bluuebunny/crossref_metadata_2025 huggingface dataset:
The N‐Terminal Ac‐EEED Sequence Plays a Role in α‐Smooth Muscle Actin Incorporation into Stress Fibers
Authors: Clément S , Hinz B , Dugina V , Gabbiani G , Chaponnier C
Published: January 2005

The main differences between the six known actin isoforms are located in their amino terminus, suggesting a crucial importance for this region in terms of actin isoform function and organization. We have recently supported this assumption by demonstrating that the α‐smooth muscle actin N‐terminal sequence AcEEED introduced in myofibroblasts as a cell‐penetrating fusion peptide causes: (i) the loss of α‐smooth muscle actin staining in stress fibers; and (ii) a decrease in cell contractility both in vitro and in vivo. Here we have further investigated the function of this sequence on stress fiber organization in living fibroblastic cells, using enhanced green fluorescent protein (EFGP)‐tagged α‐smooth muscle actin. Approximately 30 minutes after application, the fusion peptide provokes gradual disappearance of α‐smooth muscle actin‐EGFP fluorescence in stress fibers correlating temporally with the formation of rod‐like structures of 2–20 μm length in approximately 60% of cells. Characterization of the α‐smooth muscle actin‐containing rod‐like structures shows that they additionally stain for β‐ and γ‐cytoplasmic actins, gelsolin and cofilin; in contrast, other major actin‐binding proteins, such as α‐actinin are absent from these structures. Interestingly rod‐like structures are visible at submembranous cytoplasmic portion of normal cells during initial phases of spreading, suggesting that they represent a physiological step in α‐smooth muscle actin organization before incorporation in stress fibers. Fluorescence recovery after photobleaching experiments suggest that the fusion peptide reduces α‐smooth muscle actin dynamics and incorporation in stress fibers, rather than releasing it from stress fibers.
PDGF‐Stimulated Fibroblast Matrix Contraction is Mediated Through Direct Stimulation of Cell Protrusion and Ruffling Activity
Authors: Dahlmann AH , Bailly M , Khaw PT
Published: January 2005

Scarring of the cornea, the transparent tissue at the front of the eyeball, is an important cause of visual impairment. A major chemokine upregulated in tear fluid after corneal injury is platelet‐derived growth factor (PDGF), which induces the stromal cells of the cornea (keratocytes) to assume a fibroblastic phenotype. We have investigated the role of PDGF in matrix contraction by human corneal fibroblasts (HCF) using the standard three‐dimensional (3D) fibroblast‐populated collagen matrix model (FPCM).

When stimulated with PDGF, HCF in monolayer (2D) display circular ruffles (CR) on their dorsal surface. Using immunolabeling, we found that CR contain proteins of the Arp2/3 complex, which is required for the assembly of filamentous actin during protrusive activity. This PDGF‐mediated ruffling ability is altered by antibodies or blocking peptides that specifically interfere with the Arp2/3‐mediated pathway of actin polymerization, as well as by the myosin light chain kinase inhibitor, ML9. Preincubation with anti‐PDGF‐BB antibody or with the tyrosine kinase inhibitor, AG1295, also inhibits CR. While PDGF efficiently stimulates HCF‐mediated cell protrusive activity and macroscopic collagen gel contraction in the 3D model, this effect is directly inhibited by all the substances which inhibit CR in 2D, demonstrating a direct involvement of the PDGF‐mediated CR pathway in tissue contraction. Thus, 3D PDGF‐induced matrix contraction by HCF involves the same players as CR, a phenomenon observed in 2D cell cultures: PDGF‐BB receptor, receptor tyrosine kinase, the machinery for filamentous actin assembly (Arp 2/3 complex, Scar and Wasp proteins), and myosin. The functional role of circular ruffling or its equivalent in three‐dimensional fibroblast‐populated collagen matrices seems to be a rapid reorganization of the actin cytoskeleton in preparation for cell‐matrix interaction.
Cytoplasmic Actins β and γ are Differentially Distributed in Stationary and Motile Cells
Authors: Dugina V , Gabbiani G , Chaponnier C
Published: January 2005

We have compared the distribution of β‐ and γ‐cytoplasmic actins in highly motile cells using laser confocal microscopy and newly developed β‐ and γ‐cytoplasmic actins monoclonal antibodies as well as the commercially available β‐actin antibody (clone Ac74).

