| Περίληψη: | Worldwide research devotes significant effort to identify new, targetable molecular mechanisms
in the field of angiogenesis, since therapeutic modulation of angiogenesis can critically alter the
progression of a number of diseases. Stimulation of angiogenesis is desirable in situations
characterized by tissue-damaging ischemia where blood supply is severely reduced, such as
lower limb diabetic arteriopathy or following myocardial infarct. In contradistinction, stemming
excessive or ectopic angiogenesis can be beneficial in situations such as solid tumor growth or
in neovascular age-related macular degeneration.
It has been previously shown that Hydrogen Sulfide (H2S), a new vasoactive gasotransmitter,
can initiate angiogenic responses which depend on the activation of ATP-sensitive potassium
channels (KATP). Intriguingly, C-type Natriuretic Peptide (CNP), which is also known to activate
KATP, has been reported to promote endothelial cell growth; however, its angiogenic properties
have not been explored at any depth. This pattern prompted us to investigate whether direct
KATP activation induces angiogenic responses and whether endothelial responses to CNP or
Vascular Endothelial Growth Factor (VEGF) indeed require KATP activation.
We undertook a dual-pronged approach, based on both in vivo and in vitro experimental
approaches. In vivo, chick embryo chorioallantoic membrane (CAM) angiogenesis was similarly
enhanced by the direct KATP channel activator SG-209 and by the polypeptides CNP or VEGF.
Two KATP inhibitors, Glibenclamide and 5-Hydroxydecanoate (5-HD), abrogated both basal and
CNP-induced CAM angiogenesis. In vitro, direct activators of KATP such as Nicorandil and SG-
209 and receptor-acting agonists such as VEGF and CNP, increased proliferation and migration
in the mouse brain endothelial cell line bEnd.3. In addition, VEGF and CNP induced
comparable capillary tube-like formation by Human Umbilical Vein Endothelial cells (HUVECs)
in low growth factor Matrigel. All these in vitro pro-angiogenic endothelial responses were
effectively abrogated by Glibenclamide or by 5-HD. Transfection of HUVECs with a siRNA
specifically targeting the inwardly rectifying potassium channel (Kir) 6.1 subunit decreased the
expression of this subunit at both the mRNA and the protein level. The resulting knock-down of
the Kir6.1 KATP subunit impaired HUVEC migration through transwells in vitro and substantially
decreased tubular network formation in Matrigel in response to either the direct KATP activator
SG-209 or the receptor-operating KATP activator CNP. Furthermore, the bEnd.3 endothelial cell
proliferation and migration responses to SG-209 required mobilization of the “classic”
endothelial pro-angiogenesis kinases Erk1/2, p38 and Akt, since the responses to SG-209 were
all abolished by each of the respective kinase inhibitors.
This work allows us to firmly conclude that:
a) direct pharmacological modulation of KATP channels affects angiogenic responses in vitro and
in vivo, b) CNP is a bona fide angiogenic factor, as potent and efficient to mobilize endothelial
cells as VEGF, c) the angiogenic effects of CNP and VEGF depend on the activation of
endothelial KATP channels and specifically require the expression of the Kir6.1 pore-forming KATP
subunit, and finally d) KATP activation may be a common molecular mechanism that underpins
angiogenesis to a wide variety of endogenous vasoactive stimuli that includes H2S, VEGF and
CNP.
The therapeutic implications of this work are significant: Sulfonylurea-type KATP channel
inhibitors, with questionable selectivity for pancreatic β-cells, are widely used drugs to treat type
II diabetes, a disease characterized by arterial dysfunction and higher incidence of myocardial
and lower limb ischemia. The outcome of cardiac ischemia in diabetic patients is worse if they
have been treated with sulfonylureas, indicating some, until now unresolved, deleterious
cardiovascular activity of this class of compounds. The present demonstration that endothelial
KATP channel activation is a common pro-angiogenic mechanism, may in part explain this
unfavorable outcome of sulfonylurea treatment in diabetics. Furthermore, it raises the need to
design new molecules which, while inhibiting the pancreatic KATP channels, should spare the
endothelial KATP channels and the ensuing angiogenesis, thus exhibiting increased therapeutic
benefit.
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