Human Umbilical Vein Vascular Endothelial Cells Research Articles

μF-hBBB Chip Together with Tetrahedral DNA Frameworks for Visualization of LPS-Mediated Inflammation.

The blood-brain barrier (BBB) is essential for maintaining central nervous system (CNS) stability, and neuroinflammation may cause the dysfunction of the BBB. MicroRNA-146a (miR-146a) is closely associated with neuroinflammation, which showed significant upregulation in response to lipopolysaccharide (LPS) induction. Elucidating the relationship between LPS-induced miR-146a expression and the BBB could decipher the mechanism of many neurological diseases. Here, we constructed an in vitro microfluidic human-BBB (μF-hBBB) chip consisting of human umbilical vein vascular endothelial cells (HUVECs) and human astrocyte (HAs) cells. A tetrahedral DNA framework (TDF-3MB) nanoprobe was used to label miR-146a in HUVECs on μF-hBBB chips before and after LPS induction, and the study revealed a significant increase in miR-146a expression after LPS induction. We believe that such a μF-hBBB chip is a promising in vitro platform for further use in understanding CNS diseases.

P-345 GM-CSF drives endometrial repair via p-STAT3 mediating angiogenesis

Abstract Study question Does granulocyte macrophage colony-stimulating factor (GM-CSF) play a role in endometrial angiogenesis and can it be used as a new application for treating endometrial regeneration? Summary answer GM-CSF improves endometrial repair via promoting endometrial angiogenesis and may provide a novel insight and therapeutics for endometrial injury. What is known already GM-CSF is a cytokine normally expressed in the female reproductive tract and plays key roles in embryo implantation and subsequent development. Our previous study found that intraperitoneally （i.p.）injection of GM-CSF can significantly improve endometrial repair by promoting endometrial glandular cells proliferation and stromal cells migration, increasing the thickness of endometrium and embryo implantation in mice. However, whether GM-CSF is involved in endometrial angiogenesis is unknown. Study design, size, duration To observe whether angiogenesis is promoted by GM-CSF, the expression of angiogenic factor CD31 was evaluated after injection of GM-CSF into endometrial injured mice model. The effect of GM-CSF on vascular g regeneration was also explored by zebrafish embryo model of intersegmental vascular injury. Human umbilical vein vascular endothelial cells (HUVECs) were cultured in vitro, and the role of GM-CSF on HUVECs were examined through EdU, Tube formation, Scratch repair and Transwell assays. Participants/materials, setting, methods 20μl 90% ethanol was used to establish endometrial injured mice model. After modeling, compared with i.p. injection of GM-CSF and saline, observing whether GM-CSF had the effect of repairing angiogenesis of injured endometrium, by evaluating expression of CD31. Real-time PCR, Western Blot were used to verify differentially expressed levels of mRNA and protein. Western Blot was used to confirm protein location. ChIP was used to analyze stat3 regulation of GM-CSF. Main results and the role of chance GM-CSF promoted expression of angiogenic factor CD31 in the mice model of endometrial injury. GM-CSF can repair and regenerate the zebrafish embryo model of intersegmental vascular injury caused by Sorafenib. GM-CSF can promote angiogenesis by HUVECs proliferation and migration, and significantly increase the formation of vascular-like network structures In GM-CSF treatment group, the mRNA expression of VEGF, MMP2, Ang1, Ang2 and Tie2 mRNA in HUVECs cells increased significantly; GM-CSF can activate the phosphorylation expression levels of p-FAK, p-Src, p-ERK 1/2, p-STAT3, p-p38 MAPK, pc-Jun, p-CREB, p-Akt, and p-eNOS. And it can increase the expression of downstream VEGF and MMP2 proteins. GM-CSF treatment for 30 min can promote phosphorylation and translocation of STAT3 from cytoplasm to nucleus, thereby regulating the expression of VEGF and MMP2 downstream. While FAK inhibitors (PF573228) and STAT3 knockdown can abrogate GM-CSF effect on HUVECs. Limitations, reasons for caution The results at the cellular and animal level can’t be completely mimic human endometrial injury. In future, well-designed clinical trials are needed to investigate the efficacy and safety of GM-CSF for treatment of human endometrial vascular injury. Wider implications of the findings GM-CSF can promote the vascular regeneration in mice model of endometrial injury through p-STAT3. Our findings provide new ideas for clinical treatment of endometrial injury. Trial registration number Not applicable

The Antimicrobial Peptide Esculentin-1a(1-21)NH2 Stimulates Wound Healing by Promoting Angiogenesis through the PI3K/AKT Pathway.

