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Biomedical subjects

D M Bradham

Publications and source records attributed to D M Bradham.

7 recordsLinked to original sources
PubMed1998

In vivo cartilage formation from growth factor modulated articular chondrocytes.

Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.

Aged↗
PubMed1995

Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors.

During development of the embryonic limb, differentiation of mesenchymal progenitor cells into chondrocytes is regulated by cell shape, extracellular matrix, and growth and differentiation factors. In this study, reconstituted basement membrane (Matrigel) prepared from mouse Englebreth-Holm-Swarm tumor tissue was found to stimulate mesenchymal cell chondrogenesis in vitro and the production of cartilage at ectopic sites in athymic mice. The rate of chondrogenesis of mesenchymal cells from chick limb bud was increased four-fold by the addition of 400 micrograms/ml Matrigel to the media of micromass cultures, and this activity was not blocked by neutralizing antibodies to transforming growth factor-beta (TGF-beta) or fibroblast growth factor (FGF). Mesenchymal cells cultured on Matrigel, but not laminin or collagen type I or IV, formed spheres of condensed cells which stained with Alcian blue. Chick limb-bud mesenchymal cells suspended in Matrigel prepared from tumors grown in C57 mice aged 3, 12, or 26 months formed disks of hyaline cartilage within 2 weeks with wet weights of 59.1 mg, 35.7 mg, and 21.4 mg, indicating that the Matrigel from the old animals was less biologically active. In agreement with the in vivo data, Alcian blue staining of proteoglycan was over two-fold higher in micromass cultures supplemented with the Matrigel from young animals than in cultures treated with the Matrigel from old mice. A high-salt wash preparation of Matrigel from tumors grown in old mice increased the rate of chondrogenesis and cartilage production, suggesting that an inhibitor of chondrogenesis is produced by the old host. Thus, Matrigel contains chondrogenic activity distinct from TGF-beta or FGF. The aged host may produce factors that are inhibitory to mesenchymal cell differentiation and adversely affect cartilage formation and repair.

Animals↗
PubMed1994

Transrepression of type II collagen by TGF-beta and FGF is protein kinase C dependent and is mediated through regulatory sequences in the promoter and first intron.

Transforming growth factor beta and basic fibroblast growth factor are multipotential factors found in bone and cartilage that may be involved in both the proliferation and differentiation of chondrocytes. It was previously reported that TGF-beta plus FGF caused a modulation of chondrocyte phenotype that included the down-regulation of steady-state level of the collagen II transcript. In this report, the results of nuclear run-off data indicate that repression of transcript initiation from the collagen II gene is the primary mechanism involved in the growth factor induced inhibition. Transient transfection assays with CAT expression vectors containing portions of the collagen II gene show that the TGF-beta/FGF induced transrepression requires a region in the first intron previously reported to have transcriptional enhancer activity and to bind chondrocyte nuclear proteins. In addition, silencer elements in the promoter also appear to play a role. Protein data as well as transient transfection experiments indicate that the activation of protein kinase C is necessary for the growth factor-induced down-regulation of collagen II expression. These studies suggest that a cascade initiating with PKC activation is responsible for modifying transcription factors that interact with regulatory sequences in the collagen II gene. A detailed understanding of the factors involved in cartilage-specific gene regulation in chondrocytes would facilitate development of therapeutic protocols for the repair of degenerated cartilage in diseases such as osteoarthritis.

Animals↗
PubMed1993

Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair.

Connective tissue growth factor (CTGF) is a cysteine-rich peptide that exhibits platelet-derived growth factor (PDGF)-like biological and immunological activities. CTGF is a member of a family of peptides that include serum-induced immediate early gene products, a v-src-induced peptide, and a putative avian transforming gene, nov. In the present study, we demonstrate that human foreskin fibroblasts produce high levels of CTGF mRNA and protein after activation with transforming growth factor beta (TGF-beta) but not other growth factors including PDGF, epidermal growth factor, and basic fibroblast growth factor. Because of the high level selective induction of CTGF by TGF-beta, it appears that CTGF is a major autocrine growth factor produced by TGF-beta-treated human skin fibroblasts. Cycloheximide did not block the large TGF-beta stimulation of CTGF gene expression, indicating that it is directly regulated by TGF-beta. Similar regulatory mechanisms appear to function in vivo during wound repair where there is a coordinate expression of TGF-beta 1 before CTGF in regenerating tissue, suggesting a cascade process for control of tissue regeneration and repair.

3T3 Cells↗
PubMed1991

Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10.

Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.

Amino Acid Sequence↗
PubMed1988

Kidney epithelial cells express c-sis protooncogene and secrete PDGF-like protein.

Nontransformed monkey kidney cells (BSC-1 line), used as a model for renal epithelium, were assayed for release of platelet-derived growth factor (PDGF)-like proteins. BSC-1 cells continuously released a mitogenic activity for fibroblasts and a chemoattractant activity for smooth muscle cells, each of which was inhibited 80-90% by an antibody to human PDGF. A cDNA probe for the PDGF B-chain gene (c-sis), but not for the A-chain gene, hybridized to mRNA obtained from growing and quiescent cells. c-sis gene expression and PDGF-like protein secretion were studied in the presence of known growth-regulatory molecules. A secreted BSC-1 cell protein identical to transforming growth factor beta 2 inhibited DNA synthesis in growing cultures and induced marked accumulation of c-sis mRNA without a corresponding increase in the release of PDGF-like activity. Adenosine diphosphate stimulated DNA synthesis in quiescent cultures and enhanced both c-sis expression and release of PDGF-like activity. However, growing and quiescent cells did not express the PDGF receptor gene or exhibit a mitogenic response to authentic PDGF. Thus the PDGF-like protein released by these kidney epithelial cells could contribute to growth control by a paracrine mechanism.

Animals↗