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The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad-spectrum MMP inhibitor BB-94 has when applied topically to full-thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming-growth factor-[beta]1 (TGF-[beta]1). BB-94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB-94 dramatically increased the level of latent and active MMP-9. The increased levels of active MMP-9 may eventually overcome the ability of BB-94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB-94 was also found to inhibit the ability of latent TGF-[beta]1 to promote the formation and function of myofibroblasts. These results suggest that BB-94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte-fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF-[beta]1. [ABSTRACT FROM AUTHOR]

Fibroblasts differentiate into myofibroblasts by acquiring new contractile function. This is important for tissue repair, but it also contributes to organ fibrosis. Platelet-derived growth factor (PDGF) promotes tissue repair and fibrosis, but the relationship between PDGF and myofibroblasts is unclear. Using mice with lineage tracing linked to PDGF receptor α (PDGFRα) gene mutations, we examine cell fates during skin wound healing. Elevated PDGFRα signaling increases proliferation but unexpectedly delays the fibroblast-to-myofibroblast transition, suggesting that PDGFRα must be downregulated for myofibroblast differentiation. In contrast, deletion of PDGFRα decreases proliferation and myofibroblast differentiation by reducing serum response factor (SRF) nuclear localization. Consequences of SRF deletion resemble PDGFRα deletion, but deletion of two SRF coactivators, MRTFA and MRTFB, specifically eliminates myofibroblasts. Our findings suggest a scenario where PDGFRα signaling initially supports proliferation of fibroblast progenitors to expand their number during early wound healing but, later, PDGFRα downregulation facilitates fibroblast differentiation into myofibroblasts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tumour metastasis suppressor KAI1/CD82 inhibits tumour cell movement. As a transmembrane protein, tetraspanin CD82 bridges the interactions between membrane microdomains of lipid rafts and tetraspanin-enriched microdomains (TEMs). In this study, we found that CD82 and other tetraspanins contain cholesterol recognition/interaction amino-acid consensus (CRAC) sequences in their transmembrane domains and revealed that cholesterol binding of CD82 determines its interaction with lipid rafts but not with TEMs. Functionally, CD82 needs cholesterol binding to inhibit solitary migration, collective migration, invasion and infiltrative outgrowth of tumour cells. Importantly, CD82-cholesterol/-lipid raft interaction not only promotes extracellular release of lipid raft components such as cholesterol and gangliosides but also facilitates extracellular vesicle (EV)-mediated release of ezrin-radixin-moesin (ERM) protein Ezrin. Since ERM proteins link actin cytoskeleton to the plasma membrane, we show for the first time that cell movement can be regulated by EV-mediated releases, which disengage the plasma membrane from cytoskeleton and then impair cell movement. Our findings also conceptualize that interactions between membrane domains, in this case converge of lipid rafts and TEMs by CD82, can change cell movement. Moreover, CD82 coalescences with both lipid rafts and TEMs are essential for its inhibition of tumour cell movement and for its enhancement of EV release. Finally, our study underpins that tetraspanins as a superfamily of functionally versatile molecules are cholesterol-binding proteins. Abbreviations: Ab : antibody; CBM: cholesterol-binding motif; CCM: cholesterol consensus motif; CRAC/CARC: cholesterol recognition or interaction amino-acid consensus; CTxB: cholera toxin B subunit; ECM: extracellular matrix; ERM: ezrin, radixin and moesin; EV: extracellular vesicles; FBS: foetal bovine serum; mAb: monoclonal antibody; MST: microscale thermophoresis; pAb: polyclonal antibody; and TEM: tetraspanin-enriched microdomain., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial-mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin-like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self-renewal and chemoresistance assays in combination with overexpression and siRNA-knockdown to study the stem cell-supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT-15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence-free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self-renewal and resistance to FDA-approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV-positive cells with the novel CBT-15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1-targeted monoclonal antibodies against renal CSCs., (© 2018 UICC.)