Delayed wound healing is a persistent medical problem mainly caused by decreased angiogenesis. Esculentin-1a(1-21)NH2 [Esc-1a(1-21)NH2], has broad-spectrum antibacterial properties which comes from frog skins. It has shown promise as a treatment for wound healing. However, its effects on angiogenesis as well as the mechanism by which esc-1a(1-21)NH2 enhanced wound healing remained unclear. In this study, we analyzed the structural properties and biocompatibility of esc-1a(1-21)NH2 and evaluated its effect on wound closure using a full-thickness excision model in mice. Our results showed that esc-1a(1-21)NH2 significantly accelerated wound healing by increasing collagen deposition and angiogenesis, characterized by elevated expression levels of platelet, endothelial cell adhesion molecule-1 (CD31) and proliferating cell nuclear antigen (PCNA). Furthermore, the angiogenic activity of esc-1a(1-21)NH2 was confirmed in vitro by various assays. Esc-1a(1-21)NH2 significantly promoted cell migration and cell proliferation in human umbilical vein vascular endothelial cells (HUVECs) via activation of the phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (AKT) pathway, and upregulated the expression of CD31 at both mRNA and protein levels. The effect of esc-1a(1-21)NH2 on angiogenesis was diminished by LY294002, a PI3K pathway inhibitor. Taken together, this study demonstrates that esc-1a(1-21)NH2 accelerates wound closure in mice by promoting angiogenesis via the PI3K/AKT signaling pathway, suggesting its effective application in the treatment of wound healing.

Ceramide induces pyroptosis through TXNIP/NLRP3/GSDMD pathway in HUVECs

BackgroundPyroptosis of endothelial cells is a new cause of endothelial dysfunction in multiple diseases. Ceramide acts as a potential bioactive mediator of inflammation and increases vascular endothelial permeability in many diseases, whether it can aggravate vascular endothelial injury by inducing cell pyroptosis remains unknown. This study was established to explore the effects of C8-ceramide (C8-Cer) on human umbilical vein vascular endothelial cells (HUVECs) and its possible underlying mechanism.MethodsHUVECs were exposed to various concentrations of C8-Cer for 12 h, 24 h, 48 h. The cell survival rate was measured using the cell counting kit-8 assay. Western blotting and Real-time polymerase chain reaction (RT-PCR) were used to detect the pyroptosis-releated protein and mRNA expressions, respectively. Caspase-1 activity assay was used to detect caspase-1 activity. Hoechst 33342/propidium iodide double staining and flow cytometry were adopted to measure positive staining of cells. Lactate dehydrogenase release assay and enzyme-linked immunosorbent assay were adopted to measure leakage of cellular contents. FITC method was used to detect the permeability of endothelial cells. ROS fluorescence intensity were detected by flow cytometry.ResultsThe viability of HUVECs decreased gradually with the increase in ceramide concentration and time. Ceramide upregulated the expression of thioredoxin interacting protein (TXNIP), NLRP3, GSDMD, GSDMD-NT, caspase-1 and Casp1 p20 at the protein and mRNA level in a dose-dependent manner. It also enhanced the PI uptake in HUVECs and upregulated caspase-1 activity. Moreover, it promoted the release of lactate dehydrogenase, interleukin-1β, and interleukin-18. Meanwhile, we found that ceramide led to increased vascular permeability. The inhibitor of NLRP3 inflammasome assembly, MCC950, was able to disrupt the aforementioned positive loop, thus alleviating vascular endothelial cell damage. Interestingly, inhibition of TXNIP either chemically using verapamil or genetically using small interfering RNA (siRNA) can effectively inhibit ceramide-induced pyroptosis and improved cell permeability. In addition, ceramide stimulated reactive oxygen species (ROS) generation. The pretreatment of antioxidant N-acetylcysteine (NAC), ROS scavenger, blocked the expression of pyroptosis markers induced by C8-cer in HUVECs.ConclusionThe current study demonstrated that C8-Cer could aggravate vascular endothelial cell damage and increased cell permeability by inducing cell pyroptosis. The results documented that the ROS-dependent TXNIP/NLRP3/GSDMD signalling pathway plays an essential role in the ceramide-induced pyroptosis in HUVECs.

