Expression Of Matrix-related Genes Research Articles

Altered Expression of Matrix-Related Molecules in the Development of Chronic Thy1.1 Nephritis

Background/Aim: Matrix production and degradation are critically important in chronic nephritis. Our aim was to investigate the precise expression of matrix-related molecules which is essential for understanding the pathogenesis of renal disease. Methods: Chronic nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. RNA was extracted from renal cortex and isolated glomeruli 4, 7, and 10 weeks after the antibody injection. Matrix-related gene expressions were measured by polymerase chain reaction. The expression of α1(IV) and α3(IV) collagens was studied by immunohistochemistry. The gelatinolytic activity in the glomeruli was assayed by gelatin zymography. Results: Polymerase chain reaction revealed an increase of α1(IV) in both glomeruli and renal cortex from nephritic rats. In contrast, the expression of α3(IV), normally a component of the glomerular basement membrane, was decreased in nephritic animals. Immunohistochemistry confirmed the finding that α1(IV) and α3(IV) were up- and downregulated, respectively, in the glomeruli. Gene expression and activity of matrix metalloproteinase 2 were enhanced, while those of matrix metalloproteinase 9 were clearly suppressed in nephritis. Conclusions: Downregulation of α3(IV) and enhancement of the matrix metalloproteinase-2 activity in the glomeruli may contribute to the glomerular damage by altering the glomerular basement membrane components. Impairment of the glomerular basement membrane integrity may possibly be implicated in irreversible renal dysfunction.

Upregulation of Apoptotic and Matrix-related Gene Expression during Fresh Osteochondral Allograft Storage

We identified changes in proapoptotic and extracellular matrix-related gene expression with prolonged storage of fresh osteochondral allografts using gene array analysis to better understand the process of graft degradation during storage. Six human distal femurs were obtained according to standard organ harvesting protocol and stored in serum-free allograft media. Each was examined at baseline (within 72 hours postmortem), 21 days (average time of implantation), and 35 days (maximum time to implantation) for proapoptotic and extracellular matrix-related gene expression using two 100-gene microarrays, cell viability using confocal microscopy, and proteoglycan synthesis via SO4 incorporation. We found numerous genes showing upregulation associated with increased storage time, including CD30, CD30 ligand, Fas, Fas ligand, tumor necrosis factor-alpha, and several caspases. Cell viability and proteoglycan synthesis also were significantly decreased with increased storage. Loss of chondrocytes via apoptosis is likely a key determinant of osteochondral allograft viability during storage, whereas extracellular matrix degeneration may occur at a later stage. These findings provide targets for future media modulation. Improved graft viability and the potential for lengthened storage periods through improved storage conditions may improve clinical outcomes and availability of fresh osteochondral allografts.

Comparison of human fibroblast ECM-related gene expression on elastic three-dimensional substrates relative to two-dimensional films of the same material

Three-dimensional elastic substrates were fabricated from a commercially available polyurethane with an internal porosity of approximately 70% and elastic modulus of 27.4±2.76 KPa and examined for suitability in vocal fold tissue engineering. Using immunohistochemistry, biomechanical testing, and RT-PCR; we examined human fibroblast viability, distribution and extracellular matrix related gene expression within substrates for periods up to 4 weeks. We found that cells were capable of colonizing the entire volume of a 5 mm wide×3 mm deep×20 mm long substrate at high viability. Histological cross-sections showed extensive extracellular matrix deposited around the cells and throughout the pore structure of the substrates, which consisted of fibronectin and type I collagen. Cell seeded substrates displayed a significantly higher elastic modulus than unseeded controls similar to native tissue. The transfer of cell growth from two-dimensional to three-dimensional culture resulted in changes in ECM-related gene expression consistent with decreasing cell migration and increasing tissue formation. We found that fibroblasts cultured in three-dimensional substrates expressed significantly higher levels of mRNA for elastin and fibromodulin, while expressing significantly lower levels of mRNA for MMP-1 and hyaluronidase relative to two-dimensional substrates of the same material. The results suggest that three-dimensionally porous, Tecoflex-derived elastic biomaterials may be suitable substrates for engineering vocal fold tissue.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Modulated bone matrix-related gene expression is associated with differences in interfacial strength of different implant surface roughness.

The objective of this study was to examine the effect of implant surface topography on biomechanical strength and expression patterns of bone extracellular matrix (ECM)-related genes during implant healing. Cylinder implants with turned or dual acid-etched (DE) surface were placed into the femurs of female Sprague-Dawley rats. At 2 and 4 weeks after the implantation, the implant push-in test was performed to measure the load-bearing strength of the bone-tissue interface. T-shaped hollow implants were separately placed and the total RNA was extracted from the ingrown tissue within the hollow chamber. The RNA was subjected to reverse transcriptase polymerase chain reaction to analyze the expression pattern of selected bone extracellular matrix-related genes. The push-in value of the DE implant is approximately 300% greater than that of the turned implant at weeks 2 and 4 (p=.0090). The expression of collagen II, osteocalcin and biglycan was higher in the week 2 DE implant group than the turned implant group. At week 4 the expression of collagen II and IX was 7- and 26-fold, respectively, greater in the DE implant group than in the turned implant group. The surface topography of implants may induce the phenotypic alteration of wound healing cells. The increased interfacial strength of the DE implant may be associated with the modulated expression of the selected set of bone extracellular matrix genes.

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes.

To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.

Wavelength-Specific Synergy Between Ultraviolet Radiation and Interleukin-1α in the Regulation of Matrix-Related Genes: Mechanistic Role for Tumor Necrosis Factor-α

Ultraviolet light causes both acute and chronic changes in extracellular matrix. We sought to examine the effects of different ultraviolet wavelengths on expression of matrix-related genes in fibroblasts. We previously reported that tropoelastin gene expression in vivo decreases with acute ultraviolet B exposure, and interleukin-1 alpha-mediated upregulation of tropoelastin is blocked in vitro after ultraviolet B radiation. In this study, we found that only ultraviolet B, but not ultraviolet A or ultraviolet A1, blocked the ability of interleukin-1 alpha to stimulate tropoelastin expression in vitro. Ultraviolet B and interleukin-1 alpha synergistically increased tumor necrosis factor-alpha secretion by fibroblasts, a finding not seen with ultraviolet B alone nor with ultraviolet A or ultraviolet A1 combined with interleukin-1 alpha. Keratinocytes showed a similar ultraviolet B-specific induction of tumor necrosis factor-alpha production. Addition of tumor necrosis factor-alpha to cultured fibroblasts blocked interleukin-1 alpha-induced stimulation of tropoelastin message, and addition of anti-tumor necrosis factor-alpha antibodies restored the responsiveness of tropoelastin and collagen messages to exogenous interleukin-1 alpha after ultraviolet B exposure. We conclude that interleukin-1 alpha in combination specifically with ultraviolet B induces fibroblasts to secrete tumor necrosis factor-alpha, and that this ultraviolet B-specific induction of tumor necrosis factor-alpha secretion is responsible for effects of ultraviolet B on the expression of matrix-related genes in the skin.