Functional changes in bladder tissue from type III collagen-deficient mice.

OBJECTIVE: Collagen fibers impart tensile strength and transfer tension from bladder smooth muscle cells. We have previously shown that fibrotic bladders are characterized by an increased type III:type I collagen ratio. To determine the effect of decreased type III collagen on bladder function, type III collagen-deficient mice (COL3A1) were studied physiologically. METHODS: Bladders from wild-type (+/+) and heterozygous (+/-) COL3A1 mice were biochemically characterized to determine total collagen (hydroxyproline analysis) and collagen subtype concentration (cyanogen bromide digestion and ELISA). Alterations in collagen fiber diameter were assessed by electron microscopy. Bladder muscle strips were used to assess physiologic function. RESULTS: Hydroxyproline content decreased in heterozygous bladders, which had 50% less type III collagen. Wild-type bladders had a biphasic distribution of collagen fiber sizes, whereas heterozygous bladder collagen fibers spanned a broad range. Physiologically, there were no differences in contractile responses between wild-type and heterozygotes when stimulated with ATP, carbachol or KCl, indicating normal contraction via purinergic and muscarinic receptors, and in response to direct membrane depolarization. In contrast, tension generation in heterozygotes was decreased after field stimulation (FS), indicating decreased synaptic transmission. Length-tension studies showed that the heterozygote muscle strips generated less tension per unit length, indicating that they were more compliant than wild-type controls. CONCLUSIONS: Critical levels of type III collagen appear to be a requirement for normal bladder tension development and contraction. Our data show that a decrease in the type III:type I collagen ratio, and altered fiber size, results in a more compliant bladder with altered neurotransmitter function.

Transforming growth factor-beta1-induced hypertrophy and matrix expression in human bladder smooth muscle cells.

OBJECTIVES: To determine whether transforming growth factor beta (TGF-beta) could activate hyperplasia, hypertrophy, and altered collagen expression in human detrusor smooth muscle cells (SMCs). METHODS: Human bladder SMCs were treated in vitro with TGF-beta1 and analyzed for changes in both proliferative and hypertrophic responses by cell number and volume measurements, as well as for alterations in extracellular matrix gene and protein expression by Northern blot and enzyme-linked immunosorbent assay. RESULTS: Proliferation of bladder SMCs was refractory to TGF-beta1, whereas the cells became hypertrophic upon TGF-beta1 treatment. The interstitial collagens, types I and III, were increased significantly in TGF-beta1-treated cultures in a dose-dependent manner. These increases were blocked in the presence of TGF-beta1 neutralizing antibody and also when cultures were treated with the protein synthesis inhibitor cycloheximide, indicating that new protein synthesis is necessary for upregulation of the interstitial collagens. Messenger ribonucleic acid transcripts for both the COL1A1 and COL3A1 genes were elevated at 4, 6, and 24 hours in TGF-beta1-treated cultures, preceding the expression of the collagenous protein, showing that TGF-beta1 effects on bladder smooth muscle occur, at least in part, at the transcriptional level. CONCLUSIONS: These results indicate that human bladder SMCs have the potential to mediate both a hypertrophic and fibrotic response upon TGF-beta1 stimulation.

Mast cell chymase is a possible mediator of neurogenic bladder fibrosis.

AIMS: Urinary bladders of patients with myelomeningocele, owing to spina bifida, are often functionally impaired, fibrotic organs. Common to this condition are repeated occurrences of bladder infection and inflammation. Since mast cells have been associated with a fibrogenic response in inflammatory conditions, we investigated the role of mast cell granule product, chymase, as a mediator of myleodysplastic bladder fibrosis. METHODS: Human control and myelodysplastic bladder tissues were stained with Unna's stain and chymase antibody to determine mast cell number and localization. Cell specific localization of collagen mRNAs was determined by in situ hybridization (ISH). In vitro, normal human bladder fibroblasts were treated with recombinant chymase, heparin and inhibitors, and collagen subtype concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: Myelodysplastic bladders were characterized by increased mast cells in the detrusor muscle layer compared to control bladders, as well as mast cell degranulation and increased connective tissue deposition. Both types I and III collagen mRNA localized to fibroblasts surrounding detrusor muscle fascicles, whereas only collagen III mRNA localized to cells within connective tissue infiltrated muscle bundles in myelomeningocele bladder tissue. Chymase treatment of bladder fibroblasts, in vitro, was dose-dependent and resulted in significant increases in both types I and III collagen. Heparin did not alter collagen protein expression, whereas heparin-chymase combination modulated type III collagen expression. Serine protease inhibitor, phenylmethylsulfonlyfluoride, did not inhibit collagen synthesis, whereas denatured chymase resulted in decreased collagenous protein levels. CONCLUSIONS: Bladder fibrosis may be mediated by mast cell chymase stimulation of collagen synthesis.

Mast cell chymase modifies cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth muscle cells.

The hallmarks of chronic, severe asthma include prominent airway inflammation and airway smooth muscle (ASM) hypertrophy and hyperplasia. One of the factors that contribute to the injury and repair process within the airway is activation of proteases and turnover of extracellular matrix components. Mast cells, which are present in increased numbers in the asthmatic airway, are a rich source of the neutral protease chymase, which can degrade several basement membrane components. Recent data suggest that proteases also play a critical role in regulating the expression of CD44, the primary receptor for the matrix glycosaminoglycan hyaluronan. In this study we investigated the effects of chymase treatment on human ASM cell function. We found that chymase degraded the smooth muscle cell pericellular matrix. This was accompanied by an increased release of fibronectin and soluble CD44, but not soluble ICAM-1 or soluble hyaluronan, into the conditioned medium. In addition, chymase inhibited T cell adhesion to ASM and dramatically reduced epidermal growth factor-induced smooth muscle cell proliferation. These data suggest that the local release of mast cell chymase may have profound effects on ASM cell function and airway remodeling.

Transforming growth factor-beta stabilizes elastin mRNA by a pathway requiring active Smads, protein kinase C-delta, and p38.

Transforming growth factors (TGFs)-beta are multipotent in their biologic activity, regulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription, but TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway that likely involves a phosphatidylcholine-specific phospholipase C, a protein kinase C, prenylated and acylated protein(s), and one or more tyrosine kinases. However, there is a 4- to 6-h lag period after the addition of TGF-beta1 before significant stimulation of elastin expression is observed and the question of whether the Smads are involved has not been addressed. In the present work, using cultured human fetal lung fibroblasts, we show through the use of specific inhibitors and transfection of a Smad 7 construct that in addition to de novo protein synthesis and active Smads, the extended activity of protein kinase C (PKC)-delta and the stress-activated protein kinase, p38, is required for TGF-beta1 to achieve elastin mRNA stabilization.