Biodegradable Mg-Sc-Sr Alloy Improves Osteogenesis and Angiogenesis to Accelerate Bone Defect Restoration.

Magnesium (Mg) and its alloys are considered to be biodegradable metallic biomaterials for potential orthopedic implants. While the osteogenic properties of Mg alloys have been widely studied, few reports focused on developing a bifunctional Mg implant with osteogenic and angiogenic properties. Herein, a Mg-Sc-Sr alloy was developed, and this alloy's angiogenesis and osteogenesis effects were evaluated in vitro for the first time. X-ray Fluorescence (XRF), X-ray diffraction (XRD), and metallography images were used to evaluate the microstructure of the developed Mg-Sc-Sr alloy. Human umbilical vein/vascular endothelial cells (HUVECs) were used to evaluate the angiogenic character of the prepared Mg-Sc-Sr alloy. A mix of human bone-marrow-derived mesenchymal stromal cells (hBM-MSCs) and HUVEC cell cultures were used to assess the osteogenesis-stimulating effect of Mg-Sc-Sr alloy through alkaline phosphatase (ALP) and Von Kossa staining. Higher ALP activity and the number of calcified nodules (27% increase) were obtained for the Mg-Sc-Sr-treated groups compared to Mg-treated groups. In addition, higher VEGF expression (45.5% increase), tube length (80.8% increase), and number of meshes (37.9% increase) were observed. The Mg-Sc-Sr alloy showed significantly higher angiogenesis and osteogenic differentiation than pure Mg and the control group, suggesting such a composition as a promising candidate in bone implants.

Effect of Nintedanib Nanothermoreversible Hydrogel on Neovascularization in an Ocular Alkali Burn Rat Model

Purpose To compare the therapeutic effects of different forms of nintedanib ophthalmic preparations on neovascularization corneal alkali burns in rats. Methods Forty rat models of left eye corneal alkali burns were constructed, and the five groups (N = 8) were treated with normal saline, dexamethasone ointment (dexamethasone), 0.2% nintedanib aqueous solution and nintedanib nano thermoreversible hydrogel (NNTH). A slit lamp microscope was used to observe the area of neovascularization. The levels of the inflammatory factors were detected by ELISA. HE staining was performed on the rat corneas. Vascular endothelial growth factor (VEGFA) was detected by immunohistochemistry, and the expression of corneal VEGFA and CD31 was detected by western blotting. An MTT assay was performed to detect the cytotoxicity of nintedanib on human corneal epithelial cells (HCECs) and human umbilical vein vascular endothelial cells (HUVECs). Cell migration was detected by a cell scratch assay, and the proportion of apoptotic cells was detected by Annexin/PI double staining. Immunofluorescence and western blotting were performed to detect the protein expression of VEGFA and CD31. Results NNTH had a stronger inhibitory effect on corneal neovascularization (CNV) in alkali-burned rats while reducing the level of inflammatory factors. NNTH had a longer drug duration of release than nanoformulations in vitro. Nintedanib at low concentrations (<8 μM) had no significant cytotoxicity to HCECs but significantly induced apoptosis and inhibited the expression of VEGFA and CD31 and the migration of HUVECs. Conclusions Nanomorphic thermoreversible hydrogel is superior among the nintedanib ophthalmic preparations, showing better inhibition of CNV in alkali-burned eyeballs and it blocked the migration and proangiogenic ability of HUVECs.

Modulation of vascular endothelial inflammatory response by proprotein convertase subtilisin-kexin type 9

