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.

The trans-membrane receptor CD47 is considered a central relay of thrombospondin 1 (TSP-1) mediated responses in human cells/tissues, and ubiquitously serves as a marker of self on hematopoietic cells. Preliminary studies of TSP-1 binding with soluble human CD-47, serving as decoy recombinant protein (rhCD47p), demonstrated molar ratio ≥ 3 folds of rhCD47p abolished all TSP1-vascular CD47 receptor signaling. Accordingly, we attached multiple rh-CD47p ligands onto liposomes (NanoLip), as clinical prototype therapy. Novel rh-CD47p-NanoLip can avoid blood mononuclear cells, while binding many circulating TSP1 molecules, thus eliminating excessive plasma TSP-1, often implicated in pulmonary arterial hypertension (PAH) and ischemia-reperfusion injuries. Direct coupling of rh-CD47p, 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 analysis. Pharmaceutically, rh-CD47p-NanoLip formulation (av. size = 86±6.0nm) engaged 67±11μg/mL of protein, while retaining 86% of native rh-CD47p activity. Western blots and TSP-1-ELISA revealed dosimetric binding of tirmeric human TSP-1: rh-CD47p-NanoLip. Activated macrophages showed substantial decrease in phagocytosis of labeled rh-CD47p-NanoLip, compared to plain- and IgG-NanoLip controls (5- and 4- folds, respectively), indicating self-marker function. Conversely, binding of exogenous TSP-1 caused significant upturn of internalized rh-CD47p-NanoLip (p<0.05, n=4), due to TSP-1/nano-complex formation. Therapeutically, in isolated mouse thoracic aorta model, mimicking high plasma levels of TSP1 in PAH patients, titrated doses of CD47p-NanoLip neutralized TSP1-impaired vasodilation, back to the same level as controls (p<0.001, vs. TSP-1, n=4). This is the first report of its kind about CD47-based immunomodulatory nano-therapy specifically targeting TSP-1. The extracellular domains of native CD47 receptors of TSP1 were successfully “nano-modified” into a candidate systemic decoy pharmaceutical, rh-CD47p-NanoLip, to scavenge and eliminate pathologically elevated TSP-1 plasma levels, to abrogate TSP-1-associated vascular complications.

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 Information The Cardiovascular Medical Research and Education Fund (CMREF), and Atrial Fibrillation Strategically Focused Research Network (AHA‐SFRN)...

