Background & Aim: The forkhead transcription factors are known to suppress growth and promote apoptosis of endothelial cells (ECs). To investigate a role of the forkhead transcription factors in EC growth and attachment in matrix, we evaluated matrix metalloproteinases (MMPs) and connective tissue growth factor (CTGF) change according to FKHRL1 overexpression by adenoviral gene transfer in ECs.
Methods: HUVECs were infected with adenoviral vectors expressing constitutively active FKHRL1-TM in the 10% FBS. Gene responses were evaluated at 16, 24 and 40 hours post-treatment by oligonucleotide microarrays and real time PCR. The enzyme expressions were confirmed by western blot of CTGF, MMP-3, -7 and TIMP-1 and by zymography.
Results:
1) Morphologically, constitutive activation of FKHRL1 signaling suppressed EC growth and induced dehiscence of EC from the plate, and increased floating dead cells with condensed and fragmented nuclei compared to the control adeno-GFP. The agarose gel electrophoresis of fragmented DNA from floating dead cells and FACS analysis showed that the major underlying mechanism of the cytotoxicity was anoikis, resulting apoptosis
2) CTGF mRNA expression was suppressed in microarrays at 16, 24 and 40 hours post-treatment by -2.07, -1.50, -7.94 compared to adeno-GFP groups respectively, and this suppression was confirmed by western blot.
3) The expressions of MMP-3 and MMP-7 mRNA were increased and the expression of TIMP-1 was decreased. In real time PCR, MMP-3 mRNA expressions were increased by 1 ± 0.3, 137 ± 40, 690 ± 250 and MMP-7 mRNA expressions increased by1 ± 0.2, 4 ± 0.2, 42 ± 0.4, respectively. TIMP-1 mRNA expression was suppressed by -2.14, -1.36, -1.24. Western blot confirmed these genomic changes correspond to protein synthesis and casein zymography showed these MMP proteins possessed enzymatic activity in EC.
Conclusion: These data show that forkhead transcription factor suppressed growth and promotes dehiscence of endothelial cell resulting anoikis via CTGF suppression and MMPs activation. Thus these data provide mechanisms how forkhead transcription signaling controls the blood vessel growth at a molecular level.
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