Background and AimsEndotoxins carried within LDL are cleared from the circulation via hepatic LDL receptor (LDLR)-mediated endocytosis. Proprotein convertase subtilisin-kexin type 9 (PCSK9) reduces this clearance by down-regulating LDLR density on hepatocytes. In addition to hepatocytes, vascular endothelial cells also express receptor targets of PCSK9, including LDLR. Therefore, we hypothesized that PCSK9 may regulate vascular endothelial cell uptake of lipopolysaccharide (LPS) and alter the vascular endothelial cell inflammatory response. Methods and resultsWe found that LPS is internalized by human umbilical vein vascular endothelial cells (HUVECs) and LPS uptake dose-dependently increased with increasing LDL concentration. Intracellular LPS co-localized with LDL. PCSK9 and, separately, blocking antibodies against LDLR, dose-dependently decreased the vascular endothelial cell uptake of LPS and, furthermore, inhibition of endocytosis using Dynasore blocked LPS uptake. In contrast, blocking antibodies against TLR4 did not alter LPS uptake. PCSK9 decreased the LPS-induced proinflammatory response (IL-6 and IL-8 gene expression and protein secretion, and VCAM-1/ICAM-1 expression) in vascular endothelial cells. In addition, a decrease in PCSK9 and increase in LDLR, mediated by triciribine or siPCSK9, increased LPS uptake and the LPS-induced proinflammatory response. Similar results were also found in aortic vascular tissue from Pcsk9−/− mice after LPS injection. ConclusionsOur data suggest that, similar to PCSK9 treatment in hepatocytes, PCSK9 reduces vascular endothelial cell uptake of LPS via LDLR-mediated endocytosis. Consequently, PCSK9 decreases the LPS-induced proinflammatory response in vascular endothelial cells. These results raise the possibility that PCSK9 inhibition may have additional effects on vascular endothelial inflammation via this alternative pathway, beyond the known effects of PCSK9 inhibition on LDL lowering and hepatic endotoxin clearance.

Endostar Synergizes with Radiotherapy to Inhibit Angiogenesis of Cervical Cancer in a Subcutaneous Xenograft Mouse Model.

To investigate the synergic effect and underlying mechanism of Endostar, a recombinant human endostatin used for anti-angiogenesis, in radiotherapy for cervical cancer. The Cell Counting Kit-8 (CCK-8) assay and plate cloning experiment were first employed to analyze the proliferation of HeLa and SiHa cervical cancer cells and human umbilical vein vascular endothelial cells (HUVECs). Flow cytometry was used to detect apoptosis and cell cycle progression. A tube formation assay was used to assess angiogenesis in vitro. The expression of gamma H2A histone family member X (γ-H2AX) and activation of the vascular endothelial growth factor receptor (VEGFR) signaling pathway were detected by immunofluorescence and western blotting, respectively. In a HeLa xenograft model, tumor tissue expression of CD31 and alpha smooth muscle actin and serum expression of VEGF-A were detected by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay, respectively. The CCK-8 and plate cloning assays showed that Endostar and radiotherapy synergistically inhibited the growth of HUVECs but not HeLa and SiHa cells. The flow cytometric results showed that Endostar only promoted radiotherapy-induced apoptosis and G2/M phase arrest in HUVECs (p < 0.05). Endostar combined with radiotherapy also significantly inhibited tube formation by HUVECs (p < 0.05). Furthermore, Endostar inhibited the radiotherapy-induced expression of γH2AX (p < 0.05) and phosphorylation of VEGFR2/PI3K/AKT/DNA-PK in HUVECs (p < 0.05). IHC showed that Endostar enhanced the inhibitory effect of radiotherapy on the microvessel density in xenograft tumor tissues (p < 0.05), as well as serum VEGF-A expression (p < 0.05). The tumor volume in the combination therapy groups (1200 mm3) was significantly lower than in the control group (2500 mm3; p < 0.05). Our findings provide experimental evidence and a theoretical basis for the application of Endostar in combination with irradiation for anti-cervical cancer treatment.

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice

Anti-angiogenic properties of rapamycin on human retinal pericytes in an in vitro model of neovascular AMD via inhibition of the mTOR pathway

PurposeChoroidal neovascularizations (CNV) are partially stabilized through a coverage of pericytes leading to a partial anti-VEGF resistence. Drugs licensed for neovascular AMD (nAMD) do not take this mechanical and growth factor-driven CNV stability into account. The purpose of this work was to see if inhibiting the mammalian target of rapamycin (mTOR) may successfully block angiogenic cellular pathways in primary human retinal pericytes in an in vitro model of nAMD.MethodsThe mTOR inhibitor rapamycin was used to treat human retinal pericytes (HRP) at doses ranging from 0.005 to 15 g/ml. A modified metabolism-based XTT-Assay was used to assess toxicity and anti-proliferative effects. A scratch wound experiment showed the effects on migration. On Cultrex basement membrane gels, the influence of rapamycin on the development of endothelial cell capillary-like structures by human umbilical vein vascular endothelial cells (HUVEC) in the absence and presence of pericytes was investigated.ResultsRapamycin showed no signs of toxicity within its range of solubility. The drug showed dose dependent anti-proliferative activity and inhibited migration into the scratch wound. Endothelial cell tube formation in a HUVEC monoculture was effectively inhibited at 45%. A co-culture of HUVEC with pericytes on Cultrex induced endothelial tube stabilization but was disrupted by the addition of rapamycin leading to degradation of 94% of the tubes.ConclusionsRapamycin allows for an efficient modulation of aspects of angiogenesis in pericytes via mTOR-modulation in vitro. Further studies are needed to elucidate whether rapamycin may have an impact on CNV in nAMD in vivo.

