Victor DeAngelis

Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ∼2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Enzyme replacement therapy for Farber disease: Proof-of-concept studies in cells and mice

A series of studies were carried out in Farber disease (OMIM #228000) cells and mice to evaluate the feasibility of enzyme replacement therapy (ERT) for this disorder. Media from Chinese hamster ovary (CHO) cells overexpressing human recombinant acid ceramidase (rhAC) was used to treat fibroblasts from a Farber disease patient, leading to significantly reduced ceramide. We also found that chondrocytes from Farber disease mice had a markedly abnormal chondrogenic phenotype, and this was corrected by rhAC as well. Acute dosing of rhAC in Farber mice confirmed the enzymes bioactivity in vivo, and showed that it could be safely administered at doses up to 50 mg/kg. These studies also revealed little or no re-accumulation of ceramide in tissues for at least 7 days after enzyme administration. Once weekly administration of rhAC moderately improved survival of the mice, which could be enhanced by starting enzyme administration at an earlier age (3 days vs. 3 weeks). Repeat administration of the enzyme also led to normalization of spleen size, significantly reduced plasma levels of monocyte chemoattractant protein 1 (MCP-1), reduced infiltration of macrophages into liver and spleen, and significantly reduced ceramide and sphingosine in tissues. Overall, we conclude that ERT should be further developed for this debilitating and life-threatening disorder.