The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours’ treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.
OBJECTIVE: To elongate the proliferation life-span of human umbilicus vein endothelial cell (HUVEC). METHODS: We synthesized the human telomerase reverse transcriptase mRNA (hTERT mRNA) by in vitro transcription, then transferred the hTERT mRNA into HUVEC in quicent stage by lipofect introduction. RESULTS: Telomerase expressed transiently in HUVEC, and the cell life-span was elongated for 7 population doublings. CONCLUSION: Telomerase can be reconstructed controllably and transiently in HUVEC by hTERT mRNA introduction, this method has the potential to be used to elongate the lifespan of cells cultured in vitro.
Objective To summarize the research progress on the regulation of hepatic sinusoidal microenvironment to promote liver regeneration based on liver sinusoidal endothelial cells (LSECs), aiming to further clarify the mechanism of liver regeneration and provide new ideas and methods for clinical promotion of liver regeneration and prevention of liver failure. Method The basic and clinical research studies on LSECs and liver regeneration at home and abroad in recent years were searched and reviewed. Results Differentiated LSECs played an important role in liver regeneration, regulated the homeostasis of hepatic sinusoid microenvironment by paracrine and autocrine, and participated in the whole process of promoting liver regeneration, such as hepatocyte proliferation and neovascularization after acute and chronic liver injury. Conclusion In the process of liver regeneration after all kinds of acute and chronic liver injury, LSECs promote liver regeneration by regulating hepatic sinusoid microenvironment, which will provide new strategies and methods for clinical promotion of liver regeneration and prevention of liver failure after hepatectomy.
Objective To investigate the effect of microRNA-22-3p (miR-22-3p) on the inflammation of human pulmonary microvascular endothelial cells (HPMEC) induced by lipopolysaccharide (LPS) by regulating the HMGB1/NLRP3 pathway. Methods miRNA microarray was taken from peripheral blood of patients with acute respiratory distress syndrome (ARDS) caused by abdominal infection and healthy controls for analysis, and the target miRNA was selected. miRNA mimics, inhibitor and their negative controls were transfected in HPMECs which were stimulated with LPS. Real time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the mRNA and protein levels of high mobility group box-1 protein (HMGB1) and nucleotide binding oligomerization segment like receptor family 3 (NLRP3). RT-qPCR and enzyme linked immunosorbent assay were used to detect the levels of inflammatory factors in the cells and supernatant. Results miRNA microarray showed that miR-22-3p was down-regulated in the plasma of patients with ARDS. Compared with the negative control group, after miR-22-3p over-expression, the protein and mRNA levels of HMGB1 and NLRP3 decreased significantly. Similarly, the level of cleaved-caspase-1 decreased significantly. At the same time, interleukin (IL)-6, IL-8 and IL-1β mRNA level in cytoplasm and supernatant were down-regulated by miR-22-3p mimics. After transfected with miR-22-3p inhibitor, the expression levels of HMGB1, NLRP3, caspase-1 protein and inflammatory factors were significantly up-regulated. Conclusion miR-22-3p is significantly downregulated in peripheral blood of ARDS patients caused by abdominal infection, which can inhibit the expression of HMGB1 and NLRP3 and its downstream inflammatory response in HPMECs.
ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
Objective To develop a new method for a tissue engineered vascular graft by combining endothelial cells and an acelluarized allogenic matrix. Methods Acellularized matrix tubes were obtained by a 0.1% trypsin and 0 02% EDTA solution for 24 hours and 1% Triton X 100 for 176 hours, respectively. Endothelial cells were isolated from alloaorta and expanded in vitro. Finally, the inner surface of acellularized matrix was reseeded with endothelial cells. Acellularity and reseeding were analysed by light microscopy and scanning electron microscopy. Results The acellularization procedure resulted in an almost complete removal of the original cells and the loose three-dimensional (3D) matrix. The acellular matrix could be reseeded with expanded endothelial cells in vitro, and endothelial cells had the potential of spread and proliferation. Conclusion Acellular matrix produces by Tritoon X-100 and trypsin possesses satisfactory biocompatibility for allogenic endothelial cell. Vascular grafts can be generated in vitro by a combination of endothelial cells and allogenic acelluarized matrix.
Serum alkaline phosphatase (ALP) has long been used as a biomarker for the liver, kidney, and bone. Currently, increasing evidence suggests a correlation between serum ALP and cardiovascular disease (CVD). Research has shown that serum ALP affects endothelial cell function and induces changes in pyrophosphate through various mechanisms to accelerate vascular calcification and promote cardiac fibrosis. Therefore, this article reviews the potential value of serum ALP in CVD through relevant research, revealing the specific relationship between serum ALP and CVD, in order to provide new ideas for the prevention and treatment of CVD.
