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J. Biol. Chem., Vol. 277, Issue 16, 13371-13374, April 19, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13371    most recent C200044200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Strömblad, S. Articles by Cheresh, D. A. Search for Related Content PubMed PubMed Citation Articles by Strömblad, S. Articles by Cheresh, D. A. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Loss of p53 Compensates for _v-Integrin Function in Retinal Neovascularization^*

Staffan Strömblad^§, Arun Fotedar^¶, Howard Brickner^¶, Chandra Theesfeld, Edith Aguilar de Diaz, Martin Friedlander, and David A. Cheresh^**

From the  Department of Microbiology, Pathology, and Immunology, Karolinska Institutet, 141 86 Huddinge, and Södertörns Högskola, 141 04 Huddinge, Sweden, the ^¶ Sidney Kimmel Cancer Center, San Diego, California 92121, and Departments of  Cell Biology and ^** Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, January 23, 2002, and in revised form, February 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

_v-Integrin antagonists block neovascularization in various species, whereas 20% of _v-integrin null mice are born with many normal looking blood vessels. Given that blockade of _v-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for _v-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an _v-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on _v-integrin-mediated suppression of p53. Blockade of _v-integrins during neovascularization resulted in an induction of p21^CIP1 in wild type and, surprisingly, in p53 null retinas, indicating that _v-integrin ligation regulates p21^CIP1 levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and _v-integrins act in concert during retinal neovascularization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously found that integrin _v3 is preferentially expressed on newly forming blood vessels and is functionally involved in controlling angiogenesis stimulated by basic fibroblast growth factor or tumor necrosis factor-, whereas another _v-integrin, _v5, is functional in vessel formation induced by vascular endothelial growth factor or transforming growth factor- (1, 2). Importantly, antagonists of _v-integrins block neovascularization in various animal models with or without exogenous angiogenic stimulation, including chick chorioallantois, in mouse retina, and in human skin trans- plants in SCID mice, causing apoptosis of proliferating, angiogenic vascular cells (3-8). This suggests that during vessel formation, _v-integrins promote signaling events ultimately promoting vascular cell survival, thereby facilitating neovascularization. However, whereas 80% of _v-integrin null mice die in mid-gestation, 20% of these mice survive until 1 day after birth (9). Similarly, combinatorial gene knockout of integrin ₃ and ₅ subunits in mice results in enhanced angiogenesis under certain conditions (10). This indicates either that mice lacking integrins _v3 and _v5 could compensate for the function of _v-integrins in blood vessel formation or that a function of vascular _v-integrins, once expressed but blocked or unligated, is to inhibit neovascularization. However, at present it is not known whether possible compensatory or redundant mechanisms can mediate blood vessel formation in the absence of functional _v-integrins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p53^+/ mice (11) were used to set up p53^+/ × p53^/ breeding pairs. Littermates from such matings were used in the neovascularization assay, and genomic DNA from mouse tails was genotyped for p53 by a 3-primer assay as described previously (12). Newborn mice were injected subcutaneously twice daily, starting within 8 h after birth with 40 µg of cyclo-RGDfV (_v antagonist peptide 66203) or cyclo-RADfV (control peptide 69601) (lowercase denotes D-amino acids) dissolved in phosphate-buffered saline, pH 7.4. The cyclo-RGDfV peptide binds specifically with high affinity to _v3- and _v5-integrins and blocks their function both in vitro and in vivo, whereas the cyclo-RADfV peptide is non-functional (3, 7, 8, 13). After 2-3 days, the eye globes were taken out, fixed in cold methanol for 10 min followed by 6 min in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, dissected, stained for collagen type IV, and photographed as previously described (7). The distance from the head of the optic nerve to the edge of the retinal vasculature at 6-8 different representative points was measured for each retina on the photographs, and the mean vascular radius was calculated. The mean vascular area between the two retinas in each animal was then calculated, assuming a circular shape of the vasculature. Without treatment, no difference in vascular areas was observed between p53 heterozygous and p53 null mice, and therefore, to standardize and compare the results from different litters, the mean retinal vascular area in p53 null mice in each litter was considered to represent a fully developed vasculature. All p53 genotyping and measurements of the retinal vasculature were performed in a double-blind fashion to avoid any bias. For measurements of wild type retinas, the mean vascular area of control treated retinas was considered as fully developed and compared with anti-_v-treated retinas within the same litters. For Western blot analysis, retinas were fixed only in cold methanol and dissected. Retinas were then lysed in a modified radioimmune precipitation buffer and analyzed by Western blot as previously described (5) using 1 µg/ml anti-p21CIP1^WAF-1/CIP-1 polyclonal antibodies (ab-5, Oncogene, Cambridge, MA), anti-_v cytoplasmic tail polyclonal antibodies (Chemicon), or anti-actin monoclonal antibody JLA20 (Developmental Studies Hybridoma Bank, University of Iowa).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Interestingly, inhibition of angiogenesis by blockade of _v-integrins is accompanied by an induction of endothelial cell p53 activity (5). Based on this, we hypothesized that loss of p53 might compensate for _v-integrin function during neovascularization. To examine this possibility, we analyzed p53 null mice, where the effect of a specific _v-integrin antagonist was studied on retinal neovascularization. Mouse retinal neovascularization occurs during the first days after birth, and therefore, newborn mice were treated with an _v-integrin antagonist as described (7). The retinal vasculature in wild type newborn mice treated for 3 days with the _v-antagonist was significantly less developed than retinas from control treated mice (Table I). When accounting for the retinal vascular area already existing when the treatment started, the inhibition was close to 100%, in accordance with our previous observations after 4 days of treatment (7). Targeted p53 null males were then mated with p53 heterozygous females. This type of mating was not only necessary for sufficient embryonic survival but also allowed for a comparison of p53 null mice with heterozygous mice within the same litter in a double-blind fashion, including animals of exactly the same age receiving identical treatment. No difference in retinal vascularization could be observed between p53 null and heterozygous mice during the first 3 days (data not shown). In addition, no difference in degree of neovascularization between p53 null and heterozygous mice could be observed after control treatment (Fig. 1B and Table I). This indicates that p53 does not influence normal vascularization of the retina. We then treated entire litters of newborn mice of a mixed genotype (see above) with the _v-integrin antagonist. Interestingly, p53 heterozygous animals had a markedly less developed retinal vasculature compared with p53 null mice when treated with the _v-antagonist (Fig. 1 and Table I). Statistical analysis of measurements performed on the vascular area of these retinas revealed that the vascular development in anti-_v-treated p53 heterozygous was suppressed, a suppression that was found to be statistically significant (p = 5.7 × 10⁷) when compared with p53 null animals in the same litters receiving identical treatment (Table I). This result closely resembled the difference seen between retinas from antagonist versus control treated wild type mice (Table I). Taken together, these findings indicate that although p53 expression does not influence normal neovascularization, loss of p53 compensates for the function of _v-integrins in neovascularization.