Protective Effect of Flavonoids from a Deep-Sea-Derived Arthrinium sp. against ox-LDL-Induced Oxidative Injury through Activating the AKT/Nrf2/HO-1 Pathway in Vascular Endothelial Cells.

Oxidized low-density lipoprotein (ox-LDL)-induced oxidative injury in vascular endothelial cells is crucial for the progression of cardiovascular diseases, including atherosclerosis. Several flavonoids have been shown cardiovascular protective effects. Recently, our research group confirmed that the novel flavonoids isolated from the deep-sea-derived fungus Arthrinium sp., 2,3,4,6,8-pentahydroxy-1-methylxanthone (compound 1) and arthone C (compound 2) effectively scavenged ROS in vitro. In this study, we further investigated whether these compounds could protect against ox-LDL-induced oxidative injury in endothelial cells and the underlying mechanisms. Our results showed that compounds 1 and 2 inhibited ox-LDL-induced apoptosis and adhesion factors expression in human umbilical vein vascular endothelial cells (HUVECs). Mechanistic studies showed that these compounds significantly inhibited the ROS level increase and the NF-κB nuclear translocation induced by ox-LDL. Moreover, compounds 1 and 2 activated the Nrf2 to transfer into nuclei and increased the expression of its downstream antioxidant gene HO-1 by inducing the phosphorylation of AKT in HUVECs. Importantly, the AKT inhibitor MK-2206 2HCl or knockdown of Nrf2 by RNA interference attenuated the inhibition effects of these compounds on ox-LDL-induced apoptosis in HUVECs. Meanwhile, knockdown of Nrf2 abolished the effects of the compounds on ox-LDL-induced ROS level increase and the translocation of NF-κB to nuclei. Collectively, the data showed that compounds 1 and 2 protected endothelial cells against ox-LDL-induced oxidative stress through activating the AKT/Nrf2/HO-1 pathway. Our study provides new strategies for the design of lead compounds for related cardiovascular diseases treatment.

Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis

BackgroundEnhanced angiogenesis can promote diabetic wound healing. Mesenchymal stem cells (MSCs)-derived exosomes, which are cell-free therapeutics, are promising candidates for the treatment of diabetic wound healing. The present study aimed to investigate the effect of exosomes derived from MSCs pretreated with pioglitazone (PGZ-Exos) on diabetic wound healing.ResultsWe isolated PGZ-Exos from the supernatants of pioglitazone-treated BMSCs and found that PGZ-Exos significantly promote the cell viability and proliferation of Human Umbilical Vein Vascular Endothelial Cells (HUVECs) injured by high glucose (HG). PGZ-Exos enhanced the biological functions of HUVECs, including migration, tube formation, wound repair and VEGF expression in vitro. In addition, PGZ-Exos promoted the protein expression of p-AKT, p-PI3K and p-eNOS and suppressed that of PTEN. LY294002 inhibited the biological function of HUVECs through inhibition of the PI3K/AKT/eNOS pathway. In vivo modeling in diabetic rat wounds showed that pioglitazone pretreatment enhanced the therapeutic efficacy of MSCs-derived exosomes and accelerated diabetic wound healing via enhanced angiogenesis. In addition, PGZ-Exos promoted collagen deposition, ECM remodeling and VEGF and CD31 expression, indicating adequate angiogenesis in diabetic wound healing.ConclusionsPGZ-Exos accelerated diabetic wound healing by promoting the angiogenic function of HUVECs through activation of the PI3K/AKT/eNOS pathway. This offers a promising novel cell-free therapy for treating diabetic wound healing.Graphic abstract

Antifungal and wound healing promotive compounds from the resins of Dracaena cochinchinensis

