Background-Emerging data suggest that P-selectin, by controlling adhesion of white blood cells, may be important in limiting the response to vascular injury. Methods and Results-We tested the hypothesis that transient inhibition of P-selectin with either anti-P-selectin monoclonal antibody (mAb) or anti-P-selectin glycoprotein ligand-1 (PSGL-1) mAb would reduce neointima formation in the setting of carotid denudation injury in atherosclerosis-prone apolipoprotein E Ϫ/Ϫ mice. Neointima formation at 28 days was reduced significantly, by 50% or 80%, by a single injection on the day of injury of 100 or 200 g P-selectin mAb RB 40.34 and by 55% by a single injection of 100 g PSGL-1 mAb 4RA10 (PՅ0.005). In addition, there was a significant reduction in neointimal macrophage content.Conclusions-These findings demonstrate that transient P-selectin or PSGL-1 blockade at the time of arterial injury significantly limits plaque macrophage content and neointima formation in a dose-dependent manner after carotid denudation injury in apolipoprotein E Ϫ/Ϫ mice. Key Words: antibodies Ⅲ arteries Ⅲ atherosclerosis Ⅲ cell adhesion molecules Ⅲ inflammation M echanical injury stimulates a cascade of events involving the interaction of platelets, leukocytes, endothelial cells, and arterial wall cells in a healing response to maintain vascular integrity. These cellular interactions are not only important in normal healing but also have been shown to play pivotal roles both in the development of spontaneous atherosclerosis and in neointima formation after arterial injury. 1,2 The inflammatory cascade characterized by the expression of cellular adhesion molecules, such as P-selectin, plays a critical role in the early interactions of platelets, endothelial cells, and leukocytes that result in leukocyte rolling on the injured arterial wall, resulting in leukocyte recruitment to the injury site. P-selectin, found in storage granules of platelets and endothelial cells, initiates capture and rolling of circulating leukocytes at sites of inflammation and atherosclerosis. 3-5 P-selectin is rapidly mobilized and expressed on activated platelets, and immobilized platelets are capable of supporting leukocyte rolling. 6,7 This interaction is mediated to a great extent by binding of platelet P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. 5 This function of supporting leukocyte capture and rolling by platelets is particularly important in the setting of arterial injury, in which endothelial denudation exposes the subendothelial basement membrane, allowing platelets to adhere and serve as a surface that supports leukocyte rolling. 6 -8 In the mouse carotid wire injury model, reendothelialization occurs by 3 to 4 weeks, and the regenerating endothelium is known to express adhesion molecules, including P-selectin. 9,10 The strategy of P-selectin blockade to limit leukocyte recruitment in the setting of arterial injury is therefore expected to be effective both early after injury, when blockade at the level of the plate...

Background-Hyperglycemia (HG) and hyperinsulinemia (HI) may be factors enhancing the atherosclerotic complications of diabetes. We hypothesized that specific feeding of C57BL/6 apolipoprotein (apo) E Ϫ/Ϫ mice would alter their metabolic profiles and result in different degrees of neointima (NI) formation. We additionally hypothesized that an insulin-sensitizing agent (rosiglitazone) would prevent the development of type 2 diabetes and reduce neointima formation after carotid wire injury measured at 28 days. Methods and Results-Fasting glucose and insulin levels were elevated in the Western diet (WD) group, with a trend toward higher insulin levels and euglycemia in the fructose diet (FD)-fed mice. NI formation was exaggerated in the WD group compared with the FD or chow control groups. In the WD mice given rosiglitazone, glucose and insulin levels remained normal and NI formation was significantly reduced, as was NI macrophage content. Conclusions-These findings demonstrate that apoEϪ/Ϫ mice fed a WD develop type 2 diabetes with an exaggerated NI response to injury. FD mice maintain euglycemia but develop insulin resistance, with an intermediate degree of NI growth compared with chow diet controls. Rosiglitazone prevents the development of hyperglycemia and hyperinsulinemia and normalizes the insulin release profile in the apoE Ϫ/Ϫ , WD-fed mouse and significantly reduces NI formation by 65% after carotid wire injury while reducing macrophage infiltration. These data support the hypothesis that type 2 diabetes in the setting of elevated cholesterol accelerates the response to vascular injury and suggest that agents that improve insulin sensitivity may have therapeutic value in reducing restenosis in type 2 diabetes.

Background: The α₄β₁ integrin (VLA-4) supports rolling and firm adhesion of leukocytes to inflamed tissues via ligation of VCAM-1 or fibronectin expressed on the activated endothelium. We tested the hypothesis that VLA-4 mediates leukocyte recruitment and neointimal growth after arterial injury in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mouse. Methods: ApoE (–/–) mice fed a Western diet underwent air desiccation injury, and the expression patterns of VLA-4 and VCAM-1 were determined by immunohistochemistry (IHC). To determine the effect of targeted VLA-4 blockade on leukocyte recruitment and neointimal growth, ApoE (–/–) mice received an intraperitoneal injection of a VLA-4 neutralizing monoclonal antibody (PS/2) at the time of injury alone or over a prolonged administration course. Additional mice received an isotype control antibody. Results: IHC demonstrated a marked increase in VLA-4 expression 7 days following injury. Prolonged administration of PS/2 resulted in a 72% reduction (p < 0.02) in neointimal growth 28 days following injury. IHC revealed a marked 95% reduction in neutrophil recruitment at 7 days and a 48% reduction in macrophage recruitment 28 days following injury with prolonged PS/2 administration. Conclusions: Prolonged VLA-4 blockade reduces leukocyte recruitment and neointimal growth following air desiccation injury in ApoE (–/–) mice. These findings demonstrate an important role for VLA-4 in the response to arterial injury.

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet a-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes. P-selectin, a cell adhesion protein that functions in leukocyte recruitment early in the inflammatory response, is targeted to secretory granules in platelets and endothelial cells, and delivered to the cell surface upon regulated granule exocytosis in response to inflammatory stimuli (1). Cell surface Pselectin is rapidly internalized in endothelial cells and transfected cell lines (2,3), then undergoes selective sorting from early endosomes to late endosomes, resulting in rapid recycling through late endosomes and the trans-Golgi network (TGN) (4). Rapid internalization followed by endosomal sorting provides temporal regulation for cell surface expression of P-selectin following granule exocytosis, and also results in rapid delivery of P-selectin to lysosomes when it is not packaged in secretory granules (5-7). These three sorting activities are critical to regulating the adhesive function of this protein.The 35-amino acid cytoplasmic domain of P-selectin ( Figure 1) mediates each of the three sorting activities. Rapid internalization of P-selectin occurs in clathrin-coated pits (3). Although a conventional internalization signal was not identifiable by alanine-scanning mutagenesis, the cytoplasmic domain of P-selectin has recently been shown to associate with the medium (m) subunit of the AP-2 adaptor through an extended tyrosine-containing motif (8). Endosomal sorting of P-selectin is mediated by a sorting determinant that functions independently of the rapid internalization activity, and can be abolished by the single amino acid substi...