View this table: [in this window] [in a new window]   Table I Statistical evaluation of the effect on retinal vascular development by treatment with an v-integrin antagonist Statistical evaluation of measured retinal vascular areas. For p53+/ and p53/, given p values represent statistical significance for the indicated groups compared to identically treated homozygous (p53/) mice according to an unpaired two-tail t-test using Microsoft Excel software. For wild type (wt) mice, the given p value represents statistical significance for treated versus control-treated retinas within the same litters, analyzed by an unpaired t-test. All measurements and genotyping were performed in a double-blind fashion.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Retinal neovascularization in genetically targeted p53 null mice is refractory to systemic treatment with an integrin v antagonist. Newborn mice were injected subcutaneously twice daily with an integrin v antagonistic or control cyclic peptides for 2-3 days. Retinas were dissected, stained for collagen type IV (vessel basement membrane), mounted flat, and photographed (10×) as described under "Experimental Procedures." A, representative retinas from newborn mice of a mating between a p53 null male and a p53 heterozygous female, where the entire litter was treated for 21/2 days with the v antagonist. Size bar is 150 µm. B, measurements of development of the retinal vasculature of 4-5 litters per group were standardized for comparison as described under "Experimental Procedures" and plotted as % undeveloped vasculature. Each point represent the mean retinal vascular area in one mouse calculated as a mean between the two retinas in each animal.

