Title: Pyrintegrin Induced Adipogenesis
Description: Adipose tissue is traditionally regarded as a source of energy storage and cushion for skeletal functions. Soft tissue injuries take place in war and peace time, as a result of tumor resection such as breast cancer, and in rare disorders such as lipoatrophy. Adipose tissue reconstruction is one of the key challenges in medicine. Here we report the robust differentiation of human adipose derived stem cells using a small molecule into adipocytes. This thesis describes the biological effects and actions of novel unknown small-molecule inhibitor of BMP signaling--Pyrintegrin, which was previously found to promote human embryonic stem cells survival. The overall objective of this thesis is to test the hypothesis that this novel drug, Pyrintegrin treatment will induce, promote and accelerate adipogenic differentiation of stem cells in vitro and in vivo. We found that Pyrintegrin promotes the adipogenesis-dependent transcriptional changes of multiple gene products involved in the adipogenic process, including peroxisome proliferator-activated receptor (PPARgamma), CCAAT/enhancer-binding protein alpha; (C/EBPalpha), adiponectin and leptin secretion, and total triglyceride secretion.

When transplanted into mice, Pyrintegrin treated adipose cells/progenitors gave rise to ectopic fat pads with the morphological and functional characteristics of white adipose tissue. This was further confirmed by higher expression of human PPARgamma gene in Pyrintegrin treated cells group than any other group. We further tested the presence of human nuclear staining that confirmed the presence of human cells in all the transplanted groups. However, the number of positive human cells was substantially low in all the groups, which was likely due to transplanted cell death because of lack of vascularization. Hence, we further tested the Pyrintegrin adsorbed scaffolds capacity to regenerate better soft tissue compared to Ptn-free scaffold. We found that Ptn adsorbed scaffolds was positive for adipocytes as evident by positive Oil Red O staining. We further investigated the signaling mechanism of Pyrintegrin. Using a human PPARgamma reporter assay system, based on non-human mammalian cells engineered to express human PPARgamma protein, we found that Pyrintegrin is not a PPARgamma agonist as witnessed by lack of any luciferase activity.

In contrast, Rosiglitazone, a known PPARgamma agonist demonstrated significant amount of luciferase activity in these reporter cells. We also found that Pyrintegrin selectively inhibits the BMP pathway and thus blocks BMP-mediated SMAD1/5 phosphorylation, target gene transcription and osteogenic differentiation. In vitro studies showed that Pyrintegrin inhibited the differentiation of stem cells into putative osteoblasts, as evident by decreased alizarin red staining. A striking finding was that Pyrintegrin up regulated markers of adipogenesis and stimulated lipid droplets accumulation in stem cells undergoing osteoblastic differentiation in vitro. This came at the expense of down regulating markers of osteogenesis and osteoblastic differentiation of stem cells, as compared to the cells undergoing osteogenic differentiation in the absence of the drug treatment. These findings show that the novel small-molecule Pyrintegrin is a potent promoter of adipogenesis and thus may have therapeutic potential for soft tissue reconstruction.