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VASCULAR BIOLOGY
1Laboratorio de Nefrología-Hipertensión, Fundación Jiménez Díaz, Madrid; and 2Departamento de Bioquímica, Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Madrid, Spain
Submitted 27 October 2005 ; accepted in final form 6 October 2006
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ABSTRACT |
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-catenin. In functional terms, this translocation is transcriptionally active, and its protective effect is further supported by the stimulation of EC cytoprotection by transfectionally induced excess of -catenin. 2) Increase of platelet endothelial cell adhesion molecule (PECAM)/CD31 levels. 3) Increase in total and phosphorylated AKT. 4) Increase in phosphorylated glycogen synthase kinase (GSK)3
/. The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in the monolayer but also circulating detached cells and involve mechanistic interactions not previously described. JAK2/STAT; cytoprotection; anoikis; AG490
Numerous studies have dealt with the subject of endothelial protection, basically employing growth factors and stimulating cytoprotective pathways, e.g., phosphatidylinositol 3-kinase (PI3K)/AKT, stress proteins, or MAP kinases (19). The Janus kinase/signal transducers and activators of the transcription (JAK/STAT) pathway transmits information received from extracellular peptide signals through transmembrane receptors directly to target gene promoters in the nucleus, therefore providing a mechanism for transcriptional regulation without identified second messengers. While signaling through the canonical JAK/STAT pathway has been extensively characterized in stimulated immune cells, emerging evidence indicates that STATs also mediate responses to forms of cellular stress as diverse as ischemia/reperfusion, endotoxin, ultraviolet light, or hyperosmolarity (9). Notwithstanding, data are less than homogeneous as to how JAK inhibition affects cell damage in different models of ischemic injury (10, 15, 20). At present, most of the information available on this subject comes from the field of cardiovascular medicine. In this regard, JAK/STAT signaling appears to have an important role in the development of the cardioprotected phenotype associated with ischemic preconditioning (15). On the other hand, double-edged effects have been reported in models of myocardial infarction, i.e., while JAK/STAT activation reduces apoptosis in the myocardial infarction border zone, JAK2/STAT inhibition by AG490 improves the increase of protein phosphatase-1 activity and decreases the levels of p16-phospholamban, which underlie myocardial dysfunction (10). Moreover, the inhibition of the JAK/STAT pathway may result in a significant reduction in the size of devitalized tissue and in the number of cardiomyocytes undergoing apoptosis (20). Furthermore, JAK2/STAT is involved in the anti-apoptotic effect of granulocyte colony-stimulating factor in severely ischemic myocardium and endothelium (13). Collectively, these data indicate that cellular stress activates STATs in ways that can be either detrimental or supportive of cell survival. Recently, a substantial, specific piece of information has been added, by demonstrating that JAK2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells (27) and astrocytes (11). As a consequence, JAK2 inhibition induces cytoprotection against oxidative aggression. In the same line of evidence, oxidized low-density lipoproteins have been recently shown to induce, by means of lipid peroxidation products, tyrosine phosphorylation and activation of JAK2, STAT1, and STAT3, therefore providing support to consider JAK2 as an oxidative stress-activated kinase (21). The actual mechanisms involved in these effects have not been sufficiently characterized; furthermore, data on models with possible applications to vascular pathology need to be generated.
To the best of our knowledge, no information has been published regarding the putative role of the JAK2/STAT pathway in cytoprotective or cytotoxic mechanisms on EC. In this sense, two particularly important types of damage are growth factor deprivation and cell detachment-related death. The latter is mainly due to detachment-dependent apoptosis, a phenomenon that has been designated as anoikis. Anoikis is crucial in determining the death of adult EC losing adherence to the monolayer (12), and its inhibition can be attained in only a few conditions. In this regard, previous literature (18) and preliminary results from our laboratory (Castilla MA, unpublished observations) have shown that inhibition of the glycogen synthase kinase (GSK)3 pathway increases the survival of EC against detachment-related cell death. In terms of mechanism, inhibition of GSK3 with LiCl increases -catenin and favors its nuclear translocation. In these circumstances, -catenin acts as a survival factor against detachment-related cell death (26, 29). Also, it is known that GSK3 is a regulator of EC proliferation and anoikis (18).
