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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 12, 2005 as doi:10.1096/fj.05-3834fje. |
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Department of Research, Cardiovascular Laboratories, Basel University Hospital, Basel, Switzerland; and
* Division of Cardiology, Luzern Kantonsspital, Luzern, Switzerland
1 Correspondence: Cardiovascular Laboratories, Basel University Hospital, Hebelstrasse 20, Basel CH 4031, Switzerland. E-mail: Therese-J.Resink{at}unibas.ch
SPECIFIC AIMS
In vascular tissue, atypical GPI-anchored T-cadherin (T-cad) is up-regulated during atherosclerosis, restenosis after balloon angioplasty, and tumor angiogenesis. These conditions are associated with oxidative stress and concomitant alterations in vascular cell migration, proliferation, and apoptosis/survival. Neither mechanism underlying the elevation of T-cad in vascular cells nor its possible participation in cell apoptosis/survival have yet been investigated. Using cultures of human umbilical vein endothelial cells (HUVEC), this study investigated whether oxidative stress up-regulates T-cadherin expression. The functional role of the increase in T-cadherin expression in endothelial cell survival and the underlying signaling pathways were analyzed.
PRINCIPAL FINDINGS
1. Serum deprivation of HUVEC increases T-cad expression via a redox-based mechanism.
We determined levels of T-cad expression in cultures of HUVEC subjected to conditions of serum-withdrawal, which in vitro induces oxidative stress through excess production of reactive oxygen species (ROS). Serum deprivation of HUVEC for up to 24 h resulted in a transient 
2-fold elevation of T-cad in adherent cells within 2–3 h (Fig. 1
A) that preceded activation of caspases (4–8 h) and cell detachment (6–24 h). The effect of serum deprivation on T-cad expression was sensitive to inhibition both by free radical-scavenging antioxidant N-acetylcysteine (NAC; 30 mM) and the flavin protein NADPH oxidase inhibitor diphenyleneiodonium (10 µM), but not by rotenone, the mitochondrial (complex I) NADH dehydrogenase inhibitor, antimycin A, the mitochondrial (complex III) cytochrome b-c1 inhibitor, or N-monomethyl-L-arginine, a nitric oxide synthase inhibitor (Fig. 1A, B
). Direct induction of oxidative stress in HUVEC by exposure to exogenous H2O2 (1 mM, 4 h) under normal serum-containing conditions also resulted in increased T-cad levels, and this response was inhibited by NAC (Fig. 1B
).
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2. T-cadherin overexpression protects HUVEC from serum withdrawal-induced apoptosis; involvement of PI3-kinase.
Since T-cad elevation in adherent HUVEC under conditions of serum-withdrawal precedes caspase activation, it is possible that T-cad serves some proapoptotic function. However, ROS can elicit both apoptosis and survival signals and our previous studies demonstrated that overexpression of T-cad in HUVEC increases their proliferative and migratory potential. Therefore we examined whether up-regulation of T-cad might rather participate in facilitating cell survival. Adenoviral mediated overexpression of T-cad in HUVEC (T-cad+) resulted in significantly lower caspase(s) activation (using fluorimetric caspase assay) following serum withdrawal as compared with control HUVEC (empty vector-infected HUVEC (E) and parental HUVEC (P). Moreover, levels of active caspase 3 were remarkably low in T-cad+-HUVEC (Fig. 2
A, B, after 6 h of serum deprivation). Overexpression of T-cad in HUVEC thus conferred resistance to serum deprivation-induced apoptosis. Overexpression of T-cad also rendered HUVEC more resistant to apoptosis induced by staurosporine (1 µM), TNF-
(40 ng/mL), or actinomycin-D (100 ng/mL). The apparent greater survival potential of T-cad+-HUVEC inferred by these findings was confirmed by cell enumeration assays whereby the numbers of cells still adherent following serum-withdrawal or exposure to staurosporine, TNF- or actinomycin-D were greater for T-cad+ HUVEC than P- and E-HUVECs. Inclusion of either the PI3-kinase inhibitor wortmannin or the mTOR inhibitor rapamycin abolished the blunted apoptosis response of T-cad+ HUVEC to serum deprivation; adherent cell numbers and caspase activities, as the respective cell survival and apoptosis indices, were comparable between T-cad+, P-, and E- HUVECs.
