenesis.

Angiogenesis refers to the process of growing new blood vessels from pre-existing capillaries or post-capillary veins. This process plays a critical role in promoting tumorigenesis and metastasis. As a result, developing antiangiogenic agents has become an attractive strategy for tumor treatment. Sirtuin6 (SIRT6), a member of nicotinamide adenine (NAD+)-dependent histone deacetylases, regulates various biological processes, including metabolism, oxidative stress, angiogenesis, and DNA damage and repair. Some SIRT6 inhibitors have been identified, but the effects of SIRT6 inhibitors on anti-angiogenesis have not been reported. We have identified a pyrrole-pyridinimidazole derivative 8a as a highly effective inhibitor of SIRT6 and clarified its anti-pancreatic-cancer roles. This study investigated the antiangiogenic roles of 8a. We found that 8a was able to inhibit the migration and tube formation of HUVECs and downregulate the expression of angiogenesis-related proteins, including VEGF, HIF-1α, p-VEGFR2, and N-cadherin, and suppress the activation of AKT and ERK pathways. Additionally, 8a significantly blocked angiogenesis in intersegmental vessels in zebrafish embryos. Notably, in a pancreatic cancer xenograft mouse model, 8a down-regulated the expression of CD31, a marker protein of angiogenesis. These findings suggest that 8a could be a promising antiangiogenic and cancer therapeutic agent.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 µg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM2.5 exposure.

Cooking oil fume-derived PM2.5 (COF-PM2.5) is a major source of indoor air contamination in China, which has been demonstrated to be a hazard factor of cardiovascular and cerebrovascular diseases. This study aimed to investigate the role of ROS-mediated PERK/ATF4 signaling activation in COF-PM2.5-inhibited extracorporeal tube formation in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 100 µg/mL COF-PM2.5 at different times, with or without 100 nM PERK activity inhibitor GSK2606414 (GSK) or 200 µM antioxidant N-acetylcysteine (NAC) pretreatment. Our results showed that COF-PM2.5 exposure can inhibit extracorporeal tube formation and down-regulate VEGFR2 expression in HUVECs. Furthermore, our data indicated that COF-PM2.5 exposure can activate the PERK/ATF4 signaling in HUVECs. Mechanistically, pretreatment with GSK interdicted PERK/ATF4 signaling, thereby reversing COF-PM2.5-downregulated VEGFR2 protein expression in HUVECs. Furthermore, NAC reversed VEGFR2 expression downregulated induced by COF-PM2.5 by inhibiting the upregulation of intracellular ROS levels and PERK/ATF4 signaling in HUVECs. As above, COF-PM2.5 exposure could induce ROS release from HUVECs, which in turn activate the endoplasmic reticulum PERK/ATF4 signaling and inhibit tube formation of HUVECs.

The proliferation and angiogenesis in hemangioma-derived endothelial cells is affected by STC2 medicated VEGFR2/Akt/eNOS pathway.

Objective: Stanniocalcin-2 (STC2), a secreted glycoprotein that is involved in the regulation of angiogenesis, was proposed as one of the mechanisms of neovascularization in hemangioma (HA). We aimed to investigate the effect of STC2 on proliferation and angiogenesis in hemangioma-derived endothelial cells. Methods: The hemangioma samples from HA patients with the median age of six months were surgically collected in the Affiliated Hospital of Weifang Medical University from October 2019 to June 2021, and divided into normal skin tissues (n=10), involuting-phase HAs (n=10) and proliferating-phase HAs (n=10) according to the Mulliken classification. The expression of STC2 was detected in involuting-phase HAs and proliferating-phase HAs. Hemangioma endothelial cells (HemEC) were transfected with small interfering RNA (siRNA) specific for STC2, and cell survival and tube formation were analyzed. Results: STC2 expression in proliferating-phase HAs was markedly higher than in the normal skin tissues and involving-phase HAs. Similarly, STC2 expression was higher in HemEC compared to the control human umbilical vein endothelial cells (HUVEC). Knockdown of STC2 slowed the proliferation of HemEC and decreased the expression of proliferating cell nuclear antigen (PCNA) in HemEC. Moreover, knockdown of STC2 in HemEC inhibited vascular endothelial cell angiogenesis and regulated the expression and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). Mechanistically, STC2 knockdown attenuated the activation of Akt/eNOS signaling, which was abolished by insulin growth factor-1 (IGF-1), the activator of Akt signaling, accompanying by increased proliferation and tube formation of HemEC. Conclusion: Inhibition of STC2 suppresses HemEC proliferation and angiogenesis by VEGFR2/Akt/eNOS pathway, which warrants further development of STC2-based strategies for HA treatment.

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.

Study of platelet-rich fibrin promoting endothelial cell differentiation and angiogenesis induced by transplantation of adipose-derived stem cells.