As a part of the interest of our laboratory on the study of endothelial protective mechanisms (4–6), the hypothesis was raised that JAK2/STAT inhibition may have relevant protective effects on cells permanently exposed to stress, of which EC are a paradigm. Preliminary evidence from our laboratory has shown that JAK2/STAT inhibition strongly protects EC against oxidative damage (24). However, no data are available to date on the effects of JAK2/STAT inhibition on other types of injury involving EC. The advent of highly specific JAK2/STAT inhibitors provides a useful tool for examining this pathway (1, 11, 30).
The present study was aimed to analyze the hypothesis that specific JAK2/STAT inhibition induces significant changes in EC viability and to identify the mechanisms involved. Specific attention was dedicated to the study of protection of EC against detachment-related death, i.e., anoikis. A set of mutually related mechanisms with known endothelial protective properties, namely, -catenin levels and active -catenin translocation, platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, AKT phosphorylation, and potential apoptotic activators and inhibitors were examined.
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MATERIALS AND METHODS |
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Bovine aorta EC (BAEC) and human umbilical vein EC (HUVEC) were obtained, characterized, and cultured as described (3, 4), in accordance with the Guiding Principles for Research Involving Animals and Human Beings of the American Physiological Society (6). BAEC were used as the primary experimental source, and HUVEC were employed for complementary confirmatory experiments.
EC Challenge
The use of confluent, monolayer, or detached conditions was a critical point of this investigation. Confluent EC were submitted to either serum deprivation or cell detachment and suspension culture. In all cases, vehicle alone (DMSO <0.1%), AG490 (Calbiochem, Barcelona, Spain), a tyrosine kinase-blocking inhibitor of JAK2 (11, 22, 27) or its control, and the structurally related non-JAK/STAT inhibitor AG82 (Calbiochem, Barcelona, Spain) were added 45 min before the experimental maneuvers were performed. The Src family protein kinase inhibitor PP2 (10 µmol/l, Calbiochem) was also used. Cell viability was assessed by lactate dehydrogenase release (LDH), as described (6). DNA fragmentation (DNA ladder) of EC in agarose gels was used for further characterization of the apoptotic changes, as described (16). Conventional hematoxylin and eosin staining was used to obtain cell images under the different treatments.
Detached Cell Survival and Reseeding Assays
For triggering detachment-related cell death, after pretreatment, BAEC were removed from the culture plates with trypsin-EDTA and resuspended in 1% FBS medium. This small amount of FBS was necessary to avoid additional damage due to serum starvation. Resuspended cells were seeded onto tissue culture plates coated with poly-2-hydroxyethyl methacrylate (Poly-HEMA) (Sigma-Aldrich, Madrid, Spain). An additional system was used as a comparative control in selected experiments, i.e., BAEC were seeded into a spinner flask (Integra Biosciences, Chur, Switzerland) coated with Sigmacote (Sigma-Aldrich). At the times indicated, BAEC were harvested and processed by flow cytometry, as described (6). Both treatments prevented cell adhesion and hence induced anoikis.
For reseeding assay, BAEC cultured in suspension were allowed to grow for 24 h onto Matrigel-coated plates (Matrigel; BD Transduction Laboratories, Madrid, Spain) as described (6). BAEC were fixed, stained with crystal violet, and microphotographed. Cells were counted on 10 randomly chosen high-power fields by two independent observers blinded for the type of specific experiment.