Five new compounds (xuejieins A–E), including three new phenolic glycosides (1, 2, and 5) and two new flavonoids (10 and 11), together with six known compounds were isolated from the resins of Dracaena cochinchinensis (Chinese dragon's blood). The structures of the new compounds were confirmed by extensive spectroscopic methods and electronic circular dichroism (ECD) data analysis. Especially, the absolute configurations of the sugar moieties in compounds 1, 2, and 5 were clarified by GC analysis after acid hydrolysis. All isolated compounds have been tested for antifungal and wound healing promoting activities, The results showed that compound 9 shows significant antifungal activities against Botrytis cinerea, Magnaporthe grisea, Penicillium digitatum, and Sclerotinia sclerotiorum. In addition, compound 4 could significantly stimulate human keratinocytes (HaCAT) proliferation, mobility, and human umbilical vein vascular endothelial cells (HUVECs) tube formation at 40 μM.

Exosomal miR-141 promotes tumor angiogenesis via KLF12 in small cell lung cancer

BackgroundAngiogenesis, a basic requirement for tumor cell survival, is considered to be a malignant characteristic of small cell lung cancer (SCLC) and is closely related to the poor outcomes of SCLC patients. miR-141 has been found to play pro- and antiangiogenic roles in different cancers, but its role in SCLC angiogenesis has never been explored.MethodsTotal RNA was isolated from plasm exosomes and serum of SCLC patients to examine the expression of miR-141 by qRT-PCR. Cell proliferation, invasion, migration, tube formation assay, aortic ring assay and mouse tumor model were used to investigate the effect of exosomal miR-141 in angiogenesis in vitro and in vivo. Dual-luciferase assay was conducted to explore the target gene of miR-141.ResultsCirculating miR-141 was upregulated in samples from 122 SCLC patients compared with those from normal volunteers and that the increase in miR-141 was significantly associated with advanced TNM stages, implying the potential oncogenic role of miR-141 in SCLC malignancy. In vitro, miR-141 that was packaged into SCLC cell-secreted exosomes and delivered to human umbilical vein vascular endothelial cells (HUVECs) via exosomes facilitated HUVEC proliferation, invasion, migration and tube formation and promoted microvessel sprouting from mouse aortic rings. Matrigel plug assays demonstrated that SCLC cell-derived exosomal miR-141 induced neoangiogenesis in vivo. Furthermore, mouse subcutaneous tumor nodules that were developed from miR-141-overexpressing SCLC cells had a higher microvessel density (MVD) and grew faster than those developed from negative control cells. KLF12 was found to be the direct target gene of miR-141 and that the proangiogenic effect of miR-141 on HUVECs was abrogated by KLF12 overexpression.ConclusionsOur results demonstrate the specific function of the exosomal miR-141/KLF12 pathway in SCLC angiogenesis for the first time and provide potential novel targets for antiangiogenic therapies for SCLC patients.

Abstract 1486: Exosomal miR-141 promoted tumor angiogenesis via KLF12 in small cell lung cancer

Abstract Background: Angiogenesis, the basic requirement for the survival of tumor cells, has been considered to be one of the malignant characteristics of small cell lung cancer (SCLC) and was closely related to the poor outcomes of SCLC patients. MiR-141 has been found to play a pro- or anti-angiogenetic role in different cancers while its role in SCLC angiogenesis has never been explored. Methods: We isolate total RNA from plasm exosomes and serum samples from two cohorts of SCLC patients respectively to detect the expression of circulating miR-141 in SCLC patients using qRT-PCR. miR-141 was overexpressed in SCLC cell-secreted exosomes to investigate its effects on cell migration, tube formation and microvessel sprouting capabilities of human umbilical vein vascular endothelial cells (HUVECs) both in vitro and in vivo. A dual-luciferase assay was conducted to validate the direct target gene of miR-141. Results: We showed that the level of circulating miR-141 was significantly upregulated in a total of 116 SCLC patients compared with normal volunteers, and the increased miR-141 was revealed to be associated with advanced patient TNM stages which implying the potential oncogenic role of miR-141 in SCLC malignances. In vitro experiments indicated that miR-141 was packaged into SCLC cell-secreted exosomes and was delivered to HUVECs via exosomes to strengthen the migration, invasion, tube formation and microvessel sprouting capabilities of HUVECs. Moreover, mouse models demonstrated that exosomal miR-141 derived from SCLC cells induced neo-angiogenesis in vivo and subcutaneous tumor nodules developed from SCLC cells with miR-141 overexpression showed higher microvessel densities and faster tumor growth than which developed from negative control cells. Combining miRNA target prediction tools with a dual-luciferase assay, we identified that KLF12 was the direct target gene of miR-141 and the angiogenetic promotion effect of miR-141 on HUVECs was abrogated by overexpressing KLF12. Conclusions: Our results elaborated the specific function of exosomal miR-141/KLF12 pathway in SCLC angiogenesis for the first time and provided potential novel targets for anti-angiogenetic therapies in SCLC patients. Our study highlighted the critical role of exosomal miRNAs in SCLC microenvironments and cell communications which offered novel clues for the future exploration of molecular mechanisms of SCLC malignances. Citation Format: Shuangshuang Mao, Zhiliang Lu, Sufei Zheng, Guochao Zhang, Jianbing Huang, Nan Sun, Jie He. Exosomal miR-141 promoted tumor angiogenesis via KLF12 in small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1486.

