Endothelial Cells ; metabolism ; pathology ; Humans ; Sepsis ; metabolism ; physiopathology

Objective To study the effect and mechanism of pearl hydrolysate on hepatic sinusoidal capillarization in liver fibrosis. Methods Hepatic sinusoidal endothelial cells (HSEC) and hepatic stellate cells (HSC-LX2) were incubated with Hepu pearl hydrolysate.The proliferation of HSEC and HSC-LX2 was examined by MTT colorimetry.The cell cycle and apoptosis of HSC-LX2 were measured by flow cytometry.The changes of the microstructures such as fenestra and basement membrane of HSEC were observed by transmission electron microscopy. Results The intervention with leptin increased the viability of HSC-LX2 (P=0.041),decreased the viability of HSEC (P=0.004),and caused capillarization signs such as decreased number and diameter of fenestrae and formation of continuous basement membrane.The treatment with pearl hydrolysate at different doses increased and expanded the fenestrae of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),disintegrated the extracellular basement membrane of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),decreased the viability of HSC-LX2 (low dose:P=0.018;medium dose:P=0.013;high dose:P=0.009),and induced the apoptosis of HSC-LX2 (low dose:P=0.012;medium dose:P=0.006;high dose:P=0.005).Pearl hydrolysate exerted therapeutic effect on capillarization in a dose-dependent manner (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032).Moreover,high-dose pearl hydrolysate showed stronger effect on capillarization of hepatic sinuses than colchicine (P=0.034) and salvianolic acid B (P=0.038). Conclusion Hepu pearl hydrolysate can increase the viability of HSEC,restore the area of fenestrae,disintegrate the basement membrane,and decrease the viability and induce the apoptosis of HSC-LX2,demonstrating significant pharmacological effects on the capillarization of HSEC and HSC-LX2.
Humans ; Endothelial Cells/metabolism* ; Liver Cirrhosis ; Liver/pathology*

Microparticles are submicron vesicles shed from plasma membranes in response to cell activation, injury and/or apoptosis. Microparticles of various cellular origins, such as platelets, leukocytes, and endothelial cells, are found in the plasma of healthy subjects, and their amount increases under pathological conditions. Recent studies show that endothelial microparticles, a kind of envelope particles derived from endothelial cells, not only constitute a marker of endothelial dysfunction, but also play a major biological role in the diagnostic and therapeutic approaches to erectile dysfunction.
Biomarkers ; Cell Membrane ; Endothelial Cells ; metabolism ; pathology ; Erectile Dysfunction ; metabolism ; pathology ; Humans ; Liposomes ; metabolism ; Male

OBJECTIVETo evaluate the effect of tissue factor (TF) in extravascular migration of fibrosarcoma cells and hematogenous metastasis.

METHODSThe expression of tissue factor in fibrosarcoma HT1080 cells was analyzed by flow cytometry. The extravascular migration of fibrosarcoma cells was observed in a constructed monolayer vascular endothelial cells and extra-cellular matrix model.

RESULTTissue factor was highly expressed in HT1080 cells. HT1080 migrated and passed through the monolayer vascular endothelial cells to the collagen gel in a time-dependent manner. Anti-TF antibody inhibited extravascular migration of fibrosarcoma cells and the inhibition was concentration-dependent (P<0.05).

CONCLUSIONTissue factor may enhance hematogenous metastasis through extravascular migration of fibrosarcoma cells.

Cell Movement ; Cells, Cultured ; Endothelial Cells ; Fibrosarcoma ; metabolism ; pathology ; Humans ; Neoplasm Metastasis ; Thromboplastin ; metabolism ; physiology

Hypertrophic scar is a pathological repair result of excessive accumulation of extracellular matrix after skin damage, which affects the appearance and function of patients with varying degrees. The degree of scar formation is directly related to the strength of inflammatory reaction during wound healing, and excessive or prolonged inflammatory response increases the incidence of hypertrophic scars. Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in regulating the fibrotic network composed of fibroblasts, macrophages, keratinocytes, and vascular endothelial cells, and is closely related to the formation of hypertrophic scars. This article reviews the role of IL-6 and its signaling pathway in hypertrophic scar formation.
Cicatrix, Hypertrophic/pathology* ; Endothelial Cells/metabolism* ; Fibroblasts/metabolism* ; Humans ; Interleukin-6 ; Skin/pathology* ; Wound Healing/physiology*

Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.
Animals ; Mice ; Endothelial Cells/metabolism* ; Lymphatic Vessels ; Osteoclasts/pathology* ; Osteolysis, Essential/pathology* ; Semaphorin-3A/metabolism*