When fibroblastic, epithelial or endothelial cells attach on a substrate, divide, move into a wound, they have to organize their actin cytoskeleton in different types of structures such as in cell protrusions, in stress fibers or in circular bundles, e.g., during spreading. Interestingly, β‐ and γ‐cytoplasmic actins get organized in a complete different way, spatially and structurally. γ‐Cytoplasmic actin is mainly organized as a network under the dorsal membrane and in motile part of the cell (e.g., in lamellipodia); on the other hand, β‐actin is detected in the basal but not in the dorsal portion of the cell, is organized in radial cables at the cell‐cells contact in epithelial cells, in stress fibers in fibroblastic and endothelial cells and in circular bundles during cell spreading.

It is known that the only sequence differences between the two cytoplasmic actins concern four amino acids located at the N‐terminus nonapeptide. Our results suggest that the N‐terminal domain of cytoplasmic actins are of major importance in allowing their differential cellular organisation in distinct cell activities, probably through an interaction with specific actin binding proteins.
Collagen Organization Contracts Wounds
Authors: Ehrlich HP , Mackay DR
Published: January 2005

Wound contraction is associated with collagen organization. Is cell contraction or cell tractional forces responsible for collagen organization? Myofibroblasts (Mfibs) are identified by αSMA stained stress fibers. Fibroblast populated collagen lattice (FPCL) contraction can occur by cell contraction or tractional forces. FPCL cast under identical conditions are either released in 1 hour after manufacture, free‐floating (FF) FPCL or released on day 4, attached delayed released (ADR) FPCL. FF‐FPCL contraction, completed in 2 days in the absence of cell contraction, shows collagen organization but no αSMA staining. ADR‐FPCL contraction, completed in less than 2 hrs had αSMA stained contracted cells, but no collagen organization. Vanadate treated FF‐FPCL contract with collagen organization. Vanadate treated ADR‐FPCL showed impaired contraction, few αSMA stained contracted cells and no organized collagen. Apparently, ADR‐FPCL contraction requires αSMA contracting cells. At confluence almost all low density (LD) plated fibroblasts express αSMA. Vanadate or a native collagen blanket placed on confluent LD plated cells eliminates αSMA staining. With tension fibroblasts in collagen express αSMA. Without tension collagen eliminates αSMA expression. By day 7 open rat wounds close by 50% with organized collagen, but no Mfibs. Vanadate treated rat wounds close at the same rate as untreated wounds, but no Mfibs ever appear. At 3 wks the wall of the rat crotin oil granuloma is exclusively populated with Mfibs. Removal of aliquots of wound fluid daily for 3 days leads to granuloma compaction. Collagen fibers show increased organization and Mfibs are replaced by fibroblasts. It is proposed that Mfibs are associated with fibroblasts entering apoptosis. Death associated protein (DAP) kinases are a family of kinases that induce programmed cell death and phosphorylate myosin light chains (MLC). MLC phosphorylation leads to αSMA expression. The triggers for generating αSMA in stress fibers is tissue placed under tension and/or induced apoptosis through a DAP kinase. The functions of Mfibs are to counteract transient changes in tensions within granulation tissue and to prepare cells to enter apoptosis. Wound contraction advances by fibroblast organization of collagen in the absence of cell contraction.
Thrombin Inhibits the Migration of Human Liver Myofibroblasts
Authors: Gillibert‐Duplantier J , Neaud V , Desmoulière A , Bioulac‐Sage P , Rosenbaum J
Published: January 2005

Liver myofibroblasts are the key cells of liver fibrogenesis. Recent data show that the serine proteinase thrombin is involved in fibrogenesis through a mitogenic effect on myofibroblasts. The aim of this study was to evaluate the effects of thrombin on the migration of human liver myofibroblasts; another major parameter in fibrogenesis.

