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Browsing by Author "Wang, Congrong"
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Item Comparative Effectiveness of Structural versus Regulatory Protein Gene Transfer on Articular Chondrocyte Matrix Gene Expression(Sage, 2019-01) Shi, Shuiliang; Wang, Congrong; Chan, Albert; Kirmani, Kashif; Eckert, George J.; Trippel, Stephen B.; Orthopaedic Surgery, IU School of MedicineOBJECTIVE: The production of extracellular matrix is a necessary component of articular cartilage repair. Gene transfer is a promising method to improve matrix biosynthesis by articular chondrocytes. Gene transfer may employ transgenes encoding regulatory factors that stimulate the production of matrix proteins, or may employ transgenes that encode the proteins themselves. The objective of this study was to determine which of these 2 approaches would be the better choice for further development. We compared these 2 approaches using the transgenes encoding the structural matrix proteins, aggrecan or type II collagen, and the transgene encoding the anabolic factor, insulin-like growth factor I (IGF-I). METHODS: We transfected adult bovine articular chondrocytes with constructs encoding type II collagen, aggrecan, or IGF-I, and measured the expression of type II collagen ( COL2A1) and aggrecan ( ACAN) from their native genes and from their transgenes. RESULTS: IGF-I gene ( IGF1) transfer increased the expression of the native chondrocyte COL2A1 and ACAN genes 2.4 and 2.9 times control, respectively. COL2A1 gene transfer did not significantly increase COL2A1 transcripts, even when the transgene included the genomic COL2A1 regulatory sequences stimulated by chondrogenic growth factors. In contrast, ACAN gene transfer increased ACAN transcripts up to 3.4 times control levels. IGF1, but not ACAN, gene transfer increased aggrecan protein production. CONCLUSION: Taken together, these results suggest that the type II collagen and aggrecan production required for articular cartilage repair will be more effectively achieved by genes that encode anabolic regulatory factors than by genes that encode the matrix molecules themselves.Item Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding(Elsevier, 2018-03) Shi, Shuiliang; Kelly, Brian J.; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J.; Trippel, Stephen B.; Orthopaedic Surgery, School of MedicineBackground Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Methods Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Results Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. Conclusion The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. General significance These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.Item Hyaluronic acid–binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein(Springer, 2020-12) Shi, Shuiliang; Wang, Congrong; Trippel, Stephen B.; Orthopaedic Surgery, School of MedicineChondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair.Item Role of sox9 in growth factor regulation of articular chondrocytes(Wiley, 2015-07) Shi, Shuiliang; Wang, Congrong; Acton, Anthony J.; Eckert, George J.; Trippel, Stephen B.; Department of Orthopaedic Surgery, IU School of MedicineChondrogenic polypeptide growth factors influence articular chondrocyte functions that are required for articular cartilage repair. Sox9 is a transcription factor that regulates chondrogenesis, but its role in the growth factor regulation of chondrocyte proliferation and matrix synthesis is poorly understood. We tested the hypotheses that selected chondrogenic growth factors regulate sox9 gene expression and protein production by adult articular chondrocytes and that sox9 modulates the actions of these growth factors. To test these hypotheses, we delivered insulin-like growth factor-I (IGF-I), fibroblast growth factor-2 (FGF-2), bone morphogenetic protein-2 (BMP-2) and/or bone morphogenetic protein-7 (BMP-7), or their respective transgenes to adult bovine articular chondrocytes, and measured changes in sox9 gene expression and protein production. We then knocked down sox9 gene expression with sox9 siRNA, and measured changes in the expression of the genes encoding aggrecan and types I and II collagen, and in the production of glycosaminoglycan, collagen and DNA. We found that FGF-2 or the combination of IGF-I, BMP-2, and BMP-7 increased sox9 gene expression and protein production and that sox9 knockdown modulated growth factor actions in a complex fashion that differed both with growth factors and with chondrocyte function. The data suggest that sox9 mediates the stimulation of matrix production by the combined growth factors and the stimulation of chondrocyte proliferation by FGF-2. The mitogenic effect of the combined growth factors and the catabolic effect of FGF-2 appear to involve sox9-independent mechanisms. Control of these molecular mechanisms may contribute to the treatment of cartilage damage.