After skin injury fibroblasts migrate into the wound and transform into contractile, extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury, their number reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. Most of these genes were linked with the extracellular region and cell periphery including genes encoding for extracellular matrix proteins. A unique panel of core matrisome and matrisome-associated genes was differentially expressed in myofibroblasts and several genes not yet known to be linked to myofibroblast-mediated wound healing were found (e.g. Col24a1, Podnl1, Bvcan, Tinagl1, Thbs3, Adamts16, Adamts19, Cxcl's, Ccl's). In addition, a complex network of G protein-coupled signaling events was regulated in myofibroblasts (e.g. Adcy1, Plbc4, Gnas). Hence, this first characterization of a myofibroblast-specific expression profile at the peak of in situ granulation tissue formation provides important insights into novel target genes that may control excessive ECM deposition during fibrotic reactions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Doublecortin-like kinase 1 (DCLK1) is a gastrointestinal (GI) tuft cell kinase that has been investigated as a biomarker of cancer stem-like cells in colon and pancreatic cancers. However, its utility as a biomarker may be limited in principle by signal instability and dilution in heterogeneous tumors, where the proliferation of diverse tumor cell lineages obscures the direct measurement of DCLK1 activity. To address this issue, we explored the definition of a miRNA signature as a surrogate biomarker for DCLK1 in cancer stem-like cells. Utilizing RNA/miRNA-sequencing datasets from the Cancer Genome Atlas, we identified a surrogate 15-miRNA expression signature for DCLK1 activity across several GI cancers, including colon, pancreatic, and stomach cancers. Notably, Cox regression and Kaplan-Meier analysis demonstrated that this signature could predict the survival of patients with these cancers. Moreover, we identified patient subgroups that predicted the clinical utility of this DCLK1 surrogate biomarker. Our findings greatly strengthen the clinical significance for DCLK1 expression across GI cancers. Further, they provide an initial guidepost toward the development of improved prognostic biomarkers or companion biomarkers for DCLK1-targeted therapies to eradicate cancer stem-like cells in these malignancies. Cancer Res; 76(14); 4090-9. ©2016 AACR., Competing Interests: Courtney Houchen and Edwin Bannerman-Menson are co-founders of COARE Biotechnology Inc., (©2016 American Association for Cancer Research.)

Renal clear cell carcinoma (RCC) is the most common type of kidney cancer and the 8th most common cancer overall in the US. RCC survival rates drop precipitously with regional and distant spread and recent studies have demonstrated that RCC presents an epithelial-mesenchymal transition (EMT) phenotype linked to increased recurrence and decreased survival. EMT is a key characteristic of tumor stem cells (TSCs) along with chemo-resistance and radio-resistance, which are also phenotypic of RCC. Targeting these factors is key to increasing the survival of RCC patients. Doublecortin-like kinase 1 (DCLK1) marks TSCs in pancreatic and colorectal cancer and regulates EMT and stemness. Analysis of the Cancer Genome Atlas' RCC dataset revealed that DCLK1 is overexpressed and dysregulated on the mRNA and epigenetic level in more than 93% of RCC tumors relative to adjacent normal tissue. Immunohistochemistry using α-DCLK1 antibody confirmed overexpression and demonstrated a major increase in immunoreactivity in stage II-III tumors compared to normal kidney and stage I tumors. Small-interfering RNA (siRNA) mediated knockdown of DCLK1 resulted in decreased expression of EMT and pluripotency factors and significantly reduced invasion, migration, focal adhesion, drug-resistance, and clonogenic capacity. These findings suggest that DCLK1 is a novel, overexpressed factor in RCC progression that may be targeted to suppress EMT, metastasis, and stemness in early-stage and advanced RCC to increase patient survival. Moreover, the possibility that DCLK1 may mark a population of tumor stem-like cells in RCC should be further investigated in light of these findings.

Wound healing, angiogenesis, and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with antiangiogenic activities, were elevated in type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound-healing deficiency via the inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair-follicle density, microvascular density, and panniculus adiposus layer thickness, as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in the dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via inhibition of glycogen synthase kinase-3β, reversed the inhibition of Wnt/β-catenin signaling by kallistatin and rescued the wound-healing deficiency in KS-TG mice. These observations suggest that elevated circulating antiangiogenic serpins in diabetic patients may contribute to impaired wound healing through inhibition of Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling, at a level downstream of Wnt receptors, may ameliorate the wound-healing deficiency in diabetic patients.