In a Boyden chamber assay, thrombin dose‐dependently (10−10–10−7 M) decreased spontaneous myofibroblast migration down to 49 ± 1% of control values (p = 5.10−10) without affecting cell adhesion or viability. Thrombin effect was blocked by its specific catalytic inhibitor hirudin and could be reproduced by using the proteinase‐activated receptor‐1 (PAR‐1) agonist SFLLRNP. Thrombin also completely inhibited migration when induced by the chemotactic agent platelet‐derived growth factor‐BB. We then investigated the signaling mechanisms involved. The COX‐2 inhibitor NS398 dose‐dependently (2.5–10 μM) blunted the inhibitory effect of thrombin on spontaneous migration. However, NS398 did not reverse the inhibitory effect of thrombin on PDGF‐BB‐induced migration. Phosphatidylinositol 3‐kinase (PI3‐K) is a major determinant of chemotaxis induced by PDGF. Whereas thrombin did not itself induce PI3‐K activity, as shown by the lack of detectable phosphorylation of Akt‐1, it inhibited PDGF‐BB‐induced Akt‐1 phosphorylation. This effect was dependent of the Rho/ROCK pathway since it was abolished in the presence of the ROCK inhibitor Y‐27632.

In summary, thrombin, acting via a proteinase‐activated receptor, inhibits human liver myofibroblasts migration. Inhibition of basal migration is dependent on COX‐2, while inhibition of PDGF‐BB‐induced migration involves decreased PI3‐K activation via a Rho/ROCK mechanism. We suggest that thrombin could thus stabilize activated myofibroblasts on the site of active fibrogenesis.
Myofibroblasts in Dense Model Collagen Matrices: Migration, Proliferation, Apoptosis
Authors: Helary C , Ovtracht L , Godeau G , Coulomb B , Giraud Guille MM
Published: January 2005

Fibrillar collagen matrices, that mimic the supramolecular organization of collagen in tissues, are obtained by using acid soluble collagen concentrated at 40 mg/ml and then forming fibrils at neutral pH. They present mechanical properties close to that of human dermis (1). We have previously shown that 150.000 human dermal fibroblasts seeded at the surface of collagen matrices migrate and attain a density of 5500 cells/mm3 after 28 days (2).

The present work studies the phenotype of the cells migrating into dense, compared to loose (5 mg/ml) collagen networks. We demonstrate that cell migration is associated to matrix hydrolysis by MT1‐MMP on the cell front and gelatinase A (MMP2) that allow a migration distance of 350 μm after 28 days. Immunodetection of alpha‐smooth muscle actin evidences a myofibroblast phenotype for all cells penetrating both 40 and 5 mg/ml matrices. In parallel, we observe collagen I and fibronectin synthesis, typical of myofibroblasts. The TUNEL test reveals 43% of apoptotic cells in dense matrices after 28 days and a total absence of apoptosis in loose matrices. BrdU incorporation in myofibroblasts shows an intense proliferation in the dense collagen matrices that compensates cell death. Indeed the number of cells after one month of culture represents three times the number initially seeded.

To conclude, in our culture conditions using 10% foetal calf serum, the stiffness of the matrix seems to be a stimulus of apoptosis; futhermore cells which express alpha‐SM actin are both able to proliferate and migrate.
Masters and Servants of the Force: Focal Adhesion Size Controls Recruitment of α‐Smooth Muscle Actin to Stress Fibers
Authors: Goffin J , Csucs G , Lussi J , Meister JJ , Hinz B
Published: January 2005

Mechanical stress is a crucial factor for modulating fibroblasts into differentiated myofibroblasts, which are characterized by expression of α‐smooth muscle actin (α‐SMA). Incorporation of α‐SMA into stress fibers generates high contractile activity and drives the formation of “supermature” focal adhesions (FA) that are considerably longer (6–30 μm) compared with “classical” FAs (2–6 μm) of α‐SMA‐negative fibroblasts. We here show that in turn, supermature FAs control myofibroblast differentiation by communicating the level of extracellular matrix stress to the cytoskeleton. Culture on compliant silicone substrates reduces the size of supermature FAs to that of classical FAs and leads to a concomitant decrease of α‐SMA expression. Incorporation of α‐SMA into stress fibers requires the formation of FAs longer than 6 μm as demonstrated by plating myofibroblasts on arrays of adhesive islets with dimensions ranging from 1.5 × 2–20 μm and spacing between 2–6 μm, which are created on rigid culture surfaces by means of microcontact printing (μCP). Stretching 6 μm islets on flexible silicone membranes to 8 μm length induces stress fiber formation of α‐SMA‐EGFP transfected fibroblasts; this was not achieved by applying the same stretch (30%) to cells initially grown on 4 μm islets. By analyzing local deformations created in deformable micropatterned substrates by paxillin‐EGFP transfected myofibroblasts, we determined a linear relationship between the size of supermature FAs and local force exertion; hence we determined the minimal tension at individual supermature FAs required for α‐SMA recruitment.
Pharmacological and Mechanical Effects on Collagen Lattice Contraction — the Oxford Experience
Authors: Cherry GW , Parish J , Hu D , Jia C , Phan TT , Hughes MA
Published: January 2005

