This study compares phenotypic changes of human umbilical endothelial vein cells cultured in three-dimensional collagen matrixes in the presence of basic fibroblast growth factor or on Matrigel ...coats. Under both conditions, endothelial cells rapidly assembled into an irregular network of tubular structures with a high frequency of intercellular or lumen-like spaces. Tubular structures were characterized and compared by phase-contrast, confocal and electron microscopy. The dominant mechanism of lumen-like formation was highly model-dependent. Ultrastructural analyses of capillary-like structures and the mechanism of lumen-like formation indicated that the in vivo angiogenesis was better reproduced in the collagen model.
To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycans in fibroblasts, we obtained stably transfected Swiss 3T3 clones. We examined the effects of stable ...syndecan-2 overexpression on programmed cell death, finding that syndecan-2 transfected cells were more sensitive to apoptosis induced by serum-withdrawal than control cells. In addition, overexpression of syndecan-2 correlates with increased membrane levels of the Fas/CD95 receptor, suggesting that the increased serum-withdrawal apoptosis observed in Swiss 3T3 cells might be Fas receptor-dependent. Differences in Fas membrane levels between both control and syndecan-2 transfected cells result from a redistribution of the Fas receptor. Our data clearly demonstrate that increased Fas levels are primarily related to lipid rafts and that this increase is a key factor in Fas/CD95-mediated apoptosis. Moreover, disruption of lipid rafts with methyl-beta-cyclodextrin or filipin significantly reduced apoptosis in response to serum withdrawal. The differences in Fas/CD95 membrane distribution could explain why syndecan-2 transfected cells have a higher susceptibility to serum-withdrawal-induced apoptosis.
Recent evidence has established different functions for the tumor suppressor protein, p16(INK4A) aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human ...umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, alphav-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the alphavbeta3-integrin-mediated migration.
Circulating endothelial progenitor cells (EPCs) promote vascular repair and maintain integrity of the endothelial monolayer. Reduced EPCs number has been associated with endothelial dysfunction in ...various cardiovascular diseases. Cardiovascular disease risk is higher in renal transplant patients (RT) than the general population. We studied EPCs number and proliferation in RT, and examined the association with other cardiovascular risk factors such as reduced glomerular filtration rate (GFR) and LDL cholesterol. EPCs concentration was determined in 94 RT and 39 control subjects (C) by flow cytometry. EPCs proliferation was also studied after 7 days in culture. EPCs concentration was significantly reduced in RT versus C (median 33.5 5–177 vs. 53 9–257 EPCs/105 PMN cells, p = 0.006). EPCs proliferation was also reduced in RT versus C (mean ± SD; 372.7 ± 229.3 vs. 539.8 ± 291.3 EPCs × field, p = 0.003). In multiple regression analysis, GFR, HDL, LDL and body weight were independent predictors of EPCs concentration in RT (r2= 0.25, p < 0.001). EPCs number is reduced in RT, particularly in patients with reduced GFR. Moreover, EPCs from RT studied in vitro, showed reduced proliferation, which is a sign of functional impairment. These alterations may be involved in increased cardiovascular risk of RT.
Lipoprotein lipase (LPL) is anchored at the vascular endothelium through interaction with heparan sulfate. It is not known how this enzyme is turned over but it has been suggested that it is slowly ...released into blood and then taken up and degraded in the liver. Heparin releases the enzyme into the circulating blood. Several lines of evidence indicate that this leads to accelerated flux of LPL to the liver and a temporary depletion of the enzyme in peripheral tissues.
Rat livers were found to contain substantial amounts of LPL, most of which was catalytically inactive. After injection of heparin, LPL mass in liver increased for at least an hour. LPL activity also increased, but not in proportion to mass, indicating that the lipase soon lost its activity after being bound/taken up in the liver. To further study the uptake, bovine LPL was labeled with 125I and injected. Already two min after injection about 33 % of the injected lipase was in the liver where it initially located along sinusoids. With time the immunostaining shifted to the hepatocytes, became granular and then faded, indicating internalization and degradation. When heparin was injected before the lipase, the initial immunostaining along sinusoids was weaker, whereas staining over Kupffer cells was enhanced. When the lipase was converted to inactive before injection, the fraction taken up in the liver increased and the lipase located mainly to the Kupffer cells.
This study shows that there are heparin-insensitive binding sites for LPL on both hepatocytes and Kupffer cells. The latter may be the same sites as those that mediate uptake of inactive LPL. The results support the hypothesis that turnover of endothelial LPL occurs in part by transport to and degradation in the liver, and that this transport is accelerated after injection of heparin.