A possible explanation for the lack of response to the _v-integrin antagonist in p53 null mice could be deficient retinal integrin _v expression. To test this possibility, retinal lysates were analyzed for _v protein levels by Western blot analysis. As shown in Fig. 2A, _v-integrin levels in p53 null retinas do not differ from that in p53 heterozygous mice. Another possibility for a lack of response to the _v antagonist in p53 null mice could be that cells lacking p53 are insensitive to this treatment because of alterations in _v-integrin function at the cell surface. To examine this possibility, we examined _v-integrin function of mouse embryonic fibroblasts lacking p53, including their responsiveness to the _v-integrin antagonist and compared them to mouse fibroblasts expressing p53. As shown in Fig. 2B, the responsiveness to the antagonist in inhibiting _v-dependent attachment to vitronectin was virtually identical in p53 null fibroblasts and the mouse fibroblast cell line NIH 3T3, demonstrating that lack of p53 does not cause a general insensitivity to _v-integrin antagonists. These findings reveal that loss of p53 does not affect expression levels or general function of _v-integrins. Instead, we conclude that intracellular events involving p53 mediate the inhibition of neovascularization by _v antagonists, events that may be related to the activation of endothelial cell p53 that we previously observed upon _v-integrin blockage during angiogenesis (5).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Retinal v-integrin expression levels, sensitivity to an v antagonist, and regulation in the retina of p21CIP1 by the v antagonist are independent of p53. A, retinas isolated and pooled from three to four p53+/ and p53/ mice, respectively, were subjected to Western blot analysis for integrin v protein expression levels using actin levels as control. B, p53/ (filled squares) and NIH3T3 (p53+/+) (open circles) mouse fibroblasts were analyzed for their sensitivity to the integrin v antagonist used in the in vivo experiments, here assayed as cell adhesive capacity to vitronectin described previously (23) allowing cells to adhere for 10-15 min. The values are expressed as percent of cell adhesion in the absence of inhibitor and represent mean values between three distinct experiments at each concentration of the v antagonist, which in turn was analyzed in triplicate within each experiment. C, retinas isolated and pooled from three to four wild type, p53+/ or p53/ mice, treated with or without v antagonist, respectively, were subjected to Western blot analysis for p21CIP1 protein levels. Presence or absence of full neovascularization in the respective retinas are indicated as + or .

p53 is a known activator of the cell cycle suppressor p21^CIP1. In fact, in addition to regulating p53, ligation of integrin _v3 in endothelial cells also suppresses p21^CIP1 protein levels during angiogenesis (5). In UV-irradiated fibroblasts, p53 exerts its functional effect on cell cycle arrest by transcriptional activation of the Cdk inhibitor p21^CIP1 (14). As shown in Fig. 2C, blockade of _v-integrin during neovascularization induces p21^CIP1 levels in wild type and in p53 heterozygous retinas. Surprisingly, whereas untreated p53 null mice display no detectable p21^CIP1, the numeric increase in p21^CIP1 levels by anti-_v treatment of these mice is similar to what is observed in wild type mice, resulting in a higher relative increase. This indicates that the regulation of p21^CIP1 by integrin _v during neovascularization is independent of p53. Furthermore, the fact that p21^CIP1 is induced in p53 null retinas upon blockade of _v-integrins while neovascularization is still active suggests that this induction of p21^CIP1 is not sufficient to block neovascularization, although we cannot exclude that the somewhat higher total levels of p21^CIP1 in heterozygous animals might contribute to this blockade.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies using targeted gene knockout mice have in some cases revealed surprising results in that expected phenotypes were not found. This is particularly surprising for molecules found to play a role in certain in vivo events by previous loss of function studies, including for integrin _v and the capacity for at least 20% of _v-integrin null mice to form blood vessels (9) and for the capacity of integrin ₃ and ₅ subunit combinatorial gene knockouts to support enhanced pathological angiogenesis (10). In some cases, combinatorial knockout of two or three related genes has demonstrated compensatory mechanisms by displaying phenotypes missing in single gene knockout mice. However, it is unclear as to how the functions of _v-integrins can be compensated for. To this end, although it does not represent the only possible mechanism of _v-integrin compensation, our finding that p53 null mice form blood vessels in the absence of functional _v-integrins that are critical in wild type mice reveals the first in vivo example of an intracellular mechanism that is able to compensate for loss of integrin function.

Alternatively, the function of _v-integrins in neovascularization in wild type animals may be to negatively regulate and balance vessel formation in an unfavorable extracellular matrix environment in order to prevent angiogenesis in inappropriate locations. Such a function of _v-integrins would then lead to enhanced angiogenesis when _v3 is lacking as suggested by Reynolds et al. (10). In support of this model, caspase-8 was activated at the cell surface in other cell types by unligated integrin _v3, thereby causing apoptosis (15). This mechanism might be related to p53, because caspase-8 plays a role in certain p53-induced apoptosis (16). Nevertheless, in both alternative models for the function of _v-integrins, our results suggest that p53 may mediate _v-integrin regulation of cell survival during neovascularization.