Wound contraction is part of the normal process in tissue repair, although the contribution it makes to wound closure, compared with that of scar deposition, can vary in different types of wounds and in different subjects. The contributions of fibroblasts and myofibroblasts to the contraction process may also vary in different types of wound. In some instances it could be beneficial to slow down contraction and in others to accelerate it. We have investigated the effect of a number of agents, pharmacological or mechanical, on the contraction of fibroblast‐populated collagen lattices. Minoxidil concentrations from 0.5 to 4 mM inhibited contraction of lattices by leg fibroblasts from a 74‐year‐old subject in a dose‐dependent manner, whereas 0.5 mM had no effect on the contraction of lattices by neonatal foreskin fibroblasts. The inhibition was reversible when the minoxidil was replaced by normal medium after six days. Minoxidil also inhibited contraction by porcine wound fibroblasts as well as that by normal porcine skin. Minoxidil glucuronide, the major metabolite, had no effect on collagen contraction, but minoxidil sulphate inhibited contraction more strongly than minoxidil itself. Tamoxifen at 10–20 μM led to a dose‐dependent reduction in contraction, while 1 to 5 μM tamoxifen had no effect and 50 or 100 μM produced complete inhibition. The reversibility of the inhibition was dose dependent and, for a given concentration, time‐dependent. Eupolin, an aqueous extract from Chromolaena odorata, a plant licensed for the treatment of soft‐tissue wounds in Vietnam, was shown to effect a dose dependent decrease in contraction at concentrations from 50–200 μg/ml. Intermittent radiant warming, as used in the clinic, accelerated the contraction of lattices by fibroblasts from 54‐year‐ and 87‐year‐old subjects, maintained at an incubator temperature of 33.2 °C. Control cards set at 38 °C or 42 °C raised the temperature of the medium to maxima of 36.2 °C and 38.1 °C after 1 hour. Warming the lattices for three periods of 1 hour per day for one week led to significantly faster contraction and increased proliferation within the lattice.
Comparison of Different Wound Dressings on Cultured Human Fibroblasts and Collagen Lattices
Authors: Humbert P , Viennet J , Bride J , Gabiot AC
Published: January 2005

Introduction:  The cellular phases (granulation, reepithelialization, and dermal remodelling) of the healing process involve many cell types. Fibroblasts and myofibroblasts are the key cells in granulation tissue formation and wound contraction.

Objective:  To compare the effects on cultured human fibroblasts of a new nonadhesive lipidocolloid wound dressing, Urgotul®, with five other wound dressings including impregnated gauzes and some other modern wound dressings.

Method:  Cultures in monolayer were used to study the morphology and growth of fibroblasts. The Bell model of cultured dermis equivalents was used to investigate myofibroblast differentiation. These cultures were labelled α‐SM actin and F‐actin.

Results:  Two of the tested dressings induced cytotoxic effects on the fibroblasts. They were found to inhibit cell growth (greater than 60%) and to disturb cell shape and cytoskeletal differentiation. Urgotul® and the remaining three dressings showed no effect on proliferation. However some of them modified fibroblast morphology and affected F‐actin distribution.