Studies on -Catenin and PECAM-1
-Catenin and PECAM-1 (also known as CD31) levels were assessed by Western blot on total and/or nuclear proteins, using specific antibodies (-catenin, BD Transduction Laboratories; PECAM-1, R&D Systems, Minneapolis, MN). -Tubulin was used as loading control as described (6).
Further assessment of -catenin and PECAM-1 was performed by confocal immunofluorescence. For this purpose, cells grown on glass coverslips were fixed with Merckofix (Merck, Barcelona, Spain), permeabilized (0.2% Triton X-100, 15 min), and incubated in PBS with 1% bovine serum albumin. Slides were incubated overnight with the primary antibodies, washed (PBS, 5 min, 3x), and incubated with a specific secondary antibody coupled to FITC (Sigma-Aldrich). For visualization of nuclei, cells were incubated for 45 min with propidium iodide. Confocal images were obtained with a Confocal System TCS SP20 (Leica, Madrid, Spain). Image quantitation was done by means of the program Image J (National Institutes of Health, Bethesda, MD). Ten randomly selected high-power fields were used for each quantitation.
A -catenin/TCF activity reporter assay was performed to assess -catenin functionality. For transfections, cells grown to 60–80% confluence were transfected with 20 ng of pTK-Renilla (Promega, Madrid, Spain) and 200 ng of either pTOPFLASH (Top) or pFOPFLASH (Fop) containing multimerized wild-type and mutated Lef1/Tcf binding sites, respectively, fused to a luciferase reporter gene (a gift of H. Clevers; University Hospital, Utrecht, The Netherlands). Transfections (4-set duplicates, 6 h) were performed with lipofectamine (Gibco, Madrid, Spain). BAEC were treated in the presence/absence of AG490 for 24 h, and afterward, luciferase and Renilla activities were measured using the Dual Luciferase Reporter Kit (Promega). When needed, 400 ng of activator plasmid encoding a metabolically stabilized -catenin S33Y (a gift of A. Ben Ze’ev; Weizmann Institute, Rehovot, Israel) were added as indicated. DNA quantities were normalized with empty pCI-neovector. To analyze whether the increased -catenin was active, additional experiments were performed employing a specific antibody against the active form of -catenin, dephosphorylated on Ser47 or Thr41 (Upstate, Lake Placid, NY).
Transient Transfections
Different plasmids were transfected in BAEC using a CaCl2-based method. Briefly, BAEC were grown near confluence (80–90%). The optimal transfection was achieved at a CaCl2-to-DNA ratio of 6 mmol/l to 10 µg/ml, respectively; the CaCl2-DNA complex was performed in HBS buffer [21 mmol/l HEPES, 0.14 mol/l NaCl, 5.5 mmol/l D(+)-glucose, 50 mmol/l KCl, 1.32 mol/l Na2HPO4 at pH 7.1]. The transfection mix remained for an additional 6 h; then, BAEC were washed with PBS (x2), and growth medium was added until confluence was reached (48 h). Cell challenge was performed as described above. To evaluate transfection efficiency, all assays were also transfected using pEGFP-N1 vector (BD). Transfection efficiency was >50% in all cases.
A wildtype form of JAK2 (pBOS-HAJAK2) and a kinase domain mutant of JAK2 (pBOS-HAdkJAK2) (a gift from Dr. Dwayne L. Barber, Ontario Cancer Institute, Toronto, Canada) were used. For -catenin transfection, a wildtype form of -catenin in a pCINeo vector was used (a gift of A. Ben Ze’ev; Weizmann Institute, Rehovot, Israel). In all cases, an empty vector was employed as control.
Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay (EMSA) was employed to further ascertain the specificity of the effects of AG490. EMSA was performed using nuclear proteins (8 µg) and different consensus oligonucleotides (dsDNA, 0.5 ng) labeled with [
-32P]dATP in binding buffer 1x. Poly(dI-dC) was included as a competitive DNA. After incubation, samples were separated by acrylamide-bisacrylamide gels. Dried gels were exposed to X-Omat films. Commercial oligonucleotides of STAT3 and TFII were used (Santa Cruz Biotechnology, Santa Cruz, CA). Vascular endothelial growth factor (VEGF, R&D Systems) was used as a positive control of STAT3 nuclear binding (31).
