Decreased expression of smooth muscle alpha-actin results in decreased contractile function of the mouse bladder.

PURPOSE: Smooth muscle alpha-actin (SMalphaA) is an important actin isoform for functional contractility in the mouse bladder. Alterations in the expression of SMalphaA have been associated with a variety of bladder pathological conditions. Recently, a SMalphaA-null mouse was generated and differences in vascular tone and contractility were observed between wild-type and SMalphaA-null mice suggesting alterations in function of vascular smooth muscle. We used SMalphaA-null mice to explore the hypothesis that SMalphaA is necessary for normal bladder function. MATERIALS AND METHODS: Reverse transcriptase polymerase chain reaction, Western blotting and immunohistochemical staining were used to confirm the absence of SMalphaA transcript and protein in the bladder of SMalphaA-null mice. In vitro bladder contractility compared between bladder rings harvested from wild-type and SMalphaA-null mice was determined by force measurement following electrical field stimulation (EFS), and exposure to chemical agonists and antagonists including KCl, carbachol, atropine and tetrodotoxin. Resulting force generation profiles for each tissue and agent were analyzed. RESULTS: There was no detectable SMalphaA transcript and protein expression in the bladder of SMalphaA-null mice. Nine wild-type and 9 SMalphaA-null mice were used in the contractility study. Bladders from SMalphaA-null mice generated significantly less force than wild-type mice in response to EFS after KCl. Similarly, bladders from SMalphaA-null mice generated less force than wild-type mice in response to pretreatment EFS, and EFS after carbachol and atropine, although the difference was not significant. Surprisingly, the bladders in SMalphaA-null mice appeared to function normally and showed no gross or histological abnormalities. CONCLUSIONS: SMalphaA appears to be necessary for the bladder to be able to generate normal levels of contractile force. No functional deficits were observed in the bladders of these animals but no stress was placed on these bladders. To our knowledge this study represents the first report to demonstrate the importance of expression of SMalphaA in force generation in the bladder.

Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.

To evaluate whether alpha-smooth muscle actin (alpha-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of alpha-SMA, with that of lung fibroblasts (LFs), expressing high levels of alpha-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of alpha-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for alpha-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFbeta1 increased alpha-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFbeta-antagonizing agents reduced alpha-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with alpha-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with alpha-cardiac and beta- or gamma-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased alpha-SMA expression is sufficient to enhance fibroblast contractile activity.

Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion.

TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB(1-556) coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB(1-556) was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

Transforming growth factor-beta1 promotes the morphological and functional differentiation of the myofibroblast.

The myofibroblast is responsible for the generation of contractile force associated with wound contraction and pathological contractures and is characterized by the presence of alpha-smooth muscle (alpha-sm) actin-containing stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils containing the ED-A splice variant. Transforming growth factor-beta1 (TGF-beta1) can promote the expression of alpha-sm actin in myofibroblasts, but the functional significance of this increased expression is unclear. In this study, we demonstrate, using the stress-relaxed collagen lattice contraction assay, that TGF-beta1 promoted a dose-dependent increase in the generation of contractile force in myofibroblasts and a concomitant increase in the expression of alpha-sm actin. We also demonstrate that TGF-beta1 enhanced the formation of the structural elements important in myofibroblast contractile force generation and transmission, including stress fibers, vinculin-containing fibronexus adhesion complexes, and fibronectin fibrils, and that this enhancement occurred prior to, and independent of, alpha-sm actin expression. This differentiated myofibroblast phenotype was not stable. Removal of TGF-beta1 resulted in reduced expression of alpha-sm actin as well as a decreased assembly of stress fibers and vinculin-containing adhesion complexes; however, there was no reduction in fibronectin fibrils. We conclude that TGF-beta1 promotes the morphological and functional differentiation of the myofibroblast by first enhancing the formation of the structural elements characteristic of the myofibroblast followed by increased expression of alpha-sm actin and contractile force generation.

Basement membrane chondroitin sulfate proteoglycan and vascularization of the developing mammalian limb bud.