Conclusion:  Depending on their nature and components, wound dressings may respect or affect in vitro fibroblast behaviour (proliferation, morphology, and α‐SM actin and F‐actin distribution). The observed in vitro findings require further investigations.
Differential Response of Fibroblasts from Type I Diabetics to Proliferative and Migratory Signals
Authors: Vallury M , Zaher H , Lindblad WJ
Published: January 2005

Type I diabetics experience significant morbidity from failure of lower extremity injuries to heal. This impaired healing response leads to chronic ulceration, infection and potential amputation of the extremity. Numerous hypotheses have been proposed to explain this impaired healing response, but little is known about intrinsic differences in the fibroblasts populating the diabetic extremity prior to wounding.

Nontransformed fibroblast cell lines from type I diabetics were obtained from the NIGMS‐NIH cell repository at the Coriell Institute. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin, streptomycin and either 450 mg/dL (high) or 100 mg/dL (low) glucose. Cell migration was measured over 72 hr by movement of cells away from a circular source of cells, deposited within cloning rings, under high or low glucose conditions. The initial (0, 6, 24 and 48 hr) proliferative response of cells was measured by 3H‐thymidine incorporation into confluent cell monolayers following partial scraping of the cell layer. Growth curves were obtained by seeding 50,000 cells into 6‐well plates and counting cells at 24, 48, 72, and 168 hrs.

Fibroblasts from diabetic individuals showed impaired migration compared to nondiabetic cells (34% vs. 67% increase in diameter) in high glucose over an initial 72 hrs. This difference was minimized if the cells were cultured and tested in low glucose medium. In contrast, diabetic derived cells proliferated more rapidly at early times (13.5%[24 hr], 97%[48 hr], 209%[72 hr] diabetic vs. 4%, 52.5%, 156% increase in cell number) when cultured under high glucose conditions. This trend continued under low glucose conditions, although the magnitude difference was diminished. These results suggest that the fibroblast population present in the type I diabetic connective tissue possesses a phenotype distinctly different from normal cells, characterized by a reduced capacity to migrate, but a normal or heightened proliferative response. This may indicate that injury to this connective tissue fails to produce the required influx of fibroblasts to deposit new matrix.
Activated Fibroblasts are Required for Extracellular Matrix Remodeling in the Human Cervix
Authors: Malmström A , Ekman‐Ordeberg G
Published: January 2005

The normal ripening of the human cervix is characterized by rapid degradation of the extracellular matrix, where fibroblasts play an important role. To accomplish this, the fibroblasts undergo phenotypic changes, generally characterized by decreased synthesis of ECM molecules and increased production of degrading enzymes. The hypothesis of this study is that fibroblasts from cervices at different stages of remodeling are of different phenotypes. Fibroblasts were obtained from nonpregnant women, women after 36 weeks of pregnancy and women directly after partus (postpartal) and kept for at least three–four passages to obtain stable conditions prior experiments were performed. The cells were immunostained for differentiation markers, screened for cytokine and MMP expression and production, followed by a global proteomic search. In accordance with in vivo data, a decrease of α‐smooth muscle actin and 4‐prolyl‐hydroxylase was noted by immunohistochemical staining with time of pregnancy. The fibroblasts established from postpartal donors had a three‐ to fourfold increase of interleukin 6 (IL‐6). The production of the chemotactic interleukin 8 (IL‐6) was increased even further, five‐ to sixfold, in postpartal fibroblast cultures. This correlates well to the events in vivo, where IL‐6 and Il‐8 is produced in high amounts by cervical fibroblast. In addition also the secretion metalloproteinase 1 and 3 are increased threefold in postpartal cultures, which is in agreement with in vivo events. Intermediate amounts of interleukins and metalloproteinases are produced in culture obtained after 36 weeks of pregnancy. A global proteomic approach resulted in regulated proteins involved in Ca‐signaling, cytoskeletal mobility and apoptosis. Taken together these data suggest that remodeling of cervix is achieved by a transient activation or recruitment or differentiation of active fibroblasts usually found in inflammatory conditions, characterized by increased MMP and cytokine expression.
Hepatitis C Virus Infection is Highly Correlated with Hepatocellular Apoptosis and Transforming Growth Factor‐β1 MRNA Expression in Patients with Chronic Hepatitis C
Authors: Manabe N , Goto Y , Anan S , Suzuki T , Matsuura Y , Miyamura T
Published: January 2005

