Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves  2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia

Abstract

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of b 2 -adrenergic receptor (b 2 AR) signaling, leading to reduced revascularization. Indeed, in vivo b 2 AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G proteincoupled receptor kinase 2 (GRK2) is a key regulator of bAR signaling, and b adrenergic receptor kinase C-terminal peptide (bARKct), a peptide inhibitor of GRK2, has been shown to prevent bAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of b 2 AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated bARKct gene transfer in an experimental model of HI and its effects on bAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant bAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the bARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and bAR density in the ischemic muscle, compared with control groups. The effect of bARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the bARKct fenoterol, a b 2 AR-agonist, induced enhanced b 2 AR proangiogenic signaling and increased EC function. Our results suggest that bARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced b 2 AR downregulation.

Sphingosine Kinases and Sphingosine 1-Phosphate Receptors: Signaling and Actions in the Cardiovascular System

“…Importantly, the activation of such signaling pathway leads to the reciprocal downregulation of β 1 AR and S1PR 1 in cardiac myocytes, leading to worse remodeling and progression toward HF ( Cannavo et al, 2013b ). This study further supported the idea that blockade of GRK2 is a valid strategy to prevent HF development and progression, but also demonstrated the cardioprotective role of S1PR 1 ( Cannavo et al, 2013a , 2016a , b ; Cannavo and Koch, 2017a ). In line with these data, we recently reported that activation of S1PR 1 in the heart is also modulated by the β 3 AR ( Cannavo et al, 2017 ).…”

Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

“…Cell migration was assessed by wound-healing scratch assay as previously described 59 . After isolation, cardiac fibroblasts (∼3 × 10 5 ) were plated in 12-well tissue culture plates.…”

Targeting β3-Adrenergic Receptors in the Heart: Selective Agonism and β-Blockade

“…In fact, activation of Akt and eNOS, and the consequent secretion of NO, has been proven to directly stimulate endothelial cell function and promote the neoangiogenesis. 50 Further, as discussed above, the eNOS-mediated generation of NO is also responsible for cGMP and PKG activation, thus directly conferring beneficial effects on the myocardium. 51 …”

“… 56 , 57 Of note, enhancement of neoangiogenesis in the failing heart is considered one of the mechanisms of protection activated by this class of drugs. 58 In this context, nebivolol through β 3 AR activation increases the generation of NO, a key mediator of endothelial function, 50 enhancing endothelial proliferation and increasing vasodilation. 56 , 57 …”

“… 43 , 44 In fact, selective β 3 AR agonism has been proven to confer protection in the failing heart through a specific cGMP/NO signaling pathway. 26 A particular role for NO has been associated with the enhancement of endothelial cell proliferation, 50 and within failing myocardium it can lead to a beneficial effect on cardiac function and remodeling. 57 , 58 As described above, β 3 ARs are expressed not only in cardiomyocytes but also in endothelial cells, thus supporting the idea that this receptor can also promote proangiogenic mechanisms.…”