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. 2021 Jun 5:900:174038.
doi: 10.1016/j.ejphar.2021.174038. Epub 2021 Mar 16.

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage

Peter J Morone  1 , Wei Yan  2 , Jamie Adcock  3 , Padmini Komalavilas  3 , J Mocco  4 , Reid C Thompson  1 , Colleen Brophy  3 , Joyce Cheung-Flynn  5
Affiliations

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage

Peter J Morone et al. Eur J Pharmacol. .

Abstract

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.

Keywords: Phosphopeptide mimetics; SAH; Vasospasm.

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Figures

Fig. 1.
Fig. 1.. Vasorelaxation is associated with phosphorylation of the VASP protein in rat aorta.
RASM rings were suspended in a muscle bath and left untreated (Basal) or pre-contracted with phenylephrine (PE) followed by sodium nitroprusside (SNP) or nimodipine (NIMO), snap-frozen and protein extracted for immunoblotting experiments (A). % relaxation was determined as a %the maximal PE-induced contraction. Levels of VASP S239 phosphorylation were calculated as a ratio of the phosphorylated protein to total protein and relative levels were calculated by comparing to basal tissues (B). Representative immunoblots shown (C). n=4; *P<0.05; n.s., statistically non-significant.
Fig. 2.
Fig. 2.. Concurrent decrease in cerebral perfusion with downregulation of NO signaling pathway elements after SAH in rats.
Modified endovascular perforation rat model of SAH (A). Ventral view of brains showing hemorrhage (B). Representative blots (C) and quantitation (D) showing decreased levels of GC. PKG, phospho-VASP S239 (ratio of the phosphorylated protein to total protein levels), and total VASP in cerebral vessels 48 h after hemorrhage. SHAM, n=5; SAH, n=9; *P<0.05 in unpaired t- test.
Fig. 3.
Fig. 3.. Vasorelaxation effects of VASP polypeptides (VP) in rat aorta.
RASM rings were suspended in a muscle bath pre-contracted with phenylephrine, followed by treatment with sodium nitroprusside (SNP; 10−9 and 10−8M) or escalating doses of the VASP peptide indicated (x10−3M). VP3 at a dose of 0.25×10−3M generated similar relaxation to that of 10−9M SNP and has the greatest vasorelaxing property among the VP peptides. ScrVP had no vasorelaxing property. % relaxation was determined as a change to the maximal phenylephrine-induced contraction. n=4-7.
Fig. 4.
Fig. 4.. Synergistic vasorelaxation to nimodipine and VP3.
Agonist-precontracted RASM were treated with calcium channel blocker nimodipine (NIMO; 10−7M); VP3 (0.25 X 10−3M); nimodipine followed by VP3 [NIMO/VP3]; VP3 followed by nimodipine [VP3/NIMO]; or simultaneous addition of nimodipine plus VP3 [NIMO+VP3]. Representative muscle bath tracings of force generated in response to phenylephrine, nimodipine and VP3. Dashed lines indicate addition of drugs to RASM (A). % relaxation was determined as a change to the maximal phenylephrine-induced contraction (n = 3-4, *P < 0.05) (B). Endothelin (ET)-precontracted RASM were treated with nimodipine (NIMO, 10−8M); VP3 (0.25×10−3M); nimodipine and VP3 (NIMO/VP3 and VP3/NIMO) (C).
Fig. 5.
Fig. 5.. Effects of VP3 on actin cytoskeleton dynamics in intact RASM.
PE(A)- or ET (B)-precontracted RASM was treated with VP3 (1 mM) and levels of globular (G-) and F-actin were measured. Top: Representative immunoblots. Bottom, quantitative analysis of % F-actin showed reduction in VP3-treated PE- or ET-precontracted RASM compared to untreated tissue, indicating depolymerization of F-actin by VP3 treatment. n=4; *P<0.05 in paired t-test.
Fig. 6.
Fig. 6.. VASP polypeptide (VP3) relaxed serotonin- and endothelin-precontracted porcine basilar artery.
Basilar artery segments (4mm) were suspended in a muscle bath pre-contracted with serotonin (5-HT; A) or endothelin (ET; B) followed by treatment with SNP (10−8 M), nimodipine (NIMO, 10−8M), or escalating doses of VP3 or scrVP indicated (x10−3 M). % relaxation was determined as a change to the maximal agonist-induced contraction. n=2-6.
Fig. 7.
Fig. 7.. Model of mechanism of SAH-induced vasospasm.
A. In the setting of SAH, NO, guanylate cyclase (GC), and cGMP-dependent protein kinase (PKG) are downregulated in the cerebral vasculature (A). Phosphorylation of PKG substrate VASP is decreased and hence actin-myosin interactions maintain a contractile state. Nimodipine and VASP peptide restore normal vasorelaxation by decreasing intracellular calcium channels (inhibiting thick filament myosin) and bypassing the NO-cGMP-PKG signaling pathway (leading to actin depolymerization; B).
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