Analysis of Protective and Apoptotic Pathways
BAEC protective pathways were analyzed by Western blot, using rabbit polyclonal anti-Bax (Santa Cruz Biotechnology), mouse monoclonal anti-Bcl2 (BD Transduction Laboratories), rabbit polyclonal anti-phospho-AKT (Ser473) and phospho-GSK3/ (Ser21/9) and their respective loading controls, anti-AKT and anti-GSK3 (Cell Signaling Technology, Beverly, MA). Specific antibodies against JAK2 and phospho-JAK2 and against STAT1, STAT3, STAT5, and STAT6 and their respective phosphorylated forms and loading controls were also used (Cell Signaling Technology). Caspase-3 activity was assayed on confluent BAEC with caspase-3 fluorogenic substrate Ac-DEVD-AMC (BD Transduction Laboratories). Activity was expressed as the signal in a luminometer (exciting wavelength 380 nm, and emission wavelength 440 nm).
Statistics
Results are expressed as means ± SE. Unless stated otherwise, each value corresponds to a minimum of five triplicate experiments. Comparisons were done by ANOVA or paired or unpaired Student's t-test, when appropriate. Fisher and Scheffé's tests for multiple comparisons were used to determine the P value, which was considered significant at <0.05. The statistical analyses were performed with the Windows SPSS 10.0 package (SPSS, Chicago, IL).
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RESULTS |
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Experiments on Monolayer, Attached EC
Endothelial cytoprotection by JAK/STAT inhibition. Treatment with the specific JAK2 inhibitor AG490 protected confluent monolayer BAEC against serum deprivation (Fig. 1). This effect was significant from the 10 µmol/l concentration. A similar effect was obtained by incubating confluent HUVEC with AG490 (50 µmol/l) (%relative cell death with respect to 100% control: 69.5 ± 5.7%, P < 0.02). The 50 µmol/l concentration was henceforth used for examining specific points.
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-catenin and PECAM-1.
On the basis of previous evidence (2, 29), the hypothesis was raised that treatment with AG490 could act on the levels and distribution of -catenin. As shown in Fig. 3A, AG490 induced a marked increase in total and nuclear -catenin. The increase of nuclear -catenin persisted in BAEC pretreated with AG490 and lately cultured in suspension (see below). Additional experiments were done to more specifically assess whether the -catenin accumulated in the nuclear extract corresponded to the active form. By means of an specific antibody, we found that treatment with AG490 induces nuclear accumulation of the active, dephosphorylated form of -catenin (Fig. 3A, lane 3, left). When the -catenin signal was further examined by confocal microscopy, treatment with media +20% FBS induced a marked increase of -catenin, either at the intercellular junctions, cytosol, or nucleus (Fig. 4A). Treatment with AG490 induced changes in -catenin with a pattern similar to that of 20% FBS, including redistribution to the nucleus. BAEC treated with fresh medium (FM) alone had markedly less amounts of -catenin, which was located only at the intercellular junctions (Fig. 4A, detail). By comparison, the GSK3 inhibitor LiCl favored the localization of -catenin to the nucleus as well as an increase of lesser magnitude in both cell junctions and cytoplasm (Fig. 4A).
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-catenin under the effect of AG490 was functional. As shown in Fig. 3B, AG490 induced a significant increase (1.95 ± 0.2 times, P < 0.01) of TOP/FOP activity, comparable with the levels obtained after -catenin transient transfection (2.28 ± 0.2 times, P < 0.01). Additionally, the combination of -catenin transfection with AG490 treatment promoted a synergistic activation of TOP/FOP activity. Collectively, all these data indicate a functional role for the translocation of -catenin observed in this system after AG490 treatment.