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on pulmonary vascular endothelial cells (PMVECs) injury in septic mice and its mechanism. Methods: The experimental research method was adopted. The primary ADSCs were isolated and cultured from the discarded fresh adipose tissue of 3 patients (female, 10-25 years old), who were admitted to the First Affiliated Hospital of Air Force Medical University undergoing abdominal surgery, and the cell morphology was observed by inverted phase contrast microscope on the 5^th day. The expressions of CD29, CD34, CD44, CD45, CD73, and CD90 of ADSCs in the third passage were detected by flow cytometry. The third to the fifth passage of ADSCs were collected, and their exosomes from the cell supernatant were obtained by differential ultracentrifugation, and the shape, particle size, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin of exosomes were detected, respectively, by transmission electron microscopy, nano-particle tracking analysis and Western blotting. Twenty-four adult male BALB/c mice were adopted and were divided into normal control group, caecal ligation perforation (CLP) alone group, and CLP+ADSC-exosome group with each group of 8 according to random number table (the same grouping method below) and were treated accordingly. At 24 h after operation, tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) levels of mice serum were detected by enzyme-linked immunosorbent assay, and lung tissue morphology of mice was detected by hematoxylin-eosin and myeloperoxidase staining, and the expression of 8-hydroxy-deoxyguanosine (8-OHdG) of mouse lung cells was detected by immunofluorescence method. Primary PMVECs were obtained from 1-month-old C57 mice regardless gender by tissue block method. The expression of CD31 of PMVECs was detected by immunofluorescence and flow cytometry. The third passage of PMVECs was co-cultured with ADSCs derived exosomes for 12 h, and the phagocytosis of exosomes by PMVECs was detected by PKH26 kit. The third passage of PMVECs were adopted and were divided into blank control group, macrophage supernatant alone group, and macrophage supernatant+ADSC-exosome group, with 3 wells in each group, which were treated accordingly. After 24 h, the content of reactive oxygen species in cells was detected by flow cytometry, the expression of 8-OHdG in cells was detected by immunofluorescence, and Transwell assay was used to determine the permeability of cell monolayer. The number of samples in above were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: The primary ADSCs were isolated and cultured to day 5, growing densely in a spindle shape with a typical swirl-like. The percentages of CD29, CD44, CD73 and CD90 positive cells of ADSCs in the third passage were all >90%, and the percentages of CD34 and CD45 positive cells were <5%. Exosomes derived from ADSCs of the third to fifth passages showed a typical double-cavity disc-like structure with an average particle size of 103 nm, and the protein expressions of CD9, CD63 and TSG101 of exosomes were positive, while the protein expression of β-actin of exosomes was negative. At 24 h after operation, compared with those in normal control group, both the levels of TNF-α and IL-1β of mice serum in CLP alone group were significantly increased (with t values of 28.76 and 29.69, respectively, P<0.01); compared with those in CLP alone group, both the content of TNF-α and IL-1β of mice serum in CLP+ADSC-exosome group was significantly decreased (with t values of 9.90 and 4.76, respectively, P<0.05 or P<0.01). At 24 h after surgery, the pulmonary tissue structure of mice in normal control group was clear and complete without inflammatory cell infiltration; compared with those in normal control group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP alone group were more obvious; compared with those in CLP alone group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP+ADSC-exosome group were significantly reduced. At 24 h after operation, endothelial cells in lung tissues of mice in 3 groups showed positive expression of CD31; compared with that in normal control group, the fluorescence intensity of 8-OHdG positive cells of the lung tissues of mice in CLP alone group was significantly increased, and compared with that in CLP alone group, the fluorescence intensity of 8-OHdG positive cells in the lung tissues of mice in CLP+ADSC-exosome group was significantly decreased. The PMVECs in the 3^rd passage showed CD31 positive expression by immunofluorescence, and the result of flow cytometry showed that CD31 positive cells accounted for 99.5%. At 12 h after co-culture, ADSC-derived exosomes were successfully phagocytose by PMVECs and entered its cytoplasm. At 12 h after culture of the third passage of PMVECs, compared with that in blank control group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant alone group was significantly increased (t=15.73, P<0.01); compared with that in macrophage supernatant alone group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant+ADSC-exosome group was significantly decreased (t=4.72, P<0.01). At 12 h after culture of the third passage of PMVECs, and the 8-OHdG positive fluorescence intensity of PMVECs in macrophage supernatant alone group was significantly increased; and compared with that in blank control group, the 8-OHdG positive fluorescence intensity of PMVECs in macrophage+ADSC-exosome supernatant group was between blank control group and macrophage supernatant alone group. At 12 h after culture of the third passage PMVECs, compared with that in blank control group, the permeability of PMVECs monolayer in macrophage supernatant alone group was significantly increased (t=6.34, P<0.01); compared with that in macrophage supernatant alone group, the permeability of PMVECs monolayer cells in macrophage supernatant+ADSC-exosome group was significantly decreased (t=2.93, P<0.05). Conclusions: Exosomes derived from ADSCs can ameliorate oxidative damage in mouse lung tissue, decrease the level of reactive oxygen species, 8-OHdG expression, and permeability of PMVECs induced by macrophage supernatant.
Animals ; Endothelial Cells/metabolism* ; Exosomes/metabolism* ; Female ; Humans ; Lung Injury/metabolism* ; Male ; Mesenchymal Stem Cells/metabolism* ; Mice ; Sepsis/pathology*