Tritiated Water Induces Toxicity in Human Umbilical Vein Vascular Endothelial Cells via IL8.

We aimed to determine the toxic effects of tritiated water (HTO) on 12 generations (T1-T12) of human umbilical vein vascular endothelial cells (HUVECs) and elucidate the underlying mechanisms. We evaluated cellular senescence, interleukin (IL) 8 concentrations, and angiogenesis using β-galactosidase staining, enzyme-linked immunosorbent assay, and in vitro assays, respectively. The adhesion properties of contaminated cells and differentially expressed genes were assessed using the xCELLigence RTCA SP system and gene chip analysis, respectively. We found that long-term exposure to low levels of HTO can reduce the adhesion of HUVECs to the cellular matrix as well as their angiogenic capacity, while increasing their permeability, senescence, and adhesion to monocytes. Interleukin 8 activated the p38 and Epidermal Growth Factor Receptor (EGFR) pathways in HTO-treated cells and hence was identified as a key candidate of biomarker. The present study clarified the toxicity of HTO in vascular endothelial cells and identified IL8 as a novel protective target with important theoretical and practical values.

Combination of Astragalus membranaceous and Angelica sinensis Ameliorates Vascular Endothelial Cell Dysfunction by Inhibiting Oxidative Stress.

Vascular endothelial dysfunction is an essential and early sign of diabetic macroangiopathy, a primary complication of diabetes mellitus. Astragalus membranaceous-Angelica sinensis is a classic medical combination applied in China to treat diabetes mellitus. The aim of this study was to investigate the effect of the granule form of the extract produced from the dried root of Astragalus membranaceous (AM) combination with the granule form of the extract produced from the dried Angelica sinensis (AS) on diabetic macroangiopathy and its underlying mechanism. Herein, rats were treated by AM-AS at a ratio of 3 : 2 via intragastric administration. High glucose-induced human umbilical vein vascular endothelial cells (HUVECs) were then treated with drug-containing serum collected from the rats. In high glucose-treated HUVECs, AM-AS combination increased cell viability (P < 0.05), decreased the percentage of apoptotic cells (P < 0.05) and the expression of the proapoptosis protein caspase 3 (P < 0.05), reduced the proportion of cells in the G0/G1 phase (P < 0.05), decreased reactive oxygen species level (P < 0.05), enhanced cell migration and invasion (P < 0.05), and reduced the level of 8-iso-prostaglandin F2alpha. These results indicate that AM-AS combination at the ratio of 3 : 2 ameliorated HUVEC dysfunction by regulating apoptosis, cell migration, and invasion, which might be mediated by their regulatory effect on reactive oxygen species production. The current study provides a theoretical basis for the treatment of diabetic macroangiopathy using AM-AS.

Simvastatin preparations promote PDGF-BB secretion to repair LPS-induced endothelial injury through the PDGFRβ/PI3K/Akt/IQGAP1 signalling pathway.