To determine whether hepatocellular apoptosis is involved in chronic liver injury in patients with chronic hepatitis C, the correlations among the rate of hepatocellular apoptosis, the rate of hepatocytes infected with hepatitis C virus (HCV) and TGF‐β1 mRNA expression, were histologically examined. HCV was visualized by in situ PCR technique. Apoptotic hepatocytes were determined by TUNEL technique. TGF‐β1 mRNA expression was quantified by Northern blotting analysis, and visualized by in situ hybridization. Fas‐antigen and proliferating cell nuclear antigen (PCNA) on hepatocytes, and α‐smooth muscle actin (αSMA) and desmin were evaluated by immunohistochemistry. In the areas of inflammation, TUNEL‐positive apoptotic hepatocytes and PCNA‐positive hepatocytes were occasionally observed around HCV‐positive hepatocyte. TGF‐β1 mRNA‐positive myofibroblast‐like Ito cells (double‐positive for αSMA and desmin) were also observed near the HCV‐positive hepatocytes. Fas‐antigen was detected on the surface of most hepatocytes in all lobules. A high correlation was found between the rate of HCV positive hepatocytes and that of apoptotic hepatocytes (r = 0.81, P < 0.05). The increase in number of HCV‐positive hepatocytes was linked to the increases in TGF‐β1 mRNA level (r = 0.79, P < 0.05) and number of TGF‐β1 mRNA‐positive Ito cells (r = 0.74, P < 0.05). These findings confirmed that the increase in HCV‐infected hepatocytes is related to the increase of hepatocellular apoptosis and the increase of TGF‐β1 mRNA in the liver of hepatitis C patients. TGF‐β1 plays an important role in apoptosis of hepatocytes infected with HCV.
The Role of Fibroblasts in Skin Substitutes
Authors: Middelkoop E , Van Den Bogaerdt A , Ulrich M
Published: January 2005

Skin substitutes are slowly finding a position in the treatment of burns, scar reconstructions and chronic wounds. Some of the substitutes consist of extracellular matrix replacement material only, such as Integra and Alloderm; some include allogeneic cells (Dermagraft, Appligraf). The ideal skin substitute has not been developed yet, since none of the presently available products can ultimately prevent scar formation.

Study of the role of autologous fibroblasts in the healing process might give further insight into the scarring process and eventually lead to improved skin substitutes.

We compared wound reepithelialization of experimental wounds treated with proliferating keratinocytes and a dermal substitute with either dermal fibroblasts, adipose tissue derived fibroblasts or no fibroblasts. We also investigated the rate of keratinocyte migration of human skin equivalents cultured in vitro in the presence of dermal or adipose tissue derived fibroblasts.

We reached successful wound closure in 8 days with transfer of proliferating keratinocytes on a dermal substitute seeded with dermal fibroblasts. However, the wounds treated with substitutes which contained adipose tissue derived fibroblasts or no fibroblasts at all were not closed even after 21 days.

Keratinocytes seeded onto collagen lattices populated with either dermal or fat‐derived fibroblasts showed similar findings: a retarded migration and/or proliferation of keratinocytes on the collagen lattices with fat‐derived fibroblasts. The collagen lattices populated with fat‐derived fibroblasts also showed a marked contraction, up till 50% of the original area.

In both models, more alpha‐smooth muscle actin positive cells were found in the fibroblast population from adipose origin.

We conclude that epidermal regeneration is negatively influenced by the presence of fat‐derived fibroblasts in a dermal matrix; possibly, myofibroblasts play a role in this.
Cartilage Formation as a Differentiation Route of Myoepithelial Cells in Canine Mammary Tumours: Involvement of Myofibroblasts?
Authors: Nederbragt H , Erdelyi I
Published: January 2005

