Stimulation of soluble guanylate cyclase activity with riociguat promotes angiogenesis and improves neovascularization after limb ischemia

Abstract

Background and aimsThe NO-cGMP pathway is essential for angiogenesis, vasculogenesis and post-natal neovascularization. The key enzyme responsible for the synthesis of cGMP following binding of NO is soluble guanylate cyclase (sGC). Riociguat is the first member of a novel class of compounds known as sGC stimulators. We tested the hypothesis that stimulation of sGC with riociguat might improve neovascularization in response to ischemia. MethodsIn vitro, the angiogenic effect of riociguat was tested in human umbilical vein endothelial cells (HUVECs). In vivo, neovascularization was investigated in a mouse model of limb ischemia. C57Bl/6 mice were treated by gavage with 3 mg/kg/day of riociguat for a total of 28 days. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. ResultsIn a matrigel assay in vitro, riociguat dose-dependently stimulates tubule formation in HUVECs. Cell migration (scratch assay) is also increased in HUVECs treated with riociguat. At the molecular level, riociguat treatment leads to rapid activation of the p44/p42 MAP kinase pathway in HUVECs. Inhibition of protein kinase G (PKG) activity supresses both p44/p42 MAP kinase activation and angiogenesis in HUVECs treated with riociguat. In vivo, treatment with riociguat improves blood flow recovery after ischemia (Laser Doppler imaging), and increases capillary density in ischemic muscles (CD31 immunostaining). Clinically, this is associated with a significant decrease of ambulatory impairment and ischemic damages. Interestingly, mice treated with riociguat also show a 94% increase in the number of bone marrow-derived pro-angiogenic cells (PACs) compared to control mice. Moreover, riociguat treatment is associated with a significant improvement of PAC functions including migratory capacity, adhesion to an endothelial monolayer, and integration into endothelial tubular networks. ConclusionsThe sGC stimulator riociguat promotes angiogenesis and improves neovascularization after ischemia. The mechanism involves PKG-dependent activation of p44/p42 MAP kinase pathway, together with an improvement of PAC number and functions. sGC stimulation could constitute a novel therapeutic strategy to reduce tissue ischemia in patients with severe atherosclerotic diseases.