KATP channel activation induces angiogenesis in vitro and in vivo

B Umaru1, A Pyriochou1, V Kotsikoris1, A Papapetropoulos2, S Topouzis1. 1University of Patras, Rio/Patras, Greece, 2University of Athens, Athens, Greece.

BACKGROUND / INTRODUCTION

Intense research is conducted to identify pro- and anti-angiogenic agents and new molecular mechanisms of angiogenesis, based on the use of specific assays in vitro and in vivo (1). This investigation was prompted by previous studies showing that the angiogenic effects of hydrogen sulphide (H2S) depend on KATP channel (KATP) activation (2), and that C-type Natriuretic Peptide (CNP), a cardiac paracrine hormone acting through KATP , elicits endothelial cell growth (3). However, the CNP angiogenic-type responses and the involvement of KATP in angiogenesis in general are both incompletely understood.

AIMS OF THE STUDY

We aimed to test whether KATP activation is a common mechanism of angiogenesis, a) by characterizing the angiogenic responses of direct pharmacological activators and inhibitors of KATP in vitro and in vivo (4) and b) by testing whether KATP activation is required for endothelial responses to CNP or VEGF.

RESULTS

Chick chorioallantoic membrane (CAM) angiogenesis was similarly enhanced (by ~20%, p<0.05), by the direct activator of KATP SG-209 (0.1-10 nmole/cm2), by CNP or VEGF (300-5000 nmole/cm2). The KATP inhibitors glibenclamide and 5-hydroxydecanoate (5-HD) (1-100 nmole/cm2) reduced basal (max:-45%, p<0.05) angiogenesis and abolished the effects of CNP.

In vitro, in bEnd.3 endothelial cells, KATP openers nicorandil (10 μM) and SG-209 (1 μM) increased proliferation (by 105% and 89%, p<0.05) and migration (by 65% and 40%, p<0.05). These responses were abrogated by glibenclamide (10 μM) and 5-HD (100 μM). Similarly to VEGF (500 pM), CNP (1-1000 pM) increased proliferation (by 38%, p<0.05) and migration (3.5-fold, p<0.05) in bEnd.3 cells and induced tube-like formation by HUVECs in Matrigel (2.5-fold, p<0.05). Responses to both VEGF and CNP were totally blocked by glibenclamide or 5-HD.

When the expression of the kir6.1 subunit of KATP was knocked-down in HUVECs by transfection with a specific siRNA (from SantaCruz) by 70% and 50% at the mRNA and protein level, respectively (p<0.05), tubular network formation and migration in response to SG-209 and to CNP were effectively abrogated.

CONCLUSIONS

1. Direct pharmacological activation of KATP or exposure to CNP can elicit angiogenic-type responses in vivo and in vitro, comparable to those of VEGF.

2. Responses induced by VEGF or CNP in CAM in vivo or in endothelial cells in vitro are abrogated by KATP channel blockers.

3. Direct KATP activators and CNP depend upon the expression of the kir6.1 KATP subunit for their activity.

4. KATP activation thus seems to be a bona fide mechanism mediating angiogenesis to a wide range of vasoactive agents, including hydrogen sulphide (H2S), VEGF and CNP.

REFERENCES

(1) Carmeliet & Jain (2011) Nature 473:298-307

(2) Papapetropoulos et al., (2009) PNAS 106:21972-7

(3) Khambata et al., (2011) Br J Pharmacol 164:584-97

(4) Pyriochou A et al., (2007) Br J Pharmacol 152:207-14

ACKNOWLEDGMENTS We thank Dr. AJ Hobbs (Queen Mary Un. of London), for the generous gift of CNP. This work was supported by a THALIS action: «Hydrogen sulphide, a new endogenous modulator of angiogenesis: investigating its signalling, (patho)physiological roles and development of novel pharmacological antagonists» Φ.K.:D554 / MIS: 380259, financed by EU and greek national funds.