Significance: Myofibroblasts are responsible for wound closure that occurs in healed acute wounds. However, their actions can result in disfiguring scar contractures, compromised organ function, and a tumor promoting stroma. Understanding the mechanisms regulating their contractile machinery, gene expression, and lifespan is essential to develop new therapies to control their function., Recent Advances: Mechanical stress and transforming growth factor beta-1 (TGF-β1) regulate myofibroblast differentiation from mesenchymal progenitors. As these precursor cells differentiate, they assemble a contractile apparatus to generate the force used to contract wounds. The mechanisms by which mechanical stress promote expression of contractile genes through the TGF-β1 and serum response factor pathways and offer therapeutic targets to limit myofibroblast function are being elucidated., Critical Issues: Emerging evidence suggests that the integration of mechanical cues with intracellular signaling pathways is critical to myofibroblast function via its effects on gene expression, cellular contraction, and paracrine signaling with neighboring cells. In addition, while apoptosis is clearly one pathway that can limit myofibroblast lifespan, recent data suggest that pathogenic myofibroblasts can become senescent and adopt a more beneficial phenotype, or may revert to a quiescent state, thereby limiting their function., Future Directions: Given the important role that myofibroblasts play in pathologies as disparate as cutaneous scarring, organ fibrosis, and tumor progression, knowledge gained in the areas of intracellular signaling networks, mechanical signal transduction, extracellular matrix biology, and cell fate will support efforts to develop new therapies with a wide impact.

The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts., (© 2012 by the Wound Healing Society.)

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 β-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.

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.

Purpose: Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility within Dupuytren's nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytren's fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytren's disease progression and suggests potential targets for treatment., Methods: We enmeshed Dupuytren's fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytren's fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB., Results: Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytren's nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia., Conclusions: Sphingosine-1-phosphate promotes Dupuytren's fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren's nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment., (Copyright © 2010 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.

Purpose: Vascular smooth muscle (SM) cells and pericytes are essential for normal vascular development. SM alpha-actin null mice were used to determine whether vascular SM and pericyte contractile functions, and not merely their presence, are necessary for vascular development, normal blood-retina barrier (BRB) permeability, and retinal function., Methods: Age-matched SM alpha-actin null and wild-type mice were analyzed. Retinal structure, vascular pattern, and SM cell and pericyte distribution were analyzed histologically. Retinal vascular permeability (RVP) was measured with the Evans blue dye method. Electroretinography (ERG) was performed to evaluate retinal function., Results: Deletion of SM alpha-actin did not result in any alterations in retinal morphology, vascular pattern, or SM cell and pericyte ensheathing of vessels in SM alpha-actin null mice. A significant increase in RVP was observed in SM alpha-actin null mice at both postnatal day (P)50 and P75 (P<0.05 and P<0.001, respectively). ERG analysis demonstrated a significant reduction in both rod and cone function in SM alpha-actin null mice at P22, P45, and P75 (P<0.01 at all ages)., Conclusions: These results demonstrate that SM alpha-actin in SM cells and pericytes is not necessary for the formation of a normal retinal vascular pattern; however, SM alpha-actin is necessary for SM cells and pericytes to interact with endothelial cells to form a fully functional BRB. These results are important in understanding the role of contractile gene expression in the maintenance and function of the BRB and may provide a model for studying pathologic conditions, such as diabetes, that alter the function of this barrier.

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.