Inflammatory response, via pro‐inflammatory (M1) infiltrating and resident macrophages, plays an important role in the development of various inflammatory cardiac pathologies, namely ischemic heart disease, myocardial infarction, and heart failure. Conversely, reparative (M2) macrophages have a vital inflammation resolution role following ischemia/reperfusion injury. Instead of mere inactivation of pathogenic M1 macrophages, as a general treatment approach, we better designed to actively polarizing them into reparative M2 phenotype ‐ via macrophage‐specific red blood cell (RBC) soluble receptor conjugated onto nanocarriers (NC). Liposomes (Lip) targeted via soluble CD47 immuno‐modulatory ligand of macrophage, can mitigate cardiac inflammation. Especially, when CD47‐Lip nano‐system further presents phosphatidylserine (PS)‐apoptotic cell signal ‐ for additional M2‐macrophage polarization. The RBC’s “self‐marker” signal, CD47, acts as a phagocytosis switch, through interactions with immuno‐receptor signal regulatory protein (SIRPα) and Thrombospondin‐1 (TSP1) on macrophages, to suppress and evade phagocytosis. Hence, we chemically coupled multiple recombinant human CD47 proteins onto anionic PS‐containing Lip surface, to produce hematopoietic and apoptotic cell dual‐mimicking CD47‐PSLip nano‐platform. Pharmaceutically, 67±11μg/mL of protein were covalently attached on CD47‐PSLip, which subsequently reduced overall anionic surface potential by approx. 9.3mV, while retaining at least 85% of original CD47 protein activity. Western blots and ELISA confirmed dose‐dependent binding of human TSP1: CD47‐PSLip. In addition, classically TNF‐α/LPS‐activated macrophages, of both human and murine origins, showed a substantial decrease in phagocytosis of labeled CD47‐PSLip, compared to unconjugated neutral or PSLip, and IgG‐PSLip controls (p<0.01, n=3–4), indicating their active self‐marker signal. Furthermore, anionic CD47‐PSLip, as a dual reprogramming strategy, induced substantial M2 polarization/reparative phenotype, evident through high levels of secreted anti‐inflammatory cytokines (IL‐10 and TGF‐β), concomitant with downregulation of pro‐inflammatory markers (IL‐12 and TNF‐α), following 24hr‐treatment of M1‐polarized human monocyte macrophages. In vivo RBC‐mimicry was imaged in C57 mice, using near infrared‐labeled Lip (av. 70–90nm), through minimal accumulation of CD47‐PSLip in liver and spleen (p<0.001 vs IgG‐Lip control), 48hr‐post intravenous injection. Non‐compartmental pharmacokinetic model analysis revealed marked increase in their mean residence plasma half‐life (MRT≈41.7hr), in comparison to standard Stealth™ Lip preparation (~15.6hr). This is the first report about CD47/PS presenting NC, selectively targeting pathogenic macrophage infiltration underlying inflammatory ischemic cardiac disorders. Our pre‐clinical data illustrate the potential of CD47‐PSLip as a dual reprogramming nanomedicine, to induce M2 polarization/reparative macrophage phenotype, as a novel therapeutic strategy to ameliorate exacerbated cardia...

Hemostatic abnormalities mainly decreased blood flow and increased stasis, and platelet hyper-aggregation increase thrombotic risk subsequent atrial fibrillation (AF) episodes. Excess elevation of extracellular glycoprotein thrombospondin 1 (TSP1) in circulation has emerged to predict prognosis of ischemic stroke. Moreover, TSP1 is found strongly associated with platelet hyper-aggregability and impaired nitric oxide response, through its cognate receptor CD47, thus exacerbating ischemic progression in AF patients. TSP1 arises as a prospective target of our candidate medicine. Our work provides “proof-of-concept” of a novel pharmacological strategy, relying on soluble recombinant human CD47 peptide (rh-CD47p) to serve as a decoy receptor to specifically bind TSP1, and consequently neutralize TSP1-impaired vasorelaxation. Our pilot studies were initially conducted with isolated mouse thoracic aorta. A pre-treatment with rh-CD47p (added 15 min prior to TSP1 incubation) showed a significant amelioration of TSP1-impaired endothelium-dependent vasodilation ( p <0.0001, rh-CD47p+TSP1 vs. TSP1, n=6), indicating a prophylactic effect by rh-CD47p against vasoconstriction enhanced by excess TSP1. A post-treatment set-up, where TSP1 incubation was started 15 min prior to rh-CD47p addition to mimic pre-existing high level of TSP1 in ischemic stroke, exhibited a marked reversal of TSP1-inhibited vasodilation back to the same level as controls ( p <0.0001, rh-CD47p+TSP1 vs. TSP1, n=4). Dose titration of rh-CD47p in molar ratios to TSP1 in both set-ups identified the dose range of rh-CD47p for prevention/recovery from TSP1-induced vascular dysfunction. Binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western Blot, and further quantified with a modified ELISA assay. A linear correlation was found between % of rh-CD47p bound to TSP1 and dose of rh-CD47p added (r 2 =0.8). Current results established the ground of rh-CD47p as a unique pharmacologic moiety for drug development to limit vasoconstriction and improve vasodilation against TSP1 in excess, thus showing great potential for proposed novel decoy CD47-coated formulation to abrogate/mitigate TSP1-associated cardiovascular complications in AF patients.