Alpha4beta1 integrin (VLA-4) blockade attenuates both early and late leukocyte recruitment and neointimal growth following carotid injury in apolipoprotein E (-/-) mice.

BACKGROUND: The alpha(4)beta(1) 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.

The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells.

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes.

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-/- 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 apoE-/- 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-/-, 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.

Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice.

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-/- 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 microg P-selectin mAb RB 40.34 and by 55% by a single injection of 100 microg PSGL-1 mAb 4RA10 (P< or =0.005). 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-/- mice.