Background/Aims: The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to ...analyze how different extracellular matrix molecules modulate fetal hepatocyte morphology, growth and differentiation.
Methods: We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-collagen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression.
Results: Cell attachment was increased by all the extracellular matrix proteins to a similar extent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immunocytochemical analysis of the cells in these structures revealed high levels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures appeared either in the absence or in the presence of the mitogen. Cells cultured on fibronectin, regardless of whether epidermal growth factor was present or not, also presented the maximal levels of expression for liver specific genes, such as albumin or alpha-fetoprotein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-1α/HNF-1β ratio, when compared with those cells that were cultured on collagen or E-C-L extracellular matrix.
Conclusions: These results suggest that fibronectin might play a differential role, as compared to other extracellular matrix proteins, in fetal hepatocyte organization and gene expression.
Beta-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. Beta-catenin signaling has been proposed to act as inducer of cell proliferation in different ...tumors. However, in some developmental contexts and cell systems beta-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of beta-catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous beta-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase-synchronized epithelial cells. Beta-catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of beta-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not beta-catenin, is detected in M phase. Interestingly, overexpression of a stable form of beta-catenin, or inhibition of endogenous beta-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for beta-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.
The low number of postnatal endothelial progenitor cells (EPC) in the circulation limits their therapeutic application in cardiovascular medicine. Processed lipoaspirate (PLA) cells differentiate ...into osteoid, adipose, muscle, and cartilaginous cells. This study examines the potential of PLA cells as a source of EPCs.
PLA cells obtained from human lipoaspirates were cultured for 1 week in serum-depleted medium to form three-dimensional cell clusters (3DCC). The phenotype of 3DCC-derived cells was assessed by immunofluorescence staining and FACS analysis.
Flow cytometry showed that 45+/-5% of cells derived from the 3DCC expressed Flk-1, a marker of early EPC, whilst only 4+/-0.5% of freshly isolated PLA were Flk-1+. The proportion of Flk-1+ cells increased to 98+/-2% during culture in hematopoietic stem cell medium. When cultured in an endothelial cell (EC)-specific medium, Flk-1+ cells also expressed Ve-cadherin, von Willebrand's factor (vW), and a lectin receptor, and took up low-density lipoprotein. Incorporation into an endothelial cell tubular network confirmed their functional activity.
This report describes the first isolation and culture of Flk-1+ cells from human adipose tissue. The feasibility of the extraction and culture of these cells in increased numbers suggests that such autologous cells will be useful for applications ranging from basic research to cell-based therapies.
Background/Aims: Loss of specific differentiation markers, adoption of a migrating morphology and progressive replacement of the cytokeratin network by vimentin intermediate filaments characterize ...the epithelial-mesenchymal transition of cultured neonatal rat hepatocytes. In a previous study (Hepatology 1997; 25: 598–606), we reported that this process can be differentially regulated by EGF and DMSO, two agents that affect hepatocyte growth and differentiation. The aim of the present study was to determine if growth activation or differential gene expression could explain the differences in EMT observed between these two factors.
Methods: We compared the effects of EGF, HGF, TGF-β
1 and DMSO on growth, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatocytes using thymidine incorporation, Northern blotting and Western blotting analysis.
Results: When TGF-β
1 or DMSO was added to the cultures supplemented with EGF and HGF, the mitogenic activity induced by these factors was inhibited. DMSO down-regulated c-
myc and c-
fos expression. mRNA levels of some liver-specific genes such as albumin, or liver-enriched transcription factors such as C/EBPδ, HNF-4 and HNF-1β were slightly different in cultures supplemented with DMSO or TGF-β
1. However, no differences were found when DMSO or TGF-β
1 was added to the cultures supplemented with EGF. Western blotting analysis showed that TGF-β
1 decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and vimentin. When DMSO or TGF-β
1 was added in combination with EGF or HGF, both factors maintained the increase in albumin and cytokeratin induced by the growth factors although DMSO, but not TGF-β
1, inhibited vimentin expression.
Conclusions: Activation of vimentin expression produced in cultures supplemented with the mitogenic factors (EGF and HGF) is independent of the activation of cell growth, because DMSO but not TGF-β
1 can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expression in these cultures seems to be independent of the mRNA levels of transcription factors associated with the differentiated liver phenotype.
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