Evaluation of therapeutic effects of FAK inhibition in murine models of atherosclerosis

Objective: Therapeutic effects of focal adhesion kinase (FAK) inhibition using a small molecule inhibitor was evaluated in apolipoprotein E (apoE) knockout (KO) and low-density lipoprotein receptor (LDLr) KO mouse atherosclerosis models. Results: The prevention trial consisted of 8-week concurrent treatment with a high fat (HF) / high cholesterol (HC) diet. The intervention trial consisted of 6- and 8-week treatment after 6- and 8-week pre-loading of a HF/HC diet in apoE KO and LDLr KO mice, respectively. The inhibitor was admixed with a HF/HC diet and mice were given free access to the admixture. The FAK inhibitor exhibited marked inhibition against the development of the atherosclerosis in both of prevention and intervention trials at a dose of 0.03% without showing any remarkable toxic properties in biochemical examinations. These results indicated that FAK inhibition might be a possible candidate for novel therapeutic targets against atherosclerosis

Heparanase expression in B16 melanoma cells and peripheral blood neutrophils before and after extravasation detected by novel anti-mouse heparanase monoclonal antibodies.

Degradation of extracellular matrix is associated with extravasation of metastatic tumor cells and inflammatory cells. Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, is a key enzyme for the matrix degradation, yet its involvement in extravasation and invasion during pathological processes was not fully clarified in vivo. In the present study, we examined heparanase expression in mouse experimental models, lung metastasis of melanoma and skin infiltration of neutrophils. Sixteen novel monoclonal antibodies specific for mouse heparanase were established by enzyme-linked immunosorbent assay with a recombinant mouse proheparanase, immunocytochemical staining of B16F10 melanoma cells cultured in vitro, and immunoprecipitation of the lysate of heparanase transfectant cells. Heparanase expression in metastatic nodules of B16F10 melanoma cells and in neutrophils localized in the inflamed skin was immunohistochemically detected using a monoclonal antibody RIO-1 that recognized the C-terminus of mouse heparanase. Homogeneous and strong heparanase staining was observed in 46% of the lung micrometastases of B16F10 melanoma cells. The staining was intensely positive on the invasive front of larger established metastasis nodules, but it was weak or heterogeneous inside the nodules. Heparanase expression in skin-infiltrating neutrophils was examined after inducing local inflammation with croton oil. The monoclonal antibody stained a significant portion of neutrophils inside and along the blood vessels, whereas it did not stain dermal neutrophils located distant from the vasculatures. The present study strongly suggests that both melanoma cells and neutrophils transiently express heparanase before and during the invasive process in vivo

Heparanase expression in B16 melanoma cells and peripheral blood neutrophils before and after extravasation detected by novel anti-mouse heparanase monoclonal antibodies.

Degradation of extracellular matrix is associated with extravasation of metastatic tumor cells and inflammatory cells. Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, is a key enzyme for the matrix degradation, yet its involvement in extravasation and invasion during pathological processes was not fully clarified in vivo. In the present study, we examined heparanase expression in mouse experimental models, lung metastasis of melanoma and skin infiltration of neutrophils. Sixteen novel monoclonal antibodies specific for mouse heparanase were established by enzyme-linked immunosorbent assay with a recombinant mouse proheparanase, immunocytochemical staining of B16F10 melanoma cells cultured in vitro, and immunoprecipitation of the lysate of heparanase transfectant cells. Heparanase expression in metastatic nodules of B16F10 melanoma cells and in neutrophils localized in the inflamed skin was immunohistochemically detected using a monoclonal antibody RIO-1 that recognized the C-terminus of mouse heparanase. Homogeneous and strong heparanase staining was observed in 46% of the lung micrometastases of B16F10 melanoma cells. The staining was intensely positive on the invasive front of larger established metastasis nodules, but it was weak or heterogeneous inside the nodules. Heparanase expression in skin-infiltrating neutrophils was examined after inducing local inflammation with croton oil. The monoclonal antibody stained a significant portion of neutrophils inside and along the blood vessels, whereas it did not stain dermal neutrophils located distant from the vasculatures. The present study strongly suggests that both melanoma cells and neutrophils transiently express heparanase before and during the invasive process in vivo