Diabetic patients are characterized by long wound healing time, and adipose stem cells (ADSCs) can secrete growth factors to promote angiogenesis and improve diabetic wound healing. In this research, we attempted to interrogate the impact of platelet-rich fibrin (PRF) on ADSCs in diabetic wound healing. ADSCs were harvested from human adipose tissues and identified through flow cytometry. After pretreatment with cultured medium supplemented with different concentrations of PRF (2.5%, 5%, and 7.5%), proliferation and differentiation capacity of ADSCs were assessed by CCK-8 assay, qRT-PCR and immunofluorescence (IF), respectively. Tube formation assay measured angiogenesis. Western blot analysis analyzed expression of endothelial markers and the extracellular signal-regulated kinase (ERK) and serine/threonine kinase (Akt) pathways in PRF-induced ADSCs. The CCK-8 experiment indicated that PRF enhanced proliferation of ADSCs in dose-dependent manner, relative to normal control group. The expression of endothelial markers and the capacity of tube formation were significantly promoted by 7.5% PRF. The release of growth factors containing vascular endothelial grow factor (VEGF) and insulin-like growth factor-1 (IGF-1) from PRF was increased with the extension of detection time. When the receptors of VEGF or/and IGF-1 were neutralized, ADSCs differentiation into endothelial cells were obviously inhibited. Additionally, PRF stimulated ERK and Akt pathways, and the inhibitors of ERK and Akt attenuated PRF-induced differentiation of ADSCs into endothelial cells. In conclusion, PRF promoted endothelial cell differentiation and angiogenesis induced by ADSCs in diabetic wound healing, which appears to give guidance for treating patients.

Restoration of coronary microvascular function by OGA overexpression in a high-fat diet with low-dose streptozotocin-induced type 2 diabetic mice.

Sustained hyperglycemia results in excess protein O-GlcNAcylation, leading to vascular complications in diabetes. This study aims to investigate the role of O-GlcNAcylation in the progression of coronary microvascular disease (CMD) in inducible type 2 diabetic (T2D) mice generated by a high-fat diet with a single injection of low-dose streptozotocin. Inducible T2D mice exhibited an increase in protein O-GlcNAcylation in cardiac endothelial cells (CECs) and decreases in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function) and capillary density accompanied by increased endothelial apoptosis in the heart. Endothelial-specific O-GlcNAcase (OGA) overexpression significantly lowered protein O-GlcNAcylation in CECs, increased CFVR and capillary density, and decreased endothelial apoptosis in T2D mice. OGA overexpression also improved cardiac contractility in T2D mice. OGA gene transduction augmented angiogenic capacity in high-glucose treated CECs. PCR array analysis revealed that seven out of 92 genes show significant differences among control, T2D, and T2D + OGA mice, and Sp1 might be a great target for future study, the level of which was significantly increased by OGA in T2D mice. Our data suggest that reducing protein O-GlcNAcylation in CECs has a beneficial effect on coronary microvascular function, and OGA is a promising therapeutic target for CMD in diabetic patients.

Exosomal miR-133a-3p promotes the growth and metastasis of lung cancer cells following incomplete microwave ablation.

PURPOSE: Exosomal miRNAs play key roles in various biological processes such as cell proliferation, angiogenesis, migration and invasion. We explored whether exosomal miRNAs can promote local recurrence (LR) of lung tumors following incomplete microwave ablation (MWA) therapy. METHODS: Exosomal miRNA profiles before and after incomplete MWA in lung cancer (LC) patients with LR (n = 3) were sequenced and compared. The differentially expressed miRNAs of interest were validated in clinical samples (n = 10) and MWA-treated cells using RT-qPCR analysis. Target genes of the miRNAs were predicted and validated. The biological functions of miRNAs in proliferation, angiogenesis and metastasis of A549 cells were evaluated in vitro and in vivo. RESULTS: A total of 270 miRNAs (243 upregulated and 27 downregulated) were differentially expressed after incomplete MWA in patients with local recurrence. Upregulation of miR-133a-3p after MWA was validated in the cells and clinical samples. Cell functional experiments suggested that miR-133a-3p overexpression derived from serum exosomes increased cell viability, migration and invasion ability, tube formation activity and proliferation of A549 cells. Sirtuin 1 (SIRT1) was identified as a target gene for miR-133a-3p. Moreover, miR-133a-3p delivered by exosomes significantly promoted tumor growth, paralleled by reduced SIRT1 expression in a subcutaneous tumorigenesis animal model and increased the number of lung nodules by tail vein metastasis in vivo. CONCLUSION: Exosomal miR-133a-3p overexpression promoted tumor growth and metastasis following MWA and could be a promising biomarker for LC recurrence after incomplete MWA.