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3. Overexpression of T-cad in HUVEC leads to hyperactivation of Akt signaling and hypoactivation of p38MAPK signaling
The consequences of overexpression of T-cad on the phosphorylation status of different apoptosis and survival signaling regulators were examined. Compared with P- and E- HUVECs, T-cad+ HUVEC exhibited a hyperphosphorylation of the survival pathway regulators Akt and p70S6K and a hypophosphorylation of death pathway regulator p38 MAPK under normal and serum deprivation conditions (Fig. 2A, B
). The differential between P- or E-HUVECs and T-cad overexpressing HUVEC with respect to phosphorylation of Akt and p38 was abolished by PI3K inhibitor wortmannin (Fig. 2A
,) and the differential with respect to phosphorylation of p70S6K was abolished by mTOR inhibitor rapamycin (Fig. 2B
). Wortmannin (Fig. 2A
) or rapamycin (Fig. 2B
) also abrogated the apoptosis resistance of T-cad overexpressing HUVEC to serum deprivation, whereby levels of active caspase 3 were comparable between P-, E-, and T-cad+ HUVECs. Levels of total Akt, p38 MAPK, and p70 were not different between P-, E-, and T-cad+ HUVECs under any of the culture conditions. No differences in phosphorylation status and/or expression level of the survival/death regulators ERK1/2, JNK/SAPK1 and FAK p21, Bcl-2, Bcl-XL, or survivin were detected (Fig. 2C
).
CONCLUSIONS AND SIGNIFICANCE
Oxidative stress is an important determinant in the pathophysiology of atherosclerosis, restenosis, and angiogenesis; and the mechanisms by which oxidative stress mediates alterations in vascular cell function are complex. The outcome of oxidative injury varies with cell type, oxidative agent, and also with the extent and duration of injury. ROS are capable of both inducing apoptosis and generating survival signals which can elicit a wide range of responses encompassing proliferation, growth arrest, cell survival and cell death. This study has revealed a novel functional relationship between oxidative stress, T-cad expression, and cell survival status in vascular cells. We conclude that 1) T-cad is up-regulated in endothelial cells in response to oxidative stress (e.g., serum deprivation) via flavin-containing oxidases; and 2) that overexpression of T-cad in HUVEC protects against stress-induced apoptosis through concomitant activation of the PI3K/Akt/mTOR survival signal pathways and suppression of the p38 MAPK pro-apoptotic pathway (Fig. 3
).
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T-cad is one of the lesser investigated members of the cadherin superfamily. Originally it was described about 10 years ago in the embryonic nervous system where it acted as a guidance repulsive cue for extending motor axons. The majority of studies since then have concentrated mostly on the relationship between T-cad expression and proliferation of tumor cells. We have focused on elucidating the functions of T-cad in the cardiovascular system where it is widely expressed. Immunohistochemical analyses of vascular tissue have shown that the progression of atherosclerosis, restenosis, and tumor neovascularization is attended with marked increases in T-cad levels on the surface of EC and SMC, suggesting a positive correlation between T-cad expression on vascular cells and their motility and growth. Subsequent in vitro studies provided evidence for the ability of T-cad to stimulate migration and proliferation. We demonstrated that T-cad can stimulate vascular cell motility by inducing cell de-adhesion, polarization, and acquisition of a promigratory phenotype; this effect of T-cad is dependent upon homophilic ligation and is mediated through activation of RhoA/ROCK and Rac signal pathways. T-cad up-regulation was demonstrated to play a facilitory role in cell cycle progression and proliferation of vascular cells; this effect is independent of homophilic ligation mechanisms. The present novel demonstrations of a ROS-based mechanism for up-regulation of T-cad in EC and antiapoptotic signaling and functional consequences of T-cad overexpression provide evidence that T-cad may contribute to regulation of tissue cellularity by promoting cell survival under stress conditions.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3834fje;
This article has been cited by other articles:
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  M. B. Joshi, D. Ivanov, M. Philippova, P. Erne, and T. J. Resink Integrin-linked kinase is an essential mediator for T-cadherin-dependent signaling via Akt and GSK3{beta} in endothelial cells FASEB J, October 1, 2007; 21(12): 3083 - 3095. [Abstract] [Full Text] [PDF]
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 M. Philippova, A. Banfi, D. Ivanov, R. Gianni-Barrera, R. Allenspach, P. Erne, and T. Resink Atypical GPI-Anchored T-Cadherin Stimulates Angiogenesis In Vitro and In Vivo Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2222 - 2230. [Abstract] [Full Text] [PDF]
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: numbers below the blots (mean values) indicate band intensities relative to that (arbitrarily taken as 1) in parental HUVEC under normal serum-containing culture conditions. C) Quantitative data not shown since there were no differences between P, E, and T. Asterisks indicate where expression level in T differed significantly (P at least <0.01) from P and E. There were no significant differences between P and E under any experimental conditions.