Endothelial barrier dysfunction is a critical pathophysiological process of sepsis. Impaired endothelial cell migration is one of the main reasons for endothelial dysfunction. Statins may have a protective effect on endothelial barrier function. However, the effect and mechanism of statins on lipopolysaccharide (LPS)‐induced endothelial barrier dysfunction remain unclear. Simvastatin (SV) was loaded in nanostructured lipid carriers to produce SV nanoparticles (SV‐NPs). Normal SV and SV‐NPs were used to treat human umbilical vein vascular endothelial cells (HUVECs) injured by LPS. Barrier function was evaluated by monitoring cell monolayer permeability and transendothelial electrical resistance, and cell migration ability was measured by a wound healing assay. LY294002 and imatinib were used to inhibit the activity of PI3K/Akt and platelet‐derived growth factor receptor (PDGFR) β. IQ‐GTPase‐activating protein 1 (IQGAP1) siRNA was used to knockdown endogenous IQGAP1, which was used to verify the role of the PDGFRβ/PI3K/Akt/IQGAP1 pathway in SV/SV‐NPs‐mediated barrier protection in HUVECs injured by LPS. The results show that SV/SV‐NPs promoted the migration and decreased the permeability of HUVECs treated with LPS, and the efficacy of the SV‐NPs exceeded that of SV significantly. LY294002, imatinib and IQGAP1 siRNA all suppressed the barrier protection of SV/SV‐NPs. SV/SV‐NPs promoted the secretion of platelet‐derived growth factor‐BB (PDGF‐BB) and activated the PDGFRβ/PI3K/Akt/IQGAP1 pathway. SV preparations restored endothelial barrier function by restoring endothelial cell migration, which is involved in the regulation of the PDGFRβ/PI3K/Akt/IQGAP1 pathway and PDGF‐BB secretion. As an appropriate formulation for restoring endothelial dysfunction, SV‐NPs may be more effective than SV.

Eupatilin inhibits angiogenesis-mediated human hepatocellular metastasis by reducing MMP-2 and VEGF signaling

Metastasis is responsible for the great majority of deaths in cancer patients. Matrix metalloproteinases (MMPs) have critical functions in cancer metastasis. Especially, MMP-2 and MMP-9 play a major role in tumor-cell migration and invasion. Therefore, to first find out the inhibitory effect of eupatilin on expression of MMPs in SNU182 cells, we used quantitative real-rime PCR to measure MMP-2 and MMP-9 mRNA levels. Eupatilin suppressed transcription of MMP-2 in SNU182 cells more than did the corresponding controls. Also, eupatilin significantly blocked tube formation when treated with a concentration of 3.125 or 6.25 μg/mL on human umbilical vein vascular endothelial cells (HUVECs). Eupatilin induced significant anti-angiogenic potential associated with down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and phosphorylated Akt expression. Thus, tube-formation inhibition and MMP-2-mediated migration are likely to be important therapeutic targets of eupatilin in hepatocellular carcinoma metastasis.

Apatinib targets both tumor and endothelial cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed malignancies worldwide with poor prognosis and tends to be hypervascular. Aberrant expression of the vascular endothelial growth factor 2 (VEGFR‐2) has been implicated in the progression of HCC and represents a valid target for anticancer therapy. Apatinib, a small molecule inhibitor of VEGFR‐2 tyrosine kinase, shows strong antitumor activity in various tumors. This study is designed to evaluate the activity of apatinib on both human umbilical vein vascular endothelial cells (HUVECs) and HCC cell lines (in vitro and in vivo), and also to investigate the characteristics and possible mechanisms underlying these effects by molecular biology methods. Following the results in our study, apatinib inhibited phosphorylation of VEGFR‐2 in HUVECs and blocked in vitro endothelial cell migration and tube formation. Concentration‐dependent antiproliferative effects of apatinib were also observed in all 6 HCC cell lines including SK‐Hep‐1, HepG2, Hep3B, Huh‐7, PLC/PRF/5, SMMC‐7721. Moreover, response to apatinib of HCC cell lines was significantly correlated with VEGFR‐2 expression level. Additionally, apatinib significantly inhibit VEGF‐triggered VEGFR‐2 phosphorylation and activation of downstream signaling molecules such as Akt and ERK1/2 in HCCs. Apatinib can also induce a cell cycle arrest at G2/M phase and promote HCC apoptosis tested in vitro. In vivo data showed that apatinib can effectively inhibit tumor growth, decreased angiogenesis, as well as induced HCC apoptosis (in some tumors), and thus prolonged animal survival in a mouse xenograft model of human HCC. Our findings suggested that apatinib is a highly potent, oral active anti‐angiogenic, and anti‐HCC agent. The results from current study provide a clear biological rationale to evaluate apatinib as a new agent in HCC in clinical setting, especially for the VEGFR‐2 overexpression ones.