A marked increase in PECAM-1 was observed under the same conditions of the -catenin changes (Fig. 4B). This increase was further confirmed by Western blot (data not shown).
Additional Western blot studies were performed to further characterize the effects of JAK2 inhibition by AG490. These studies suggest that, at least in baseline conditions, AG490 may improve BAEC resistance by means of AKT rather than by other anti-apoptotic proteins, as follows. 1) A significant increase in AKT protein was observed (4.5 ± 0.3-fold increase in AG490 with respect to FM, P < 0.05). 2) No significant changes were found in the Bcl2/Bax ratio (FM 1.00 ± 0.18 vs. AG490 1.19 ± 0.21, P = 0.07, n = 5). In the same regard, no changes by AG490 were detected on caspase-3 activity (FM 100 ± 8.1 vs. AG490 113.91 ± 17.3 arbitrary units, P = NS).
Experiments in Detached Conditions
Because EC survival is critically dependent on cell-matrix and cell-cell adhesion (23), a set of experiments was performed to assess the role of JAK2 pathway inhibition in cell death related to disrupted adhesion and culture in suspension.
A clear-cut protective effect of JAK2 inhibition was evident on BAEC cultured in suspension. BAEC were pretreated with FM containing no FBS or 20% FBS or FM + AG490 at 50 µmol/l. As shown in Fig. 5A, pretreatment with AG490 induced a marked increment in BAEC viability at different times after detachment. Furthermore, inhibition of apoptotic death in detached cells was assessed by examining DNA degradation. As shown in Fig. 5B, treatment with AG490 blocked DNA laddering, thus indicating that inhibition of apoptosis was an important component of AG490-induced protection against the effects of detachment.
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Because of the putative effects of AG490 on the Src pathway, further experiments were done to analyze the possible participation of Src in the observed protective actions against detachment-related cell death. These experiments showed no effect of the Src inhibitor PP2 on the survival of detached BAEC, as follows: %cell death after 8 h of suspension culture, FM 32.9 ± 5.7 vs. PP2 35.1 ± 6.2 (n = 4, P = NS).
The effect of AG490 was not only relevant to BAEC damage, as assessed by flow cytometry, but was also functionally significant in terms of the BAEC growth capability in reseeding (Fig. 6). Pretreatment with AG490 (50 µmol/l) induced a marked increase in the yield of reseeding on Matrigel after culture in suspension (no. of cells 7.8 ± 1.2, 9.4 ± 0.8, and 12.3 ± 1.1 times higher in AG490-treated samples after 8, 24, and 48 h, respectively; all P < 0.001 with respect to vehicle-treated controls, n = 5, triplicate experiments).
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-catenin levels are increased in BAEC pretreated with AG490 and lately cultured in suspension compared with FM or 20% FBS. This result posed the question of whether the increased -catenin has a significant role in improving BAEC survival in detached conditions. This issue was examined by transfecting BAEC with a plasmid that increases the expression of -catenin. In these conditions, a marked increase in detached BAEC survival was observed. (Fig. 7B), thereby supporting the importance of -catenin as a major mechanism.
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, i.e., inactivated GSK3. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-catenin, and by higher levels of total and active phosphorylated forms of a key defensive protein, AKT. Furthermore, the present experiments reveal that a series of mechanisms are activated on JAK2/STAT inhibition. Although the mechanistic distinction between apoptotic and necrotic forms of cell death is not a central issue of the present investigation, our results, using both DNA laddering and LDH release, indicate that AG490 exerts protective effects on both types of EC elimination. In this regard, the relevant point is that JAK2/STAT inhibition protects BAEC against death triggered by either serum deprivation or detachment.
