Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 microg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene-transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.

Atherosclerosis in aged mice over-expressing the reverse cholesterol transport genes

We determined whether over-expression of one of the three genes involved in reverse cholesterol transport, apolipoprotein (apo) AI, lecithin-cholesterol acyl transferase (LCAT) and cholesteryl ester transfer protein (CETP), or of their combinations influenced the development of diet-induced atherosclerosis. Eight genotypic groups of mice were studied (AI, LCAT, CETP, LCAT/AI, CETP/AI, LCAT/CETP, LCAT/AI/CETP, and non-transgenic) after four months on an atherogenic diet. The extent of atherosclerosis was assessed by morphometric analysis of lipid-stained areas in the aortic roots. The relative influence (R²) of genotype, sex, total cholesterol, and its main sub-fraction levels on atherosclerotic lesion size was determined by multiple linear regression analysis. Whereas apo AI (R² = 0.22, P < 0.001) and CETP (R² = 0.13, P < 0.01) expression reduced lesion size, the LCAT (R² = 0.16, P < 0.005) and LCAT/AI (R² = 0.13, P < 0.003) genotypes had the opposite effect. Logistic regression analysis revealed that the risk of developing atherosclerotic lesions greater than the 50th percentile was 4.3-fold lower for the apo AI transgenic mice than for non-transgenic mice, and was 3.0-fold lower for male than for female mice. These results show that apo AI overexpression decreased the risk of developing large atherosclerotic lesions but was not sufficient to reduce the atherogenic effect of LCAT when both transgenes were co-expressed. On the other hand, CETP expression was sufficient to eliminate the deleterious effect of LCAT and LCAT/AI overexpression. Therefore, increasing each step of the reverse cholesterol transport per se does not necessarily imply protection against atherosclerosis while CETP expression can change specific athero genic scenarios.