We used immunocytochemistry to study the basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) distribution in mammalian limb bud and its relationship to and possible role in limb development. Anti-BM-CSPG immunostaining was examined in the developing limb buds of 24 Sprague-Dawley rats at embryonic days 12 to 14 and 19. BM-CSPG immunostaining was present in 3 regions. The first region was located peripherally in the limb bud ectodermal basement membrane (BM) that separates ectoderm from mesoderm and was present at all embryonic stages examined. The second region was in the mesenchymal extracellular matrix independent of the vascular system. This staining pattern was diffuse, granular, and often homogeneous, except for clustering adjacent to developing vessels, and was observed distally in the limb bud. In the mesenchymal extracellular matrix adjacent to the distal BM this staining pattern formed fibrils that were perpendicular and connected to the limb bud BM and extended into the underlying mesenchyme. The third region was localized to the BM of developing blood vessels of the limb bud. Blood vessel staining allowed analysis of limb bud vessel formation. The early developing blood vessels at the proximal limb bud were organized differently from those located distally. Large central vessels were present proximally, whereas a rich plexus of smaller vascular channels was present at the distal margin. A subectodermal avascular zone was observed at the margin of the limb bud, except beneath the apical ectodermal ridge where immunostained blood vessels extended from the distal vascular plexus toward the apical ectodermal ridge. The formation of central larger vessels occurs proximally, whereas formation of peripheral smaller vessels seems to take place locally and distally under the influence of the apical ectodermal ridge. BM-CSPG plays an important role in blood vessel formation and mammalian limb bud development. (J Hand Surg 2000; 25A:150-158.

Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase.

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.

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.

Distribution of type IV collagen during avian limb bud development.

Normal limb development is dependent on an epithelial-mesenchymal interaction between the overlying apical ectodermal ridge (AER) and the underlying mesenchyme. The basement membrane between the epithelium and the mesenchyme has been proposed to play an important role in regulating epithelial-mesenchymal interactions during development. To explore the role basement membrane type IV collagen may play during limb development we investigated the distribution of type IV collagen by immunolocalization. Developing avian leg buds were examined at 2 developmental stages: stage 23, when the AER is inductively active, and stage 28, when the AER is regressing. The proximal basement membrane in stage 23 limb buds stained much more intensely than the distal basement membrane. This proximal-distal immunostaining difference was less in stage 28 limb buds. We used the monoclonal antibody IIB12, which recognizes an epitope adjacent to the initial collagenase cleavage site on the type IV collagen molecule, to explore whether this proximal-distal difference in basement membrane staining could result from the loss of type IV collagen. The distal basement membrane of stage 23 limb buds demonstrated little immunostaining with the IIB12 antibody, suggesting enhanced collagenase-associated degradation. The immunostaining was increased in stage 28 limb buds. Consistent with a loss of type IV collagen, we also found that unfixed stage 23 leg bud cryostat sections stored at 4 degrees C lost their immunostaining for type IV collagen, in contrast to stored stage 28 limb bud cryostat sections. These results demonstrate that type IV collagen is distributed in a proximal-distal pattern in the basement membrane of the developing chick limb bud and suggest that this pattern may be the result of a selective degradation of type IV collagen in the basement membrane underlying the active AER. These results are consistent with the hypothesis that the basement membrane plays a role in regulating the epithelial-mesenchymal interaction responsible for induction of limb outgrowth.

Cellular structure and biology of Dupuytren's disease.

Numerous studies support the idea that the myofibroblast is a key cell responsible for the tissue contraction in Dupuytren's disease. In vitro models have been developed to study the underlying cellular basis of myofibroblast differentiation and contraction. Studies suggest that the growth factor TGF-beta 1 combined with mechanical stress can promote the differentiation of fibroblasts into myofibroblasts. Agonists, such as LPA and thrombin, can promote the contraction of myofibroblasts through specific intracellular signaling pathways that regulate levels of phosphorylated myosin light chain. Agents that can affect these intracellular signaling pathways hold promise as a means to decrease contraction of the myofibroblast and of the palmar fascia in Dupuytren's disease. Finally, the recent finding that IFN-gamma can suppress both the differentiation of the myofibroblast and the generation of contractile force, together with preliminary clinical results using IFN-gamma, suggest the potential use of IFN-gamma for nonsurgical therapy of Dupuytren's disease. Future studies into the cellular basis of tissue contraction should provide alternative methods to improve management of Dupuytren's contracture.