IL-6 deficient (IL-6KO) mice display significantly delayed cutaneous wound closure. Myofibroblasts are the primary mediators of wound closure, and alpha-smooth muscle actin (alpha-SMA) is a marker of fibroblast differentiation to the myofibroblast phenotype. Wounds from IL-6KO, and wild-type mice were collected up to 6 days following wounding. Expression of alpha-SMA mRNA was found to be increased in wounds of IL-6KO mice up to 48 hours post wounding, but decreased below wild-type levels by 72 hours. Recombinant IL-6 treatment of IL-6KO dermal fibroblasts showed an induction of alpha-SMA mRNA and protein peaking at 1 ng/ml cytokine, but declining at higher concentrations. Actinomycin-D treatment of fibroblast cultures indicated that recombinant mouse IL-6 (rmIL-6) induction of alpha-SMA mRNA appeared to be primarily transcriptionally regulated, and extracellular signal-regulated kinase 1/2 kinase, but not signal transducers and activators of transcription 3 was readily phosphorylated in rmIL-6 treated IL-6KO fibroblasts. A dose-response increase in the mRNA expression of the IL-6R signaling inhibitor protein suppressors of cytokine signaling (SOCS) 3 was also noted in rmIL-6-treated IL-6KO fibroblasts. These data indicate that alpha-SMA expression is dysregulated in IL-6KO mice. The expression of alpha-SMA induced by rmIL-6 in fibroblasts from IL-6KO mice appears to be transcriptionally modulated, dependent on JAK1 kinase, and possibly downregulated as a result of increased SOCS3 expression.

Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of alpha-smooth muscle actin (alpha-SMA); however, little is known regarding molecular mechanisms that control activation of alpha-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for alpha-SMA expression in myofibroblasts in vivo and to determine whether activation of alpha-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)6GG] and a transforming growth factor-beta1 control element (TCE) that are required for alpha-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 alpha-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-beta1 in cultured transgenic fibroblasts. These same elements were also critical in regulating alpha-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 alpha-SMA expression in myofibroblasts in granulation tissue.

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.

In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm2) on the back of 22 young female Sprague-Dawley rats, 12 treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered keratinocyte proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myofibroblasts in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming growth factor-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal fibroblasts. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that keratinocyte resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes., (Copyright 2004 Elsevier Inc.)

Purpose: Clinically bladder cells used in tissue engineering techniques will come from neuropathic bladders and not normal bladders. We determined if neuropathic bladder smooth muscle (SM) cells (SMCs) retain functional differences when cultured in vitro., Materials and Methods: Primary cultures of SMCs were established from patients with a neuropathic bladder (5) and a normal bladder (5). Expression of alpha-SM actin and SM myosin heavy chain was determined using immunocytochemical staining and Western blot analysis. Baseline cell proliferation and the mitogenic response to angiotensin II was assessed by cell counting and cell viability assays. Cell contractility was determined for normal and neuropathic SMCs using an in vitro collagen lattice assay. Cell adherence was measured assessed using partial and complete trypsinization assays., Results: Normal and neuropathic SMCs showed similar morphology in culture, and similar patterns of alpha-SM actin and SM myosin expression. Following 10 days of plating under optimal growth conditions the number of neuropathic SMCs was 170% more than normal SMCs. In response to angiotensin II neuropathic SMCs reached 54% of maximal growth capacity as opposed to 30% for normal SMCs (p <0.01). Neuropathic SMCs contracted significantly less in 10% serum and calcium ionophore (p <0.05), as determined by in vitro contractility assays. Neuropathic SMCs had 19% and 30% less adherent cells than normal SMCs (p <0.01) following isotonic solution washes and trypsinization, respectively., Conclusions: These results demonstrate that cultured neuropathic bladder SMCs possess and maintain different characteristics than normal SMCs in vitro. The potential clinical implications of using these cells in conjunction with tissue engineering techniques for the promotion of bladder regeneration requires further investigation.

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.

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.

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.

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.

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., (Copyright 2000 Academic Press.)

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., (Copyright 2000 by the American Society for Surgery of the Hand.).)

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.

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.

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.

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.

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 (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.

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.

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.

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 (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.

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.

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.

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.

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.

