Assessing the effects of transforming growth factor-beta1 on bladder smooth muscle cell phenotype. I. Modulation of in vitro contractility.

PURPOSE: Modulation of the bladder smooth muscle cell phenotype contributes to the resulting bladder dysfunction in many pathological bladder conditions. Transforming growth factor-beta1 is an important regulator of cellular phenotype in fibrotic diseases that has specific effects on bladder smooth muscle cells associated with phenotypic changes. We verified transforming growth factor-beta1 expression in neurogenic bladder tissue and investigated its effects on bladder smooth muscle cell collagen gel contraction. MATERIALS AND METHODS: Transforming growth factor-beta1 immunostaining was performed on tissue sections from spinalized rats and quantified based on the ratio of fluorescence to total detrusor area. Rat bladder smooth muscle cells were seeded at different densities on anchored collagen gels and the effect of transforming growth factor-beta1 on contractility was assessed by measuring changes in the collagen gel area with time. Phenotypic changes induced by transforming growth factor-beta1 were detected by immunostaining for caldesmon and the specific isoform high molecular weight caldesmon. RESULTS: Transforming growth factor-beta1 immunostaining revealed increased levels specifically in the detrusor of spinal cord injured rats. Rat bladder smooth muscle cell contraction increased with larger cell populations and was inhibited by transforming growth factor-beta1. Transforming growth factor-beta1 induced a decrease in high molecular weight caldesmon expression in bladder smooth muscle cells. CONCLUSIONS: Increased transforming growth factor-beta1 expression in the detrusor of spinal cord injured rats implies up-regulation and localized signaling in response to injury. Bladder smooth muscle cells showed a loss of contractility in response to transforming growth factor-beta1 in all cell populations. A shift in phenotype was confirmed by high molecular weight caldesmon immunostaining. These results suggest that transforming growth factor-beta1 can modulate bladder smooth muscle cell function and may be a crucial regulator of bladder smooth muscle cell phenotype in pathological bladder conditions.

Assessing the effects of transforming growth factor-beta1 on bladder smooth muscle cell phenotype. II. Modulation of collagen organization.

PURPOSE: Pathological alterations in the relationship between cells and the extracellular matrix have a profound effect on tissue morphology and function. Transforming growth factor-beta1 is thought to have a role in bladder pathology by modulating the bladder smooth muscle cell phenotype and, thus, interactions with the extracellular matrix. We investigated the effects of transforming growth factor-beta1 on the organization of an in vitro extracellular matrix by bladder smooth muscle cells. MATERIALS AND METHODS: Rat bladder smooth muscle cells were seeded at different densities (5 x 10(4), 1 x 10(5) and 2.5 x 10(5) cells) on anchored collagen gels and allowed to contract for 18 or 24 hours. Transforming growth factor-beta1 effects on collagen organization were assessed by analyzing collagen fibril orientation using small angle light scattering. Phase contrast microscopy was used to correlate changes in bladder smooth muscle cell morphology to areas of high fibril orientation. Bladder smooth muscle cells were trypsinized from the gels to confirm altered collagen architecture. RESULTS: Transforming growth factor-beta1 altered collagen fibril organization locally but this was only significant in the highest cell population. Transforming growth factor-beta1 induced a population dependent effect, in which bladder smooth muscle cells formed bundles or aggregates. These aggregates corresponded with local areas of high collagen fibril alignment. These changes in collagen architecture were maintained macroscopically after removing the bladder smooth muscle cells. CONCLUSIONS: Changes in collagen architecture organization in response to transforming growth factor-beta1 indicate changes in the bladder smooth muscle cell phenotype, resulting in altered cell/extracellular matrix interactions. Changes in this relationship at the microscopic level could be an important component of tissue remodeling and subsequent dysfunction, and indicate a possible role for transforming growth factor-beta1 in bladder pathology cases.

Synergistic effects of cyclic tension and transforming growth factor-beta1 on the aortic valve myofibroblast.

BACKGROUND: Phenotypically, aortic valve interstitial cells are dynamic myofibroblasts, appearing contractile and activated in times of development, disease, and remodeling. The precise mechanism of phenotypic modulation is unclear, but it is speculated that both biomechanical and biochemical factors are influential. Therefore, we hypothesized that isolated and combined treatments of cyclic tension and transforming growth factor-beta1 would alter the phenotype and subsequent collagen biosynthesis of aortic valve interstitial cells in situ. METHODS AND RESULTS: Porcine aortic valve leaflets received 7- and 14-day treatments of 15% cyclic stretch (Tension); 0.5 ng/ml transforming growth factor-beta1 (TGF); 15% cyclic stretch and 0.5 ng/ml transforming growth factor-beta1 (Tension+TGF); or neither mechanical nor cytokine stimuli (Null). Tissues were homogenized and assayed for aortic valve interstitial cell phenotype (smooth muscle alpha-actin) and collagen biosynthesis (via heat shock protein 47, which was further verified with type I collagen C-terminal propeptide). At both 7 and 14 days, smooth muscle alpha-actin, heat shock protein 47, and type I collagen C-terminal propeptide quantities were significantly greater (P<.001) in the Tension+TGF group than in all other groups. Additionally, Tension alone appeared to maintain smooth muscle alpha-actin and heat shock protein 47 levels that were measured on Day 0, while TGF alone elicited an increase in smooth muscle alpha-actin and heat shock protein 47 compared to Day 0 levels. Null treatment revealed diminished proteins at both time points. CONCLUSIONS: Elevated transforming growth factor-beta1 levels, in the presence of cyclic mechanical tension, resulted in synergistic increases in contractile and biosynthetic proteins in aortic valve interstitial cells. Since cyclic mechanical stimuli can never be relieved in vivo, the presence of transforming growth factor-beta1 (possibly from infiltrating macrophages) may result in overly biosynthetic aortic valve interstitial cells, leading to altered extracellular matrix architecture, compromised valve function, and, ultimately, degenerative valvular disease.

The role of MMP-I up-regulation in the increased compliance in muscle-derived stem cell-seeded small intestinal submucosa.

We have previously observed that muscle-derived stem cells (MDSC) seeded onto porcine small intestinal submucosa (SIS) increase the mechanical compliance of the engineered tissue construct [Lu SH, Sacks MS, Chung SY, Gloeckner DC, Pruchnic R, Huard J, et al. Biaxial mechanical properties of muscle-derived cell seeded small intestinal submucosa for bladder wall reconstitution. Biomaterials 2005;26(4):443-9]. To date, however, the initial remodeling events which occur when MDSC are seeded onto SIS have yet to be elucidated. One potential mechanism responsible for the observed increase in mechanical compliance is the release of matrix metalloproteinase-I (MMP-I). To investigate this finding, MDSC ( approximately 1x10(6)) were cultured on single-layer SIS cell culture inserts (4.7 cm2) for 1-10 days. MDSC MMP-I activity on SIS in the supernatant at 1, 3, 5, 7, and 10 days was determined using a collagenase assay kit. MMP-I activity of the MDSC/SIS was significantly higher (p<0.0025) after one day in culture compared to specimens collected from subsequent time points and the unseeded control. To further study the initial remodeling events, the impact of MMP-I on mechanical compliance was examined. SIS was incubated with 0.16 U/mL collagenase-I for 3, 4.5, 5, and 24h, then biaxial mechanical testing was performed. After 5h of digestion with collagenase-I, mechanical compliance under 1 MPa peak stress was increased by 7% in the circumferential direction, compared to control SIS. These findings suggest that the release of MMP-I in response to initial seeding on SIS and subsequent breakdown of collagen fibers is the mechanism responsible for an increase in mechanical compliance.