Internalized plasma membrane cholesterol passes through an endosome compartment that is distinct from the acid vesicle-lysosome compartment.

Cholesterol from the plasma membrane of MA-10 Leydig tumor cells is internalized into the cell and either esterified or used as substrate for steroid hormone synthesis. In the present studies we show that chloroquine and sphinganine cause LDL cholesterol and cholesteryl esters to accumulate in the cells. A lysosome fraction contained the excess cholesterol and cholesteryl esters. Both inhibitors blocked the conversion of plasma membrane cholesterol into intracellular cholesteryl esters and caused dose-dependent inhibition of dibutyryl-cAMP-stimulated progesterone synthesis. Radiolabeled cholesterol applied to the plasma membrane of MA-10 cells accumulated in the lysosome fraction of chloroquine and sphinganine-treated cells. Evidence that these inhibitors did not require the Golgi was provided by experiments using brefeldin A. Experiments utilizing a fluorescent cholesterol analogue and a lysosomal marker indicated that cholesterol entered the cells in structures that were different than the acidic vesicle-lysosome compartment. Consistent with this observation was the observation that the peak fluorescence fractions of cells subjected to density gradient centrifugation was of lower density than the lysosome fraction.

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.

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.

Pharmacologic regulation of Dupuytren's fibroblast contraction in vitro.

Dupuytren's disease is associated with contraction of specialized fibroblasts present in the diseased palmar fascia. Pharmacologic agents were evaluated for their ability to promote or inhibit contraction of Dupuytren's fibroblasts in vitro using a collagen lattice contraction assay. In the first part of the study, lysophosphatidic acid (LPA), serotonin, angiotensin II, and prostaglandin F2 alpha were tested for their ability to promote Dupuytren's fibroblast contraction. Lysophosphatidic acid was found to significantly promote Dupuytren's fibroblast contraction as compared with controls. This response to LPA is dose dependent, with a half-maximal response of 0.07 microM. Angiotensin II, serotonin, and prostaglandin F2 alpha at 1 mM, induced a significant amount of contraction as compared to controls, but the amount of contraction was at least six times less than that observed for LPA. In the second part of the study, prostaglandins E1 and E2 or the calcium blockers nifedipine and verapamil were tested for their ability to inhibit LPA-promoted contraction. It was found that both types of inhibitors partially block LPA-promoted contraction of Dupuytren's fibroblasts. The effect of the various pharmacologic agents on normal palmar fibroblasts was not evaluated. The focus of this study was to examine the regulation of contraction of Dupuytren's fibroblasts. This study demonstrates that LPA is a potent agonist of Dupuytren's fibroblast contraction and that this contraction can be inhibited by specific pharmacologic agents. These findings provide a rational basis for investigating further the clinical use of the calcium channel blockers nifedipine or verapamil and prostaglandins E1 and E2 to control Dupuytren's disease and possibly other fibrotic conditions.

Cellular contraction of collagen lattices is inhibited by nonenzymatic glycation.

High glucose concentrations associated with diabetes have been shown to cause the nonenzymatic modification of proteins. Reducing sugars covalently bind to free amine groups, undergo Amadori rearrangements, and crosslink with other glucose-modified proteins. Crosslinking of type I collagen by incubation with different concentrations of glucose 6-phosphate for up to 5 days resulted in a nondeformable collagen lattice as assayed by physical compaction analysis. Nonglycated collagen was fully compactible. Fibroblasts cultured on nonglycated collagen lattices were able to contract the lattice over a 5-day period, while fibroblasts on collagen glycated with 50 mM or more glucose 6-phosphate were unable to do this. Cells on both nonglycated and glycated collagen lattices initially lacked organized bundles of actin microfilaments or stress fibers. Over time, the cells on glycated lattices formed stress fibers, suggesting that they were still exerting mechanical force on a nondeformable matrix. These results suggest that crosslinking of collagen fibrils by nonenzymatic glycation alters the physical properties of the extracellular matrix, resulting in changes in the organization of the intracellular actin cytoskeleton.

