Oxidative stress upregulates Wnt signaling in human retinal microvascular endothelial cells through activation of disheveled

Abnormal retinal neovascularization associated with various retinopathies can result in irreversible vision loss. Although the mechanisms involved in this occurrence is unclear, increasing evidence suggests that aberrant Wnt signaling participates in the pathogenesis of abnormal neovascularization. Because Wnt signaling upregulation can be induced by oxidative stress through the activation of disheveled (DVL), a key molecule in the Wnt signaling pathway, we investigated whether oxidative stress can activate Wnt signaling and induce angiogenic phenotypes in human retinal microvascular endothelial cells (HRMECs). We found that increased Wnt signaling activity, as well as enhanced angiogenic phenotypes, such as tube formation and cell migration, were detected in the hydrogen peroxide-treated HRMECs. Moreover, these effects were effectively suppressed by a small-molecule Wnt inhibitor targeting the PDZ domain of DVL. Therefore, we propose that targeting abnormal Wnt signaling at the DVL level with a small-molecule inhibitor may represent a novel approach in retinal neovascularization treatment and prevention.     

Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic differentiation

Wnt signaling is involved in developmental processes and in adult stem cell homeostasis. This study analyzes the role(s) of key Wnt signaling mediators in the maintenance and osteogenesis of mesenchymal stem cells (MSCs). We focus specifically on the involvement of low-density lipoprotein-related protein 5 (LRP5), T-cell factor 1 (TCF1), and Frizzled (Fz) receptors, in the presence or absence of exogenous, prototypical canonical (Wnt3a), and non-canonical (Wnt5a) Wnts. In undifferentiated MSCs, LRP5 and TCF1 mediate canonical Wnt signal transduction, leading to increased proliferation, enhanced synergistically by Wnt3a. However, LRP5 overexpression inhibits osteogenic differentiation, further suppressed by Wnt3a. Wnt5a does not affect cell proliferation but enhances osteogenesis of MSCs. Interestingly, Wnt5a inhibits Wnt3a effects on MSCs, while Wnt3a suppresses Wnt5a-mediated enhancement of osteogenesis. Flow cytometry revealed that LRP5 expression elicits differential changes in Fz receptor profiles in undifferentiated versus osteogenic MSCs. Taken together, these results suggest that Wnt signaling crosstalk and functional antagonism with the LRP5 co-receptor are key signaling regulators of MSC maintenance and differentiation.     

A simplified in vitro model of oxidant injury using vascular endothelial cells

Summary Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant injury induced by hydrogen peroxide (H₂O₂) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase (LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H₂O₂ with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production. MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric assay for measuring MDA production in endothelial cells used 6-well plates instead of 80-cm² flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular level.     

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.     

Culturing of human vascular endothelial cells strongly affects their endothelin-1 and prostacyclin production

Cultured human umbilical vein endothelial cells (HUVECs) are a widely used model to study the regulation of endothelial production of vasoactive substances such as endothelin-1 (ET-1) and prostacyclin (PGI₂) in human. As even short term culturing is known to affect the function of many cell types, we studied whether there are differences in the production of ET-1 and PGI₂ between freshly isolated HUVECs and HUVECs cultured for two passages, and whether variation in cell density affects the production of ET-1 and PGI₂ by these cells. At confluency, freshly isolated HUVECs produced only from one-tenth to one-fifth of ET-1, but 46-86 times more PGI2 (p < 0.001), when compared to respective productions by similar amounts of cultured HUVECs. When the cell density of freshly isolated HUVECs was lowered either by diluting the cell suspension or by plating the same amount of cells on different size wells, the production of ET-1 increased: lowering cell density to one-tenth led to 18 fold increase in ET-1 production (p < 0.001). PGI₂ production was not affected by cell density. Thus our data imply that the production of both ET-1 and PGI₂ are differently regulated in freshly isolated and cultured HUVECs, and that cell density is an important determinant in the regulation of ET-1 production.     

Hear the Wnt Ror: how melanoma cells adjust to changes in Wnt

The interplay between canonical and non‐canonical Wnt pathways in development and tumorigenesis is tightly regulated. In this review we will describe the yin and the yang of canonical and non‐canonical Wnt signaling pathways during melanocyte development, and melanoma genesis. Canonical Wnt signaling, represented by Wnts such as Wnt1 and Wnt3A, signals via β‐catenin to promote melanocyte differentiation and tumor development. Non‐canonical Wnt signaling, specifically Wnt5A, regulates canonical pathways, and signals to induce melanoma metastasis. This review will focus on the role of Wnt5A during melanoma progression, and its relationship to canonical Wnt signaling.     

