Human solCD39 inhibits injury-induced development of neointimal hyperplasia

Abstract

Blood platelets provide the initial response to vascular endothelial injury, becoming activated as they adhere to the injured site. Activated platelets recruit leukocytes, and initiate proliferation and migration of vascular smooth muscle cells (SMC) within the injured vessel wall, leading to development of neointimal hyperplasia. Endothelial CD39/NTPDase1 and recombinant solCD39 rapidly metabolise nucleotides, including stimulatory ADP released from activated platelets, thereby suppressing additional platelet reactivity. Using a murine model of vascular endothelial injury, we investigated whether circulating human solCD39 could reduce platelet activation and accumulation, thus abating leukocyte infiltration and neointimal formation following vascular damage. Intraperitoneally-administered solCD39 ADPase activity in plasma peaked 1 hour (h) post-injection, with an elimination half-life of 43 h. Accordingly, mice were administered solCD39 or saline 1 h prior to vessel injury, then either sacrificed 24 h post-injury or treated with solCD39 or saline (three times weekly) for an additional 18 days. Twenty-four hours post-injury, solCD39-treated mice displayed a reduction in platelet activation and recruitment, P-selectin expression, and leukocyte accumulation in the arterial lumen. Furthermore, repeated administration of solCD39 modulated the late stage of vascular injury by suppressing leukocyte deposition, macrophage infiltration and smooth muscle cell (SMC) proliferation/migration, resulting in abrogation of neointimal thickening. In contrast, injured femoral arteries of saline-injected mice exhibited massive platelet thrombus formation, marked P-selectin expression, and leukocyte infiltration. Pronounced neointimal growth with macrophage and SMC accretion was also observed (intimal-to-medial area ratio 1.56 +/- 0.34 at 19 days). Thus, systemic administration of solCD39 profoundly affects injury-induced cellular responses, minimising platelet deposition and leukocyte recruitment, and suppressing neointimal hyperplasia.

Fig. 1. Pharmacokinetics of IP-administered human solCD39 in mice

The activity of solCD39 in murine plasma was measured using our ADPase radio-TLC assay, as described in “Materials and Methods”. The enzymatic activity of each sample was expressed as picomoles ADP metabolized per min per μl plasma. Each data point represents the mean ADPase activity (± SD) in plasma samples from 2-4 mice; values for each plasma sample were obtained from 2-3 separate assays. The dashed line, which signifies the ADPase activity of 17.1 μg/ml solCD39 in murine plasma, is shown as a comparison. SolCD39 in vivo half-life was determined using two phase exponential decay nonlinear regression analyses.

Fig. 2. Immunohistochemical analysis of mouse femoral arteries 24 hr after wire-induced endothelial injury

Immunohistochemical analysis was performed on representative cross-sections of femoral arteries of mice injected with human solCD39 or saline to identify specific cell types or adhesion molecule expression. Sections were stained with hematoxylin-eosin (A, B; 40x) as well as visualized under bright-field illumination (C, D; 20x). Sections were also immunofluorescently stained for platelets (E, F; anti-CD41 antibody, 20x), adhesion molecule P-selectin (G, H; anti-CD62P antibody, 20x), leukocytes (I, J; anti-CD45 antibody, 20x), and macrophages (K, L; anti-Mac-3 antibody, 20x). Images of the corresponding IgG isotype control to each specific antibody are labeled with lower case letters (e–l). Bar = 50 μm.

Fig. 3. Immunohistochemical analysis of mouse femoral arteries 24 hr after wire-induced endothelial injury

Representative cross-sections of femoral arteries of mice injected with human solCD39 or saline were visualized under bright-field illumination (A, B; 20x) as well as immunohistochemically probed for adhesion molecule ICAM-1 (C, D; anti-CD54 antibody, 40x) and for SMC (K, L; anti-actin, α-SM antibody, 40x), followed by colorimetric staining. In addition, sections were immunofluorescently stained for adhesion molecule VCAM-1 (E, F; anti-CD106 antibody, 20x) and for EC (G, H; anti-CD31 (PECAM-1) and I, J; anti-panendothelial cell antigen antibodies, 20x). Arrowheads indicate ICAM-1 (C) and VCAM-1 (E) expression on the luminal surface of the injured vessel in saline-treated control mice. Images of the corresponding IgG isotype control to each specific antibody are labeled with lower case letters (c-l). Bar = 50 μm.

Fig. 4. Immunohistochemical analysis of mouse femoral arteries 19 days after wire-induced endothelial injury

Immunohistochemical analysis was performed on representative cross-sections of femoral arteries of mice injected with human solCD39 or saline to identify specific cell types. Sections were stained with hematoxylin-eosin (A, B; 40x) as well as visualized under bright-field illumination (C, D; 20x). Sections were also immunofluorescently stained for leukocytes (E, F; anti-CD45 antibody, 20x), and EC (G, H; anti-CD31 (PECAM-1) and I, J; anti-panendothelial cell antigen antibodies, 20x). Images of the corresponding IgG isotype control to each specific antibody are labeled with lower case letters (e-j). Bar = 50 μm.

Fig. 5. Immunohistochemical analysis of mouse femoral arteries 19 days after wire-induced endothelial injury

Representative cross-sections of femoral arteries of mice injected with human solCD39 or saline were visualized under bright-field illumination (A, B; 20x). Sections were also immunohistochemically probed for adhesion molecule ICAM-1 (C, D; anti-CD54 antibody, 40x) and for SMC (I, J; anti-actin, α-SM antibody, 40x), followed by colorimetric staining. In addition, sections were immunofluorescently stained for adhesion molecule VCAM-1 (E, F; anti-CD106 antibody, 20x) and for macrophages (G, H; anti-Mac-3 antibody, 20x). Images of the corresponding IgG isotype control to each specific antibody are labeled with lower case letters (c–j). Bar = 50 μm.