James J. Tomasek

Myofibroblasts and mechano-regulation of connective tissue remodelling

During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast — its origins, functions and molecular regulation — will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.

Gelatinase A Activation Is Regulated by the Organization of the Polymerized Actin Cytoskeleton

Gelatinase A (GL-A) is a matrix metalloproteinase (MMP) involved in both connective tissue remodeling and tumor invasion. GL-A activation is mediated by a membrane-type MMP (MT-MMP) that cleaves the GL-A propeptide. In this study, we examined the role of the actin cytoskeleton in regulating GL-A activation and MT-MMP-1 expression. Human palmar fascia fibroblasts and human fetal lung fibroblasts were cultured on a planar substratum or within different types of collagen lattices. Fibroblasts that formed stress fibers, either on a planar substratum or within an attached collagen lattice, showed reduced GL-A activation compared with fibroblasts lacking stress fibers, within either floating or stress-released collagen lattices. To determine whether changes in the organization of the actin cytoskeleton could promote GL-A activation, fibroblasts with stress fibers were treated with cytochalasin D. Within 24 h after treatment, GL-A activation was dramatically increased. Associated with this GL-A activation was an increase in MT-MMP-1 mRNA as determined by Northern blot analysis. Treatment with nocodazole, which induced microtubule depolymerization and cell shape changes without affecting stress fibers, did not promote GL-A activation. These results suggest that the extracellular matrix and the actin cytoskeleton transduce signals that modulate GL-A activation and regulate tissue remodeling.

Nonuniform distribution of fibronectin during avian limb development

Abstract Using indirect immunofluorescence we have examined the distribution of the cell surface and extracellular matrix glycoprotein fibronectin at the epithelial-mesenchymal interface and in the mesenchyme of developing chick and duck wing buds. At all stages examined, in both species, staining for fibronectin is greatly enhanced in the basement membrane subjacent to the apical ectodermal ridge (AER), a site of inductive tissue interaction, relative to the epithelial basement membranes in the noninductive dorsal and ventral limb epithelial-mesenchymal interfaces. In stage 23, 25, and 28 chick limb buds, staining for fibronectin is uniform in the least mature distal mesenchyme, retained between more proximal cells undergoing precartilage condensation and lost in those regions undergoing myogenesis, and persistent in all but the most mature cartilage present at the latest stage examined. These results are consistent with a role for fibronectin in AER-induced limb outgrowth, and with a transient role for the glycoprotein in the formation of the skeletal pattern of the limb.

Correlation of α-smooth muscle actin expression and contraction in Dupuytren's disease fibroblasts

We studied 11 nodules from patients with Dupuytrens contracture to determine whether alpha-smooth muscle actin expression in Dupuytrens fibroblasts is related to the generation of contractile force. Tissue was placed into explant culture and fibroblast strains were obtained. The mean percent of cultured Dupuytrens fibroblasts expressing alpha-smooth muscle actin, as determined by immunofluorescence, was 14 +/- 8 and ranged from 1% to 26%. The ability of Dupuytrens fibroblasts to generate contractile force was determined by using a previously described collagen lattice contraction assay. We observed a significant positive correlation between the expression of alpha-smooth muscle actin and the generation of contractile force in cell strains of Dupuytrens fibroblasts. In addition, six fibroblast strains from palmar fascia of individuals undergoing carpal tunnel release were examined. In six strains of palmar fibroblasts the mean percent of cells expressing alpha-smooth muscle actin was 5 +/- 3 and ranged from 1% to 9%. Six Dupuytrens fibroblast strains, in which more than 15% of the cells expressed alpha-smooth muscle actin, were significantly more contractile than the palmar fibroblasts. These results suggest that Dupuytrens fibroblasts can acquire smooth muscle characteristics and that the acquisition of a smooth muscle-like phenotype correlates with increased contractility.

Regulation of α-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-β1 control element

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of α-smooth muscle actin (α-SMA); however, little is known regarding molecular mechanisms that control activation of α-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for α-SMA expression in myofibroblasts in vivo and to determine whether activation of α-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-β1 control element (TCE) that are required for α-SMA expression in smooth muscle cells. A Lac Z transgene construct from −2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous α-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-β1 in cultured transgenic fibroblasts. These same elements were also critical in regulating α-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of α-SMA expression in myofibroblasts in granulation tissue.

Myocardin-Related Transcription Factors-A and -B are Key Regulators of TGF-β1-Induced Fibroblast to Myofibroblast Differentiation

Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor beta-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors-A and -B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively-active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1 differentiated myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of myofibroblast differentiation in response to TGF-β1.

IDENTIFICATION AND CLONING OF THE MEMBRANE-ASSOCIATED SERINE PROTEASE, HEPSIN, FROM MOUSE PREIMPLANTATION EMBRYOS

Previous studies have suggested the existence of a membrane-associated serine protease expressed by mammalian preimplantation embryos. In this study, we have identified hepsin, a type II transmembrane serine protease, in early mouse blastocysts. Mouse hepsin was highly homologous to the previously identified human and rat cDNAs. Two isoforms, differing in their cytoplasmic domains, were detected. The tissue distribution of mouse hepsin was similar to that seen in humans, with prominent expression in liver and kidney. In mouse embryos, hepsin expression was observed in the two-cell stage, reached a maximal level at the early blastocyst stage, and decreased subsequent to blastocyst hatching. Expression of a soluble form of hepsin revealed its ability to autoactivate in a concentration-dependent manner. Catalytically inactive soluble hepsin was unable to autoactivate. These results suggest that hepsin may be the first serine protease expressed during mammalian development, making its ability to autoactivate critical to its function.

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

PURPOSE The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. MATERIALS AND METHODS Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. RESULTS Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. CONCLUSIONS In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.

Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell‐like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+‐induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)‐mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin‐1 (ET‐1), serotonin, and angiotensin‐II and isometric force generation was measured. We here investigated ET‐1 in depth, because it was the only agonist that produced a long‐lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET‐1–promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long‐lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET‐1. Moreover, K+‐induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long‐duration contraction of myofibroblasts necessary for wound closure.

The effects of anabolic steroids on rat tendon. An ultrastructural, biomechanical, and biochemical analysis.

Forty-eight male rats were randomly separated into four groups: a control group, a group treated with anabolic steroids, a group treated with daily exercise, and a group treated with both steroids and exercise. At 6 weeks, biomechanical, ultrastructural, and biochemical testing was performed on the Achilles tendons of half of the rats in each group. The remaining rats continued in the experimental protocol, but steroid administration was discontinued. Similar testing was then performed on the remaining rats at 12 weeks. Testing showed ana bolic steroids produced a stiffer tendon that absorbs less energy and fails with less elongation; tendon strength was unaffected. Effects were entirely revers ible on discontinuation of the steroids. Light microscopic analysis revealed no changes in the appearance of the fibrils. No change in fibril diameter or shape was noted on electron microscopic analysis. Biochemical testing revealed no change in qualitative immunofluorescence staining with Type III collagen or fibronectin. Abuse of anabolic steroids is a widespread problem among com petitive athletes; consequently, complications after their use are seen with increasing frequency. Knowledge of the effects of these drugs on tendon and the muscu lotendinous unit may prove helpful in counseling ath letes who use anabolic steroids.