Saturday, September 19, 2009

B2A peptide

B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

"This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for sulfated glycosaminoglycan and immunochemical detection of collagen type II. In vivo, osteoarthritis was chemically induced in knees of adult rats by an injection of mono-iodoacetate (MIA) into the synovial space. Treatment was administered at 7- and 14 days after the MIA by injection into the synovial space of B2A or saline and terminated at 21 days, after which knee cartilage damage was determined and scored by histological analysis. In murine C3H10T1/2 micromass culture, B2A induced the expression of more than 11 genes associated with growth factors/receptors, transcription, and the extracellular matrix, including PDGF-AA. B2A also significantly increased the sulfated glycosaminoglycan and collagen of murine- and human micromass cultures. In the knee osteoarthritis model, B2A treatment enhanced cartilage repair compared to untreated knees as determined histologically by a decrease in damage indicators. These findings suggest that B2A induces stem cells chondrogenic differentiation in vitro and enhances cartilage repair in vivo."

"B2A is a positive BMP-2 receptor modulator whose design is modeled on BMP and incorporates three domains: a heparin-binding domain, a hydrophobic domain, and a receptor-targeted domain. B2A-type peptides bind to type I and type II receptors, and appear to have a selectivity for BMPR-Ib{upregulated by LSJL}. B2A stimulation of cells increases ERK1/2 activation and, in the presence of BMP-2, augments Smads and alkaline phosphatase activities."

"Compared to the controls, 11 genes were found up-regulated in the B2A-treated micromass samples and one gene was down-regulated. Genes that were up-regulated in B2A treatment micromasses can be categorized into three groups: (1) genes associated with growth factors and growth factor receptors, (2) genes associated with transcription factors and gene regulation, and (3) genes of matrix proteins. In the first group, Fgfr1{upregulated in LSJL} and Fgfr2 were found significantly increased in the B2A-treated samples. We also detected a moderate up-regulation of growth factors Fgf1. In the second gene group, the Smad1, Smad4, and Twist1 were up-regulated. Twist1, it should be noted, is required to promote and maintain chondrogenic differentiation in immature chondrocytes. Among the matrix genes of the third gene group, collagens Col11a1{upregulated in LSJL} and Col3a1{upregulated in LSJL} were up-regulated, with Col3a1 being the most pronounced. In contrast, Col5a1, a collagen that is not associated with cartilage, was noticeably down-regulated by B2A. Phex and Serpinh1, genes that regulate extracellular matrix stability, were found to be increased. The Bmp1 gene, encoding a metalloproteinase, which induces ectopic cartilage formation, was found significantly increased in the B2A-treated cells. The microarray-base expression analyses also revealed that Sox9 and Col2 were up-regulated but did not reach statistical significance. In individual quantitative real-time PCR, Sox9 and Col2 were found increased in the B2A-treated micromasses with 1.3 (p < 0.05) and 1.6 (p < 0.0001) fold of control, respectively."

"PDGF-AA has been found to be a specific PDGF isoform associated with chondrogenic phenotype. It is a potent mitogenic and chemotactic factor for mesenchymal stem cells and chondrocytes, and can also increase proteoglycan production in chondrocytes. The increased Pdgfa might serve as an autocrine loop to propel the chondrogenic differentiation"

"Bmp1, a member of the BMP1/TLD metalloproteinases, was found significantly up-regulated (∼500 fold). In addition to playing critical roles in regulating the formation of various extracellular matrixes, BMP1 also regulates activity of TGF-β family members. BMP1 activates BMP2/4 by cleaving their antagonist, chordin. In vivo, BMP1 is able to induce ectopic cartilage."

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