In BAEC, -catenin acts both as a vascular endothelial (VE)-cadherin-associated protein in the adherens junction complex and also as a transcriptional activator; the latter role is mediated through its interaction with members of the TCF/Lef family of transcription factors in Wnt signaling (14, 26, 29). In adherent nonstimulated cells, -catenin is localized to the adhesion complexes; simultaneously, its cytoplasmic levels are kept low, by association with other proteins, e.g., adenomatous poliposis coli (APC) or GSK3, which drive cytoplasmic -catenin to proteasome-mediated degradation. The mechanisms identified in the present study are time related to the protective effect of JAK2 inhibition by AG490. Changes in quantity and distribution of -catenin, but especially the demonstration of the nuclear localization of its active dephosphorylated form, and the presence of -catenin-dependent transcriptional activity support a significant role of this protein in the BAEC protective effects; in fact, the pattern found is coincident with that described in death-resistant hemangioma-derived cell lines (17).
Our results involving PECAM-1 are of importance concerning the -catenin increase and redistribution to the nucleus. Accordingly, as found by Biswas et al. (2), PECAM-1 is determinately associated with protection from degradation and with increased nuclear -catenin. In consequence, the simultaneous increase of PECAM-1 and -catenin accompanying JAK2 inhibition is compatible with the existence of an operational sequence of BAEC protection. Of specific interest, our data go beyond the existing information, all in adherent BAEC, and provide a first piece of evidence on the relationship of -catenin with the fate of BAEC in suspension. The present results strongly suggest that activation and nuclear translocation of -catenin can be key elements of survival after detachment.
In resting cells, GSK3 is highly active, and in growth factor deprivation conditions, GSK3 activation is able to increase EC apoptosis (18). Conversely, inhibition of GSK3 signaling, mainly through phosphorylation, protects cells from apoptosis under conditions of mitogen deprivation and regulates cell death by anoikis (18). Inhibition of GSK3 activity results in blockade of -catenin phosphorylation and subsequent stabilization of cytoplasmic -catenin, which eventually translocates to the nucleus (8). Our results showing increased levels of phosphorylated GSK3 in BAEC maintained in suspension after pretreatment with AG490 are inversely related to the increase in -catenin observed in the same circumstances.
Since results showing protective effects of GSK3 inhibition have been previously published (18), we focused our studies only on the comparison of -catenin patterns of response to LiCl and AG490. In this setting, relevant similarities concerning -catenin turnover were detected. In fact, the increased phosphorylation of GSK3 and -catenin increase and translocation under the effect of AG490 are hereby newly described.
Of specific interest, pretreatment of BAEC with AG490 induced a major increase in survival of detached BAEC. This effect was even stronger than that obtained by means of growth factor-rich media, i.e., 20% FBS, and had significant consequences on BAEC reseeding. In fact, no reliable pharmacological tools are available to both prolong life of detached BAEC and enhance their reseeding efficacy, as done by means of AG490. This result can have several practical applications in the field of EC protection.
In terms of mechanism, a strong support for the protective role of -catenin in the present conditions was provided by the results obtained in -catenin-transfected BAEC; these cells had a markedly higher survival capability than their empty vector counterparts. To the best of our knowledge, this is the first demonstration that increased -catenin has a relevant role in prolonging life of BAEC detached from the endothelial monolayer. Furthermore, our results indicate that at least one well-known protective pathway, i.e., PI3K/AKT, is persistently activated in detached conditions under the influence of AG490. The actual functionality of this activation needs further study, by analyzing specific targets of PI3K/AKT activation. On the contrary, the data obtained in the presence of the Src inhibitor, PP2, suggest the absence of a relevant involvement of the Src pathway in BAEC death after detachment and therefore make improbable any possible role of Src inhibition in the effects of AG490. Moreover, it should be noted that the Src inhibitory capability of AG490, albeit in a different experimental system, appears to be rather weak (25).
The present results provide a set of new findings, with relevant potential in terms of practical applications. The strength of the protective effect of JAK2 inhibition against diverse injuries, and particularly against cell detachment-induced death, is particularly valuable.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* F. Neria and C. Caramelo contributed equally to this study. 