Thrombin promotion of isometric contraction in fibroblasts: its extracellular mechanism of action.

Isometric force generated by fibroblasts plays an essential role in tissue contraction during normal wound healing and pathologic contractures. Thrombin, a serine protease present in all wounds, has been shown to promote wound healing. The purpose of this study was to determine the extracellular mechanism by which thrombin promotes isometric contraction by fibroblasts in an in vitro collagen lattice model of tissue contraction. The amount of isometric force generated by human fibroblasts can be measured directly with a stabilized collagen lattice attached to a force transducer. Thrombin promoted isometric contraction by human fibroblasts in a dose-dependent manner. In addition, thrombin-promoted isometric contraction is dependent on the enzymatic and anionic binding activity of thrombin, as demonstrated by inhibition with specific enzymatic and anionic binding inhibitors. These results suggest that thrombin may promote isometric contraction by fibroblasts through the enzymatic cleavage of its cell surface receptor, resulting in a new amino terminus that serves as a "tethered ligand" to activate the receptor directly. To test this mechanism of action, a synthetic peptide (SFLLRN) representing the "tethered ligand" region of the activated thrombin receptor was synthesized and examined for its ability to promote isometric contraction by fibroblasts. This peptide promoted fibroblast contraction in a dose-dependent manner. In contrast, a control isomer peptide (FSLLRN), in which the two amino-terminal amino acids were reversed, failed to promote this response. These findings demonstrate that human alpha-thrombin promotes isometric contraction by human fibroblasts and that binding to and cleavage of its cell surface receptor are integral to this response.

Mechanical properties of the extracellular matrix influence fibronectin fibril assembly in vitro.

Mechanical properties of the extracellular matrix (ECM) are proposed to influence cell behavior and biological activity. The influence of the mechanical environment on fibronectin fibril assembly was evaluated. Fibroblasts were cultured in hydrated collagen gels with two distinctly different mechanical properties. Cells cultured within a stabilized collagen gel generate stress that is transmitted throughout the matrix (stressed gel). In contrast, cells that are cultured within a collagen gel that is floating freely in media do not generate stress (relaxed gel). Fibroblasts in the stressed collagen gel develop large bundles of actin microfilaments and associated fibronectin fibrils, while fibroblasts within relaxed gels do not form stress fibers or assemble fibronectin into fibrils. In addition, we have evaluated the mechanism of fibronectin fibril assembly employed by fibroblasts cultured within a stressed three-dimensional collagen matrix and the role of fibronectin fibrils in transmission of cell-generated forces to the surrounding matrix. Fibronectin fragments (70-kDa amino terminal fragment, 110-kDa cell-adhesive fragment, and GRGDS peptide) and a monoclonal antibody body blocked fibronectin fibril assembly in stressed three-dimensional collagen gels. These results suggest that the features of fibronectin required for fibronectin fibril assembly by cells in collagen gels is similar to those required by cells cultured on a planar substratum. Although fibronectin fibril assembly was blocked by these inhibiting fragments and antibody, the cells displayed prominent actin bundles and developed isometric tension, indicating that stress fiber formation and contractile force transmission is not dependent on the presence of fibronectin fibrils.

Generation of contractile force by cultured Dupuytren's disease and normal palmar fibroblasts.