OBJECTIVETo investigate the influence of hypoxia on the proliferation and activity of human umbilical vein vascular endothelial cells (EA. hy926).

METHODSEA. hy926 cells were cultured in vitro and divided into normal control and hypoxia groups. The cells in hypoxia group were placed into hypoxic jar and treated with mixed gases(94% N2 +5% CO2 + 1% O2) for 1,3,6 and 12 hours. Then the total proteins were extracted for the determination of the expression of vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA). The cell cycle and growth curve were determined with flow cytometry and MTT method, respectively.

RESULTSThe expression of PCNA protein began to increase at 3 post-hypoxia hour (PHH), peaked at 6 PHH, but without obvious difference compared with that at 12 PHH. The expression of VEGF began to increase at 1 PHH, peaked at 6 PHH, and decreased at 12 PHH, though it was still markedly higher than that of normoxia at 12 PHH. MTT results showed that the cell activity began to increase at 1 PHH, and it was still to increased at 3 PHH, then decreased at 6 PHH, and it was lower than that in control group at 12 PHH. The number of cells in G0/G1 phase was decreased, but the cells in S and G2/M phase was increased at 1, 3, 6 PHH when compared with those in normal controls. The proliferation index (PI) of cells in hypoxia group at 1PHH (43 +/- 9)%, 3PHH (39 +/- 11)%, 6 PHH (40 +/- 11))% were higher than that before hypoxia (32 +/- 9)% and 3 (39 +/- 11) % and 6 hours (40 +/- 11)% after hypoxia (P < 0.05). The PI was obviously lower at 12 PHH (27 +/- 4))% compared with that of cells under normoxic condition (P < 0.05).

CONCLUSIONShort-term hypoxia is beneficial to promote the proliferation of the cells, but this effect will be inhibited with the prolongation of hypoxia.

Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Hypoxia ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; metabolism

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.
Ascitic Fluid/*metabolism ; Cells, Cultured ; Endometriosis/*metabolism ; Macrophages, Peritoneal/pathology ; Vascular Endothelial Growth Factor A/*biosynthesis

To examine the changes in number and function of endothelial progenitor cells (EPCs) from peripheral blood (PB) in hypertension disorder complicating pregnancy (HDCP), 20 women with HDCP and 20 normal pregnant women at the third trimester were studied. Mononuclear cells (MNCs) from PB were isolated by Ficoll density gradient centrifugation. EPCs were identified by positive expression of both CD34 and CD133 under fluorescence microscope and positive expression of factor VIII as shown by immunocytochemistry. The number of EPCs was flow-cytometrically determined. Proliferation and migration of EPCs were measured by MTT assay and modified Boyden chamber assay, respectively. The adhesion activity of EPCs was detected by counting the number of the adherent cells. The results showed that, compared with normal pregnant women, the number of EPCs was significantly reduced in HDCP (4.29%+/-1.21% vs 15.32%+/-2.00%, P<0.01), the functional activity of EPCs in HDCP, such as proliferation (13.45%+/-1.68% vs 18.45%+/-1.67%), migration (37.25+/-7.28 cells/field vs 67.10+/-9.55 cells/field) and adhesion activity (20.65+/-5.19 cells/field vs 34.40+/-6.72 cells/filed) was impaired (P<0.01). It is concluded that the number and function of EPCs are significantly decreased in HDCP.
Antigens, CD/metabolism ; Antigens, CD34/metabolism ; Case-Control Studies ; Cell Adhesion ; Cell Count ; Cell Movement ; Endothelial Cells/pathology ; Endothelial Cells/*physiology ; Glycoproteins/metabolism ; Hypertension, Pregnancy-Induced/*pathology ; Peptides/metabolism ; Stem Cells/pathology ; Stem Cells/*physiology