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REFERENCES |
|---|
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Biswas P, Canosa S, Schoenfeld J, Schoenfeld D, Tucker A, Madri JA. PECAM-1 promotes beta-catenin accumulation and stimulates endothelial cell proliferation. Biochem Biophys Res Commun 303: 212–218, 2003.[CrossRef][Web of Science][Medline]
3. Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 119: 629–641, 1992.
4. Castilla MA, Arroyo MV, Aceituno E, Aragoncillo P, Gonzalez-Pacheco FR, Texeiro E, Bragado R, Caramelo C. Disruption of cadherin-related junctions triggers autocrine expression of vascular endothelial growth factor in bovine aortic endothelial cells : effects on cell proliferation and death resistance. Circ Res 85: 1132–1138, 1999.
5. Castilla MA, Caramelo C, Gazapo RM, Martin O, Gonzalez-Pacheco FR, Tejedor A, Bragado R, Arroyo MV. Role of vascular endothelial growth factor (VEGF) in endothelial cell protection against cytotoxic agents. Life Sci 67: 1003–1013, 2000.[CrossRef][Web of Science][Medline]
6. Castilla MA, Neria F, Renedo G, Pereira DS, Gonzalez-Pacheco FR, Jimenez S, Tramon P, Deudero JJ, Arroyo MV, Yague S, Caramelo C. Tumor-induced endothelial cell activation: role of vascular endothelial growth factor. Am J Physiol Cell Physiol 286: C1170–C1176, 2004.
7. Dejana E. Endothelial cell-cell junctions: happy together. Nat Rev Mol Cell Biol 5: 261–270, 2004.[CrossRef][Web of Science][Medline]
8. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116: 1175–1186, 2003.
9. Dudley AC, Thomas D, Best J, Jenkins A. The STATs in cell stress-type responses. Cell Commun Signal 2: 8, 2004.[CrossRef][Medline]
10. El Adawi H, Deng L, Tramontano A, Smith S, Mascareno E, Ganguly K, Castillo R, El Sherif N. The functional role of the JAK-STAT pathway in post-infarction remodeling. Cardiovasc Res 57: 129–138, 2003.
11. Gorina R, Petegnief V, Chamorro A, Planas AM. AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway. J Neurochem 92: 505–518, 2005.[CrossRef][Web of Science][Medline]
12. Grossmann J. Molecular mechanisms of "detachment-induced apoptosis–Anoikis." Apoptosis 7: 247–260, 2002.[CrossRef][Web of Science][Medline]
13. Harada M, Qin Y, Takano H, Minamino T, Zou Y, Toko H, Ohtsuka M, Matsuura K, Sano M, Nishi J, Iwanaga K, Akazawa H, Kunieda T, Zhu W, Hasegawa H, Kunisada K, Nagai T, Nakaya H, Yamauchi-Takihara K, Komuro I. G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes. Nat Med 11: 305–311, 2005.[CrossRef][Web of Science][Medline]
14. Hatsell S, Rowlands T, Hiremath M, Cowin P. Beta-catenin and Tcfs in mammary development and cancer. J Mammary Gland Biol Neoplasia 8: 145–158, 2003.[CrossRef][Web of Science][Medline]
15. Hattori R, Maulik N, Otani H, Zhu L, Cordis G, Engelman RM, Siddiqui MA, Das DK. Role of STAT3 in ischemic preconditioning. J Mol Cell Cardiol 33: 1929–1936, 2001.[CrossRef][Web of Science][Medline]
16. Hortelano S, Castilla M, Torres AM, Tejedor A, Bosca L. Potentiation by nitric oxide of cyclosporin A and FK506-induced apoptosis in renal proximal tubule cells. J Am Soc Nephrol 11: 2315–2323, 2000.
