chiniofon and pyrintegrin

Focal adipose deficiency, such as lipoatrophy, lumpectomy or facial trauma, is a formidable challenge in reconstructive medicine, and yet scarcely investigated in experimental studies. Here, we report that Pyrintegrin (Ptn), a 2,4-disubstituted pyrimidine known to promote embryonic stem cells survival, is robustly adipogenic and induces postnatal adipose tissue formation in vivo of transplanted adipose stem/progenitor cells (ASCs) and recruited endogenous cells. In vitro, Ptn stimulated human adipose tissue derived ASCs to differentiate into lipid-laden adipocytes by upregulating peroxisome proliferator-activated receptor (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), with differentiated cells increasingly secreting adiponectin, leptin, glycerol and total triglycerides. Ptn-primed human ASCs seeded in 3D-bioprinted biomaterial scaffolds yielded newly formed adipose tissue that expressed human PPARγ, when transplanted into the dorsum of athymic mice. Remarkably, Ptn-adsorbed 3D scaffolds implanted in the inguinal fat pad had enhanced adipose tissue formation, suggesting Ptn's ability to induce in situ adipogenesis of endogenous cells. Ptn promoted adipogenesis by upregulating PPARγ and C/EBPα not only in adipogenesis induction medium, but also in chemically defined medium specifically for osteogenesis, and concurrently attenuated Runx2 and Osx via BMP-mediated SMAD1/5 phosphorylation. These findings suggest Ptn's novel role as an adipogenesis inducer with a therapeutic potential in soft tissue reconstruction and augmentation.

Topics: Adipocytes; Adipogenesis; Adipose Tissue; Animals; CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Cell Lineage; Cells, Cultured; Humans; Hydroxyquinolines; Male; Mice; Mice, Nude; PPAR gamma; Stem Cell Transplantation; Stem Cells; Sulfonamides; Tissue Scaffolds; Tissue Transplantation

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an β1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active β1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active β1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.

Topics: Actins; Albuminuria; Animals; Cell Movement; Epithelial Cells; Focal Adhesions; High-Throughput Screening Assays; Hydroxyquinolines; Integrin beta1; Kidney Diseases; Kidney Glomerulus; Lipopolysaccharides; Mice; Microscopy, Confocal; Phenotype; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Sulfonamides