Evaluation of Decoy CD47‐Nanomedicine as Novel Therapeutic Strategy for Idiopathic Pulmonary Arterial Hypertension Specifically Abrogating Thrombospondin 1‐Induced Vascular Dysfunction

Abstract

Thrombospondin‐1 (TSP1), a multifunctional matrix protein, influences a range of functions such as platelet activation and angiogenesis. Its innate trans‐membrane receptor, the integrin‐associated protein CD47, is considered a central relay of TSP1‐mediated responses, along with its role as a marker of self on hematopoietic cells, inhibiting phagocytosis.Earlier, we have established dosimetric TSP1 binding with soluble human CD47, serving as decoy recombinant protein (rhCD47p), which abolished TSP1 vascular CD47 receptor communications, starting at 3x‐molar excess. Current pharmaceutical design, successfully cross‐linking multiple rhCD47p ligands onto liposomes (NanoLip), avoids mononuclear cells in blood, while specifically binding many circulating TSP1 molecules. Thus, our prototype therapy, rhCD47p‐NanoLip, can eliminate excessive plasma TSP1, often implicated in pulmonary arterial hypertension (PAH) and ischemia‐reperfusion injuries.Direct bio‐conjugation of rhCD47p, onto NanoLip surface was achieved using a sulfo‐NHS‐reaction between amino groups on rh‐CD47p and corbodiimide‐activated NanoLip, followed by column purification, and physico‐chemical characterization. Prototype anti‐TSP1 nano‐formulation (av. particle size = 86±6.0nm) had 67±11μg/mL of protein and retained 86% of native rhCD47p activity. The specific binding of rhCD47p‐NanoLip to human TSP1, was confirmed qualitatively via western blots, and quantitatively using modified TSP1‐based ELISA, showing linear dose‐dependent binding of monomeric human TSP1: rhCD47p‐NanoLip across the tested 1:2–1:6 molar ratio range.In vitro, activated mouse and human blood macrophages showed substantial decrease in phagocytosis of rhCD47p‐NanoLip, compared to plain‐ and IgG‐NanoLip controls (5‐ and 4‐folds, respectively), indicating active self‐marker function. In contrast, binding of exogenous human TSP1 restored phagocytosis of rhCD47p‐NanoLip (p<0.05, n=4), due to TSP1‐scavenging nano‐complex, mediating immuno‐mediated elimination. Following IV administration in mice, our rhCD47p‐NanoLip demonstrated prolonged mean circulation time (Cal.≥46±5hr, vs. 15±3hr for commercial PEG‐NanoLip), mimicking circulating blood cells. Pharmacologically, in isolated mouse thoracic aorta model, mimicking pathological high TSP1‐plasma levels in PAH patients, rhCD47p‐NanoLip treatment completely neutralized TSP1‐inhibited vasodilation, back to the same level as controls (p<0.001, vs. TSP1, n=4).Our pre‐clinical data represent successful early stage development of a unique CD47‐based immuno‐modulatory nano‐therapy, specifically targeting TSP1. The extracellular domains of native CD47 receptors of TSP1 were successfully “nano‐modified” into a systemic decoy pharmaceutical prototype, rh‐CD47p‐NanoLip, capable of ameliorating pathologically elevated TSP1 plasma levels, to abrogate TSP1‐induced cardiovascular disorders.Support or Funding InformationThe Cardiovascular Medical Research and Education Fund (CMREF), and Atrial Fibrillation Strategically Focused Research Network (AHA‐SFRN) Award.