KDELC2 Upregulates Glioblastoma Angiogenesis via Reactive Oxygen Species Activation and Tumor-Associated Macrophage Proliferation.

Glioblastoma is notorious for its rapid progression and neovascularization. In this study, it was found that KDEL (Lys-Asp-Glu-Leu) containing 2 (KDELC2) stimulated vasculogenic factor expression and induced human umbilical vein endothelial cell (HUVEC) proliferation. The NLRP3 inflammasome and autophagy activation via hypoxic inducible factor 1 alpha (HIF-1α) and mitochondrial reactive oxygen species (ROS) production was also confirmed. The application of the NLRP3 inflammasome inhibitor MCC950 and autophagy inhibitor 3-methyladenine (3-MA) indicated that the above phenomenon activation correlated with an endothelial overgrowth. Furthermore, KDELC2 suppression decreased the endoplasmic reticulum (ER) stress factors' expression. The ER stress inhibitors, such as salubrinal and GSK2606414, significantly suppressed HUVEC proliferation, indicating that ER stress promotes glioblastoma vascularization. Finally, shKDELC2 glioblastoma-conditioned medium (CM) stimulated TAM polarization and induced THP-1 cells to transform into M1 macrophages. In contrast, THP-1 cells co-cultured with compensatory overexpressed (OE)-KDELC2 glioblastoma cells increased IL-10 secretion, a biomarker of M2 macrophages. HUVECs co-cultured with shKDELC2 glioblastoma-polarized THP-1 cells were less proliferative, demonstrating that KDELC2 promotes angiogenesis. Mito-TEMPO and MCC950 increased caspase-1p20 and IL-1ß expression in THP-1 macrophages, indicating that mitochondrial ROS and autophagy could also interrupt THP-1-M1 macrophage polarization. In conclusion, mitochondrial ROS, ER stress, and the TAMs resulting from OE-KDELC2 glioblastoma cells play important roles in upregulating glioblastoma angiogenesis.

Histamine promotes angiogenesis through a histamine H1 receptor-PKC-VEGF-mediated pathway in human endothelial cells.

Histamine is a well-known inflammatory mediator, but how histamine induces angiogenesis remains poorly understood. In the present study, we demonstrated a dose-dependent dynamic tube formation in the human endothelial cell line EA.hy926 in the presence of histamine that was completely blocked by histamine H1 receptor (H1R) and protein kinase C (PKC) inhibitors. However, histamine H2, H3, and H4 receptor inhibitors did not inhibit tube formation, suggesting that H1R-PKC signaling is involved in histamine-induced tube formation. Moreover, we found an H1-specific induction of vascular endothelial growth factor (VEGF) expression. Inhibition of VEGF receptor 2 (VEGFR2) suppressed the histamine-induced tube formation, indicating that VEGF is downstream of histamine signaling. Additionally, we demonstrated that histamine stimulation induces the expression of critical regulators of angiogenesis such as matrix metalloproteinase (MMP)-9 and MMP-14 metalloproteases, as histamine-induced tube formation is blocked by MMP inhibitors. In summary, our study indicates that histamine can activate the H1R in human endothelial cells and thereby promote tube formation through the PKC, MMP, and VEGF signaling pathways.

USP14 Regulates ATF2/PIK3CD Axis to Promote Microvascular Endothelial Cell Proliferation, Migration, and Angiogenesis in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness in diabetic patients. However, the pathogenesis of DR is complex, and no firm conclusions have been drawn so far. It has become a hot spot in ophthalmology research to deeply study the mechanism of DR pathological changes and find effective treatment options. Human retinal microvascular endothelial cells (HRMECs) were induced by high glucose (HG) to construct DR cell model. CCK-8 assay was used to detect the viability of HRMECs. Transwell assay was used to detect the migration ability of HRMECs. Tube formation assay was used to identify the tube formation ability of HRMECs. The expressions of USP14, ATF2 and PIK3CD were detected by Western blot analysis and qRT-PCR assay. Immunoprecipitation (IP) was used to ascertain the relationship of USP14 and ATF2. To explore the regulatory relationship between ATF2 and PIK3CD by dual-luciferase reporter gene assay and Chromatin immunoprecipitation (ChIP) assay. High glucose treatment promoted the proliferation, migration, and tube formation of HRMEC, and the expressions of USP14, ATF2 and PIK3CD were significantly up-regulated. USP14 or ATF2 knockdown inhibited HG-induced HRMECs proliferation, migration, and tube formation. USP14 regulated the expression of ATF2, and ATF2 promoted PIK3CD expression. PIK3CD overexpression attenuated the inhibitory effectiveness of USP14 knockdown on proliferation, migration and tube formation of DR cell model. Here, we revealed that USP14 regulated the ATF2/PIK3CD axis to promote proliferation, migration, and tube formation in HG-induced HRMECs.