Neovascularization is a critical component of tumor growth, where a tumor is unable to grow beyond a minimal size without new blood vessels (17). In fact, we previously observed that _v-integrin antagonists could block the growth of human tumors in animal models (3, 4). In addition, uncontrolled ocular neovascularization is a major cause of blindness in various ocular diseases, including diabetic retinopathy, presumed ocular histoplasmosis syndrome, and age-related macular degeneration. Integrins _v3 and _v5 may be involved in the regulation of neovascularization of these diseases as systemic treatment with _v-integrin antagonists block retinal neovascularization (7, 8). This suggests that _v-integrin antagonists constitute a potential therapy for ocular diseases and cancer. Our findings indicate that the molecular mechanism for this potential anti-angiogenic treatment actively involves p53, similar to what was recently indicated for angiostatin and TNP-470 (18-20).

We were unable to detect apoptosis in the retinal vasculature because of an obscuring background with a large number of apoptotic cells in the whole mounts of developing retinas with no apparent differences between the groups (data not shown). However, previous studies in other models clearly demonstrate that blocking of _v-integrins during neovascularization leads to vascular cell apoptosis (3, 5). This suggests that the inhibition of vessel formation by _v antagonists may be caused by induction of apoptosis of the forming vascular cells, and the fact that vascular formation in p53 null mice is refractory to _v antagonist treatment suggests that these vessels do not undergo apoptosis (Fig. 3). Whereas p53 may mediate _v-integrin-regulated apoptosis in vascular cells, induction of p53 by loss of integrin ligation does not constitute a generic mechanism for regulation of cell survival in all cells and by all integrins. For example, ligation of integrin ₃₁ in an in vitro model of mammary epithelial cells lead to apoptosis only in the absence of functional p53 (21), a mechanism that appears to be the opposite of our findings on vascular cell integrin _v3 and p53 during neovascularization. In future studies, it will be interesting to elucidate whether downstream integrin signaling pathways such as activation of ERKs¹ might be involved in the regulation of p53, because ERK1/2 signaling was identified as another critical _v-integrin-mediated event during angiogenesis (22). In addition, it remains to be elucidated whether the p53-mediated response to _v antagonist treatment in vascular cells is functionally related to activation of caspase-8 by unligated _v3 (15).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Hypothetical model for role of p53 in compensating for v-integrin function during blood vessel formation. In neovascularization of wild type animals (left), v3-and/or v5-integrins are activated (1, 2). The v-integrins are then allowed to ligate to their provisional matrix, a ligation that is necessary to keep endothelial cell p53 inactive and cells surviving (vascularization is facilitated (3, 5)). When v-integrins are blocked in wild type animals (middle) and thereby prevented from forming clusters, endothelial cell p53 activity is induced, and the vascular cells undergo apoptosis leading to a block of blood vessel formation (5). However, when p53 is absent during vascularization, inhibition of v-integrins does not affect the formation of viable vessels (right). Taken together, this suggests that p53 and v-integrins are linked into the same pathway in the control of blood vessel formation. VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; ECM, extracellular matrix.

In conclusion, we demonstrate that loss of p53 compensates for the function of _v-integrins in retinal neovascularization, possibly by interfering with _v-integrin regulation of vascular cell apoptosis. This indicates a critical function for _v-integrin ligation during neovascularization in suppressing p53 and that p53 constitutes an important part of the control of neovascularization.

	ACKNOWLEDGEMENT

We thank Dr. Klas Wiman for providing p53 null mouse embryonic fibroblasts.

	FOOTNOTES

^* This work was supported by grants from the Swedish Cancer Society, The Swedish Medical Research Council, and the Magnus Bergvall Foundation (to S. S.), National Institutes of Health Grants CA74435 (to A. F.) and CA 502289 and CA 45726 (to D. A. C.), and NEI, National Institutes of Health Grant EY11254 (to M. F.).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.

^§ To whom correspondence should be addressed: Karolinska Institutet, Huddinge University Hospital F46, SE-141 86 Huddinge, Sweden. Tel.: 46-8-585-81032; Fax: 46-8-585-81020; E-mail: Staffan.Stromblad@ impi.ki.se.

Published, JBC Papers in Press, February 20, 2002, DOI 10.1074/jbc.C200044200

	ABBREVIATIONS

The abbreviation used is: ERK, extracellular signal-regulated kinase.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/16/13371    most recent C200044200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Strömblad, S. Articles by Cheresh, D. A. Search for Related Content PubMed PubMed Citation Articles by Strömblad, S. Articles by Cheresh, D. A. Social Bookmarking                      What's this?