Contractile fibroblasts are believed to be responsible for palmar fascia contracture in Dupuytren's Disease. An in vitro collagen lattice model was used to examine the contractile properties of Dupuytren's fibroblasts from 10 patients undergoing partial fasciectomy, and palmar fascia fibroblasts from 6 patients undergoing carpel tunnel release. Dupuytren's and palmar fascia fibroblasts cultured within a stabilized collagen lattice acquired morphological characteristics similar to those of 'myofibroblasts' in Dupuytren's diseased fascia. Both types of fibroblasts generated contractile forces that resulted in rapid collagen lattice contraction after release of the lattice from points of stabilization. Generation of contractile force by the fibroblasts was inhibited by disruption of the actin cytoskeleton, lack of cells, or serum removal. Afferent neuropeptides (substance P, galanin and neurokinin A) did not promote lattice contraction. These results demonstrate that normal palmar fascia fibroblasts can modulate into Dupuytren's-like fibroblasts and that cultured fibroblasts, from either Dupuytren's diseased or normal palmar fascia, can generate contractile forces that are transmitted to extracellular matrix. In addition, fibroblast contraction is an actin based process which requires specific factor(s) present in serum. It is suggested that in Dupuytren's disease extracellular cues trigger the modulation of fibroblasts to Dupuytren's fibroblasts and the promotion of contractile forces responsible for palmar fascia contrature.

Distribution of ED-A and ED-B containing fibronectin isoforms in Dupuytren's disease.

Different fibronectin (FN) isoforms arise via alternate splicing of a single gene transcript in a cell- and tissue-specific manner. Antibodies were used to evaluate the presence and distribution of FN and its isoforms in Dupuytren's diseased and normal palmar fascia. Immunolocalization studies show extracellular FN fibrils, including FN isoforms containing extra domains A (A-FN) and B (B-FN), in proliferative and involutional stage Dupuytren's diseased tissue. However, B-FN appears less abundant and more restricted in its distribution as compared to A-FN or total FN. Total FN and A-FN are significantly reduced in residual tissue, while B-FN is not present. A-FN and B-FN are not present in normal palmar fascia, while total FN staining is slight and restricted to the loose connective tissue surrounding the large, parallel bundles of collagen fibers. The presence of A-FN and B-FN in Dupuytren's diseased palmar fascia represents a disease-induced appearance of these FN isoforms and further evidence of an association between Dupuytren's disease and wound healing.

Thrombin stimulates fibroblast-mediated collagen lattice contraction by its proteolytically activated receptor.

Fibroblast contraction is proposed to play an important role in tissue contraction during events such as wound healing. Thrombin has been implicated to promote force generation in fibroblasts; however, its extracellular mode of action is unclear. The purpose of this study was to determine the role thrombin and the activation of its receptor plays in promoting the contraction of human fibroblasts in an in vitro collagen lattice contraction assay. Human alpha-thrombin promoted fibroblast contraction in a dose-dependent manner with maximal activity at 0.2 nM. In contrast, both hirudin-alpha-thrombin and D-phenylalanyl-L-propyl-L-arginyl chloromethyl ketone-alpha-thrombin, which lack enzymatic activity, failed to elicit fibroblast contraction. Thus, the enzymatic activity of thrombin appears to be necessary for promotion of fibroblast contraction. Northern analysis confirmed that these human fibroblasts expressed mRNA for the human alpha-thrombin receptor. Moreover, the synthetic peptide (SFLLRNPND-KYEPF) representing the "tethered ligand" portion of the activated alpha-thrombin receptor promoted fibroblast contraction, while a control isomer peptide, in which the first two amino acids were reversed, failed to elicit this response. These findings indicate that alpha-thrombin promotes the contraction of adult human fibroblasts and that cleavage of the human alpha-thrombin receptor is sufficient to produce this response.

Diabetic and age-related enhancement of collagen-linked fluorescence in cortical bones of rats.

Nonenzymatic collagen cross-linking occurs in a variety of connective tissues as a result of formation of advanced glycosylation end products. Diabetes and aging significantly increase levels of nonenzymatic collagen cross-linking in connective tissues. This study was undertaken to determine whether nonenzymatic collagen cross-linking occurs in rat cortical bone and if these levels are increased in diabetic and aged rats. Collagen-linked fluorescence, a measurement of nonenzymatic collagen cross-linking, was significantly increased in rat cortical bone with diabetes and age. In addition, incubation of bone powder with glucose resulted in a similar increase in collagen-linked fluorescence. These changes in bone collagen may contribute to alterations observed in bone with diabetes and age by influencing bone cell function and the ability of the matrix to be responsive to bone cells.