Fibroblasts cultured within free-floating collagen gels can bind to and reorganize the surrounding collagen fibrils into a more dense and compact arrangement. Collagen gel contraction provides an in vitro model for studying fibroblast-collagen interactions important in wound healing, fibrosis, scar contraction, and connective tissue morphogenesis. We have assessed the role of fibronectin and its interaction with the alpha 5 beta 1 "high affinity" fibronectin-specific integrin receptor in collagen gel contraction. A variety of agents, which specifically inhibit fibronectin-alpha 5 beta 1 interactions, were tested for their abilities to inhibit fibroblast-mediated collagen gel contraction. These included anti-alpha 5 beta 1 monoclonal antibodies, the synthetic peptide GRGDSP, the cell adhesive fragment of fibronectin, and an antibody against the cell adhesive region of fibronectin. None of these agents inhibited collagen gel contraction. Therefore, it is concluded that fibronectin-alpha 5 beta 1 interactions are not necessary for collagen gel contraction. However, collagen gel contraction is dependent on a member or members of the beta 1 subfamily of integrin matrix receptors. A polyclonal antiserum and a monoclonal antibody, both directed against the beta 1 subunit of integrin matrix receptors, inhibited the spreading of fibroblasts in the collagen gel and inhibited collagen gel contraction. This study demonstrates that fibroblast-mediated collagen gel contraction is independent of fibronectin-alpha 5 beta 1 interactions but dependent on an interaction of beta 1 integrin matrix receptors with collagen fibers.

The generation of tension in granulation tissue undergoing contraction is believed to be a cell-mediated event. In this study we used attached collagen lattices as a model system for studying the cellular mechanisms of tension generation by fibroblasts in an extracellular matrix. Fibroblasts in attached collagen lattices developed stress fibers, surface associated fibronectin fibrils, and a fibronexus-like transmembrane association interconnecting the two structural components. Release of the attached collagen lattice from its points of attachment resulted in a rapid, symmetrical contraction of the collagen lattice. Rapid contraction occurred within the first 10 minutes after release of the lattice from the substratum, with greater than 70% of the contraction occurring within the first 2 minutes. Rapid contraction resulted in a shortening of the elongate fibroblasts and compaction of the stress fibers with their subsequent disappearance from the cell. Cytochalasin D treatment prior to release disrupted the actin cytoskeleton and completely inhibited rapid contraction. The removal of serum prior to release inhibited rapid contraction, while the re-addition of serum restored rapid contraction. These results demonstrate that fibroblasts can develop tension in an attached collagen lattice and that upon release of tension the fibroblasts undergo contraction resulting in a rapid contraction of the collagen lattice. Fibroblast contraction is dependent upon an organized actin cytoskeleton and is promoted by the presence of serum.

The fibronexus is a close transmembrane association between fibronectin filaments and actin microfilaments. It has been found at the surfaces of fibroblasts in tissue culture, as well as within contracting granulation tissue. This specialized connection has been proposed to play an important role in the adhesive properties of fibroblasts. The purpose of this study is to determine whether the fibronexus is present in other contracting tissues besides granulation tissue, specifically in Dupuytren's diseased tissue. Dupuytren's disease is a pathologic condition in which the palmar aponeurosis becomes shortened leading to irreversible flexion of the digits. Shortening of the aponeurosis is believed to be an active cellular process. Extracellular filaments and actin microfilaments form close transmembrane associations at the surfaces of actin-rich fibroblasts in Dupuytren's disease. Extracellular filaments extend from the cell surface into the surrounding tissue connecting fibroblasts with collagen fibrils and adjacent cells. In this study we have used immunoelectron microscopy to demonstrate that the extracellular filaments that participate in these close transmembrane associations contain fibronectin. High voltage electron microscopy has been used to examine the three-dimensional relationships between the cytoskeleton and fibronectin filaments in Dupuytren's diseased tissue. We propose that the fibronexus is a dominant adhesive structure at the surface of fibroblasts in Dupuytren's diseased tissue. The fibronexus, by mediating cell-to-cell and cell-to-matrix attachments, may serve to transmit contractile forces generated by actin microfilaments in these cells throughout the diseased tissue.

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.

Cells of metazoan organisms produce and react to complex macromolecular microenvironments known as extracellular matrices. Assembly in vitro of native, compositionally nonuniform collagen-fibronectin matrices caused translocation of certain types of cells or polystyrene-latex beads from regions lacking fibronectin into regions containing it. The translocation process was not due to diffusion, convection, or electrostatic distribution effects, but may depend on nonequilibrium phenomena at the interface of contiguous collagen matrices formed in the presence and absence of fibronectin or particles. Extracellular matrix formation alone was sufficient to drive translocation by a biophysical process that may play a role in cellular migration during embryogenesis, as well as in other types of tissue reorganization such as inflammation, wound healing, and tumor invasion.