Transport of L-cystine in human umbilical vein endothelial cells in culture

The uptake of L-cystine into cultured human umbilical vein endothelial cells has been shown to occur by a Na⁺-independent system which is inhibited by L-glutamate and L-homocysteine, but not by other amino acids. It is likely that the system transporting L-cystine is shared by L-glutamate. Thiol groups associated with membrane bound components appear to be essential for L-cystine uptake but it is not yet evident whether these constitute an integral part of the transporterper se.     

Glyoxal causes inflammatory injury in human vascular endothelial cells

To explore mechanisms of diabetes-associated vascular endothelial cells (ECs) injury, human umbilical vein ECs were treated for 24 h with high glucose (HG; 26 mM), advanced glycation end-products (AGEs; 100 μg/ml) or their intermediate, glyoxal (GO: 50-5000 μM). HG and AGEs had no effects on ECs morphology and inflammatory states as measured by vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase (COX)-2 expressions. GO (500 μM, 24 h) induced cytotoxic morphological changes and protein expression of COX-2 but not VCAM-1. GO (500 μM, 24 h) activated ERK but not JNK, p38 or NF-κB. However, ERK inhibitor PD98059 was ineffective to GO-induced COX-2. While EUK134, synthetic combined superoxide dismutase/catalase mimetic, had no effect on GO-mediated inflammation, sodium nitroprusside inhibited it. The present results indicate that glyoxal, a metabolite of glucose might be a more powerful inducer for vascular ECs inflammatory injury. Nitric oxide but not anti-oxidant is preventive against GO-mediated inflammatory injury.     

5-Hydroxytryptamine as a potent migration enhancer of human aortic endothelial cells

The purpose of the present study was to investigate whether 5-hydroxytryptamine (5-HT, serotonin) affects migration of vascular endothelial cells. 5-HT significantly enhanced migration of human aortic endothelial cells (HAECs), and this enhancement was completely inhibited by GR 55562, a 5-HT1 receptor antagonist, and fluoxetine, a 5-HT transporter inhibitor, but was not affected by ketanserin, a 5-HT2 receptor antagonist. 5-HT stimulation increased RhoA and ERK activity of HAECs, and inhibitors of RhoA (Y-27632 and H-1152) and inhibitors of MEK (U0126 and PD98059) abolished the 5-HT-induced increase in migration velocity. Inhibition of Rho kinase by Y-27632 blocked stress fiber formation and rear release of HAECs. Thus, 5-HT has a potent enhancing action on migration of HAECs through activating the RhoA and ERK pathways following 5-HT1 receptor stimulation.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

The p38 mitogen-activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor-dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2-induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2-induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2-induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2-induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2-driven angiogenesis.     

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.     

Sfrp1a and Sfrp5 function as positive regulators of Wnt and BMP signaling during early retinal development

Axial patterning of the developing eye is critically important for proper axonal pathfinding as well as for key morphogenetic events, such as closure of the optic fissure. The dorsal retina is initially specified by the actions of Bone Morphogenetic Protein (BMP) signaling, with such identity subsequently maintained by the Wnt-β catenin pathway. Using zebrafish as a model system, we demonstrate that Secreted frizzled-related protein 1a (Sfrp1a) and Sfrp5 work cooperatively to pattern the retina along the dorso-ventral axis. Sfrp1a/5 depleted embryos display a reduction in dorsal marker gene expression that is consistent with defects in BMP- and Wnt-dependent dorsal retina identity. In accord with this finding, we observe a marked reduction in transgenic reporters of BMP and Wnt signaling within the dorsal retina of Sfrp1a/5 depleted embryos. In contrast to studies in which canonical Wnt signaling is blocked, we note an increase in BMP ligand expression in Sfrp1a/5 depleted embryos, a phenotype similar to that seen in embryos with inhibited BMP signaling. Overexpression of a low dose of sfrp5 mRNA causes an increase in dorsal retina marker gene expression. We propose a model in which Sfrp proteins function as facilitators of both BMP and Wnt signaling within the dorsal retina.