17. Ilan N, Tucker A, Madri JA. Vascular endothelial growth factor expression, beta-catenin tyrosine phosphorylation, and endothelial proliferative behavior: a pathway for transformation? Lab Invest 83: 1105–1115, 2003.[CrossRef][Web of Science][Medline]
18. Kim HS, Skurk C, Thomas SR, Bialik A, Suhara T, Kureishi Y, Birnbaum M, Keaney JF Jr, Walsh K. Regulation of angiogenesis by glycogen synthase kinase-3beta. J Biol Chem 277: 41888–41896, 2002.
19. Liu W, Davis DW, Ramirez K, McConkey DJ, Ellis LM. Endothelial cell apoptosis is inhibited by a soluble factor secreted by human colon cancer cells. Int J Cancer 92: 26–30, 2001.[CrossRef][Web of Science][Medline]
20. Mascareno E, El Shafei M, Maulik N, Sato M, Guo Y, Das DK, Siddiqui MA. JAK/STAT signaling is associated with cardiac dysfunction during ischemia and reperfusion. Circulation 104: 325–329, 2001.
21. Maziere C, Conte MA, Maziere JC. Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein. Free Radic Biol Med 31: 1334–1340, 2001.[CrossRef][Web of Science][Medline]
22. Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM. Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature 379: 645–648, 1996.[CrossRef][Medline]
23. Michel JB. Anoikis in the cardiovascular system: known and unknown extracellular mediators. Arterioscler Thromb Vasc Biol 23: 2146–2154, 2003.
24. Neria F, Caramelo C, Peinado H, Gonzalez-Pacheco FR, Deudero JJ, de solis AJ, Fernandez Sanchez R, peñate S, Cano A, Castilla MA. Endothelial protection by inhibition of the JAK/STAT pathway in monolayer and detached EC (Abstract). Nefrología 25: 7, 2005.
25. Oda Y, Renaux B, Bjorge J, Saifeddine M, Fujita DJ, Hollenberg MD. cSrc is a major cytosolic tyrosine kinase in vascular tissue. Can J Physiol Pharmacol 77: 606–617, 1999.[CrossRef][Web of Science][Medline]
26. Orford K, Orford CC, Byers SW. Exogenous expression of beta-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest. J Cell Biol 146: 855–868, 1999.
27. Sandberg EM, Sayeski PP. Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells. J Biol Chem 279: 34547–34552, 2004.
28. Uchimura H, Marumo T, Takase O, Kawachi H, Shimizu F, Hayashi M, Saruta T, Hishikawa K, Fujita T. Intrarenal injection of bone marrow-derived angiogenic cells reduces endothelial injury and mesangial cell activation in experimental glomerulonephritis. J Am Soc Nephrol 16: 997–1004, 2005.
29. Weng Z, Xin M, Pablo L, Grueneberg D, Hagel M, Bain G, Muller T, Papkoff J. Protection against anoikis and down-regulation of cadherin expression by a regulatable beta-catenin protein. J Biol Chem 277: 18677–18686, 2002.
30. Yadav A, Kalita A, Banerjee K. JAK/STAT3 pathway is involved in survival of neurons in response to insulin like growth factor and negatively regulated by suppression of cytokine signaling-3. J Biol Chem 280: 31830–31840, 2005.
31. Yahata Y, Shirakata Y, Tokumaru S, Yamasaki K, Sayama K, Hanakawa Y, Detmar M, Hashimoto K. Nuclear translocation of phosphorylated STAT3 is essential for vascular endothelial growth factor-induced human dermal microvascular endothelial cell migration and tube formation. J Biol Chem 278: 40026–40031, 2003.
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  I. V. Libani, E. C. Guy, L. Melchiori, R. Schiro, P. Ramos, L. Breda, T. Scholzen, A. Chadburn, Y. Liu, M. Kernbach, et al. Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in {beta}-thalassemia Blood, August 1, 2008; 112(3): 875 - 885. [Abstract] [Full Text] [PDF]
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-P, dephosphorylated.