Objective To investigate the molecular signal mechanism of transform growth factor (TGF)-β induced arterial endothelial-mesenchymal transition. Methods Rat arterial endothelial cells were primarily cultured by ex-transplant method. The endothelial cells were incubated by combinant TGF-β (10 ng/mL) for 48 hours and then were detected by immunofluorescence staining and western blotting to observe the cell surface marker expression profile and Akt/mTOR signal activation. On the other hand, the endothelial cells were preincubated by Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the Akt/mTOR signal, and then the cells were further treated with TGF-β (10 ng/mL) for 48 hours to observe the cell surface marker expression profile without Akt/mTOR signal activation. Results Rat artery endothelial cells by TGF-β after incubation, the expressions of smooth muscle cell markers α-smooth muscle actin (α-SMA) and smooth muscle-22α (SM-22α) were up-regulated, and the endothelial cell markers CD31 and vW factor were significantly down-regulated, at the same time, the expressions of phosphorylated Akt and mTOR were also up-regulated. However, after preincubation of Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the phosphorylation of Akt and mTOR signal, above TGF-β-induced expressions of α-SMA and SM-22α in arterial endothelial cells were significantly suppressed and the expressions of CD31 and vWF were preserved. Conclusion TGF-β-induced arterial endothelial-mesenchymal transition is dependent on activation of Akt/mTOR signal, suggesting that Akt/mTOR-dependent arterial endothelial-mesenchymal transition would be one of the mechanisms for intima hyperplasia in transplant arteriosclerosis.
Objective To investigate the effects of adipose-derived stem cells (ADSCs) and endothelial cells (ECs) on the survival and neovascularization of fat tissue transplants. Methods The ADSCs were isolated by collagenase digestion from the adipose tissues voluntarily donated by the patients undergoing mastectomy, and subcultured. The passage 3 ADSCs were used for subsequent experiments. The residual fat tissues were used to prepare fat particles (FPs). The human umbilical vein endothelial cells (HUVECs) were used as ECs for subsequent experiments. Eighty healthy male nude mice, aged 4-6 weeks, were randomly divided into 4 groups (n=20). The mice were received subcutaneous injection at the dorsum of 1 mL FPs+0.3 mL normal saline (NS) in control group, 1 mL FPs+2×106 ECs+0.3 mL NS in ECs group, 1 mL FPs+2×106 ADSCs+0.3 mL NS in ADSCs group, and 1 mL FPs+1×106 ECs+1×106 ADSCs+0.3 NS in ADSCs+ECs group. General observations of the injection sites were performed, and the survival of the mice was recorded. At 2, 4, 8, and 12 weeks after injection, grafted fat tissues were firstly assessed by ultrasonography, then they were collected for volume measurement (water displacement method) and histology observation (HE staining and immunofluorescence staining). Results All mice survived until the end of experiment. At each time point, no significant difference was noted between groups in ultrasonography assay. There was no significant blood flow signal in the grafted fat tissues, or cysts, calcification, solid occupying in recipient area. Generally, the volume of grafted fat tissues decreased with time in all groups. Specifically, the volumes of grafted fat tissues were larger in ADSCs group and ADSCs+ECs group than that in control group and ECs group (P<0.05) at each time point, and in ADSCs group than in ADSCs+ECs group (P<0.05) at 8 and 12 weeks. HE staining showed that all groups had similar tendencies in general histology changes, and remodeling in ADSCs group was the fastest than in the other groups. By immunofluorescence staining for neovascularization, the new vessels in all groups were increasing with time. The vessel densities were higher in ECs group, ADSCs group, and ADSCs+ECs group than in control group (P<0.05) at each time point, in ADSCs group than in ECs group and ADSCs+ECs group (P<0.05) at 4 weeks, in ADSCs group and ADSCs+ECs group than in ECs group (P<0.05) at 8 and 12 weeks. Conclusion ADSCs can significantly increase the survival of transplanted fat tissue, which may be related to promoting the neovascularization.
To study the potential molecular mechanism of tumor angiogenesis in its microenvironment, we investigated the effects of HepG2 conditioned medium on the proliferation of vascular endothelial cell and vascular angiogenesis in our laboratory. Human umbilical vein endothelial EA.hy926 cells were co-cultured with HepG2 conditioned medium in vitro. The proliferation and the tubulogenesis of EA.hy926 cells were detected by teramethylazo salt azole (MTT) and tube formation assay, respectively. The results showed that the survival rate of the EA.hy926 cells was significantly increased under the co-culture condition. HepG2 conditioned medium also enhanced the angiogenesis ability of EA.hy926 cells. In addition, the expressions of intracellular VEGF and extracellular VEGFR (Flk-1) were regulated upward in a time-dependent manner. In conclusion, the proliferation of vascular endothelial cells and Vascula angiogenesis were improved under the condition of indirect co-culture.