CXCL10 is a novel anti-angiogenic factor downstream of p53 in cardiomyocytes

Abstract

Tumor suppressor protein p53 plays crucial roles in the onset of heart failure. p53 activation results in cardiac dysfunction, at least partially by suppressing angiogenesis. Though p53 has been reported to reduce VEGF production by inhibiting hypoxia-inducible factor, the anti-angiogenic property of p53 remains to be fully elucidated in cardiomyocytes. To explore the molecular signals downstream of p53 that regulate vascular function, especially under normoxic conditions, DNA microarray was performed using p53-overexpressing rat neonatal cardiomyocytes. Among genes induced by more than 2-fold, we focused on CXCL10, an anti-angiogenic chemokine. Real-time PCR revealed that p53 upregulated the CXCL10 expression as well as p21, a well-known downstream target of p53. Since p53 is known to be activated by doxorubicin (Doxo), we examined the effects of Doxo on the expression of CXCL10 and found that Doxo enhanced the CXCL10 expression, accompanied by p53 induction. Importantly, Doxo-induced CXCL10 was abrogated by siRNA knockdown of p53, indicating that p53 activation is necessary for Doxo-induced CXCL10. Next, we examined the effect of hypoxic condition on p53-mediated induction of CXCL10. Interestingly, CXCL10 was induced by hypoxia and its induction was potentiated by the overexpression of p53. Finally, the conditioned media from cultured cardiomyocytes expressing p53 decreased the tube formation of endothelial cells compared with control, analyzed by angiogenesis assay. However, the inhibition of CXCR3, the receptor of CXCL10, restored the tube formation. These data indicate that CXCL10 is a novel anti-angiogenic factor downstream of p53 in cardiomyocytes and could contribute to the suppression of vascular function by p53.

Keywords: CXCL10; angiogenesis; cardiomyocyte; hypoxia; p53.

FIGURE 1

p53 induced CXCL10 expression in cardiomyocytes. DNA microarray was performed using p53‐overexpressing NRCMs. (a) Scatter plot shows the genes colored blue with >2‐fold upregulate in p53‐overexpressing NRCMs compared with β‐gal. (b) Heat map shows genes colored red with >2‐fold change and green with <0.5‐fold change between β‐gal and p53. (c) Gene ontology analysis. (d, e) The expression of (d) CXCL10 and (e) p21 transcripts was measured by qPCR (n = 4). Experiments were performed three times with similar results. (f) The protein expression of CXCL10 in culture media of p53‐overexpressing NRCMs was measured by ELISA (n = 3). *p < 0.05, **p < 0.01. by Student's t‐test.

FIGURE 2

Doxorubicin induced the expression of CXCL10 in cardiomyocytes through p53 in cardiomyocytes. (a–h) NRCMs were stimulated with Doxo for indicated concentration and time. (i–l) NRCMs were transfected with siRNA for p53 (sip53) or control (siCon) for 48 h, followed by stimulation with Doxo for 24 h. The expression of p53 (a, e, i) and CXCL10 (b, f, j) mRNA was measured by qPCR. (n = 3). (c, g, k) The expression of p53 protein was analyzed by immunoblotting. Representative data are shown. (d, h, l) The band intensities of p53 were measured (n = 3). Experiments were performed three times with similar results. (a–h). *p < 0.05, **p < 0.01 by one‐way ANOVA followed by Dunnett test. (i–l) *p < 0.05, **p < 0.01 by one‐way ANOVA followed by Tukey–Kramer test.

FIGURE 3

Hypoxia synergistically elevated the expression of CXCL10. p53‐overexpresisng NRCMs were treated with CoCl2 for 24 h. (a) The expression of p53 and GAPDH protein was analyzed by immunoblotting. Representative data were shown. The expression of (b) VEGF and (c) CXCL10 transcripts was measured by qPCR (n = 4). Experiments were performed three times with similar results. *p < 0.05, **p < 0.01 by one‐way ANOVA followed by Tukey–Kramer test.

FIGURE 4

p53‐induced CXCL10, secreted from cardiomyocytes, inhibited the tube formation of endothelial cells. Culture media were collected from p53‐ or β‐gal‐overexpressing NRCMs. RAOECs were cultured with the conditioned media in the presence of AMG487, an inhibitor of CXCR3. Angiogenesis assay was performed. (a) Representative images were shown. (b) The number of tube formation was measured (n = 6). *p < 0.05, **p < 0.01 by one‐way ANOVA followed by Tukey–Kramer test.