Novel Pharmaceutical Strategy for Selective Abrogation of TSP1-Induced Vascular Dysfunction by Decoy Recombinant CD47 Soluble Receptor in Prophylaxis and Treatment Models.

Molly Yao,Tamer Elbayoumi,Samayita Ganguly,Jalicia Sturdivant,Aren Ebrahimi

doi:10.3390/biomedicines9060642

Molly Yao, Tamer Elbayoumi + Show 3 more

Open Access

https://doi.org/10.3390/biomedicines9060642

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Journal: Biomedicines	Publication Date: Jun 3, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Midwestern University

Abstract

Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our “proof-of-concept” pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.

Highlights

Under physiologic circumstances, homotrimeric glycoprotein thrombospondin 1(TSP1) is ubiquitously expressed in extracellular matrix and stored in the α-granules of platelets, remaining very low in local sites and circulation [1]
The binding of rh-CD47p to TSP1 was first verified via immunoblotting, in which the functionally smaller template, the monomeric form of TSP1, was coincubated with decoy receptor protein (DRP) in a cell-free setting to avoid a complicated protein reduction of the larger trimeric TSP1, trimeric form of TSP1 (tTSP1)
Plasma TSP1 in patients with pulmonary hypertension can be as high as 1114 ± 136 ng/mL compared to controls at 82 ± 16 ng/mL [39] and 571 ± 226 ng/mL in acute ischemic stroke (AIS) versus 146 ± 50 ng/mL in control [40]

Summary

Introduction

Homotrimeric glycoprotein thrombospondin 1(TSP1) is ubiquitously expressed in extracellular matrix and stored in the α-granules of platelets, remaining very low in local sites and circulation [1]. Substantial elevation in circulatory TSP1—up to a thousand-fold higher—has been recognized as a biomarker in multiple pathologic conditions, including coronary artery disease (CAD), pulmonary arterial hypertension (PAH), type II diabetes, and cardiac allografts [2,3,4]. Other than a risk indicator, excess plasma TSP1 has been heavily involved in pathologic modulation and progress of these conditions [5,6]. In PAH patients and diseased murine models, overexpressed TSP1 has been shown to promote oxidative stress in lung tissues and to contribute towards simultaneous vascular dysfunction and subsequent vasculopathy [7,8]. Upregulated TSP1 was revealed to crosslink cardiac and renal fibrosis in obesity and metabolic dysfunction and correlated with insulin resistance as well as adipose inflammation in nondiabetic subjects.

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