In canine mammary tumours the presence of large areas of spindle cells with myxoid differentiation (so‐called complex mammary tumours) or areas with cartilage and bone (mixed mammary tumours) is a frequent finding. In most cases these tumours are benign. It has been suggested that this route of differentiation may be a tumor suppressor route and this warrants a closer investigation of this route in canine mammary mixed tumours. The origin of the cartilage is supposed to be the myoepithelial cell which may have undergone an epithelial to mesenchymal transition. In this study we concentrated on the composition of the extracellular matrix (ECM) in the stroma of these tumours because this may shed light on the phenotypes of the different cells involved. The immunohistochemical expression of ECM components (aggrecan, versican, collagen II, link protein, 3B3/chondroitin sulphate‐6 epitope) of the tumor stroma indicates a close relationship between the myxoid spindle cells in complex and mixed tumours and the precartilageneous and cartilage tissues in mixed tumors. Semiquantitative PCR analyses of different types of normal tissue and tumours showed, next to a rather general expression of versican, expression of aggrecan and collagen type II in complex and mixed tumours (as expected because they are (pre)cartilage components) and in epitheliomas, but also of collagen type II in simple (i.e., noncomplex) tumors and, although faintly, in normal glands. Staining of cytoskeletal proteins in the spindle cells of complex tumors demonstrates expression of cytokeratin 14, α‐smooth muscle actin and vimentin in these cells, raising the possibility that they may be modified myofibroblasts. We hypothesize that myofibroblasts in the tumor stroma may be the precursors of cartilage and that in the stroma of normal glands they may differentiate into myoepithelial cells.
About the Level of Stress Between Myofibroblasts
Authors: Pittet P , Meister JJ , Hinz B
Published: January 2005

Fibroblast to myofibroblast modulation is characterized by expression of α‐smooth muscle actin (α‐SMA) and represents a crucial step in granulation tissue contraction during wound healing. We have recently demonstrated that myofibroblasts develop cadherin‐type cell‐cell adherens junctions (AJs), which join stress fibers over the cell membrane. Cadherin expression changes from N‐cadherin to OB‐cadherin upon TGF‐β induced myofibroblast differentiation in culture and coincides with the enlargement of AJs. We here show that myofibroblast AJs exhibit a higher mechanical resistance compared with that of α‐SMA‐negative fibroblasts by measuring the binding strength between two cells. (1) By separating two suspended cells placed in contact with laser tweezers, we revealed two types of Ca2+‐dependent adhesion bonds with different strength. The first bond exhibited a breaking force of 5.5 ± 1.5 nN and existed in both, fibroblasts and myofibroblasts. The strength of the second bond differed between the two cells, breaking at 18.2 ± 0.8 nN in fibroblasts and at 23.0 ± 1.5 nN in myofibroblasts. Measuring the adhesion force between two plated cells by atomic force microscopy after short contact grossly confirmed these observations. Since no cyoskeletal reinforcement was involved in these experimental conditions, we suggest that these differences represent the different cadherins involved in (myo)fibroblast adhesion. (2) Subjecting suspended cells that were attached to plated cells to hydrodynamic forces in a flow chamber demonstrated ∼18% higher adhesion of myofibroblasts over fibroblasts at shear forces of 4 Nm−2. This higher adhesion was reduced to the level of fibroblast attachment by a peptide that inhibits α‐SMA‐mediated contraction, indicating AJ reinforcement by α‐SMA‐positive stress fibers. To conclude, high cell‐cell adhesion of myofibroblasts appears to be determined by their specific cadherin pattern and by α‐SMA‐mediated mechanical reinforcement of AJs.
Fetal Cutaneous Myofibroblasts — Fact or Fallacy?
Authors: Rolfe K , Vigor C , Richardson J , Sanders R , Linge C
Published: January 2005

It has been known for several decades that early gestational human fetuses will regenerate cutaneous wounds resulting in a scarless repair. In postnatal humans, myofibroblasts are known to play a central role in wound healing and in the pathology of fibrosis. The role of the profibrotic growth factor TGF‐β1 in human fetal fibroblasts remains unclear, with recent data now showing that TGF‐β1 is present in fetal wounds but for shorter periods. We have therefore examined the effects of TGF‐β1 on fibroblasts isolated from early human fetuses (<14week EGA). We have identified that stimulation with 5 ng/ml TGF‐β1 induces a significant proportion of human fetal fibroblasts to differentiate into myofibroblasts (approximately 40%), as identified by staining of α‐smooth muscle actin (n = 5). Interestingly, this response was earlier (peaking at day 2–3) and more transitory (over by day 4–5) than that seen with postnatal dermal fibroblasts (peaking at day 6–8 and dropping at day 10). We examined the effects of blocking various intracellular pathways involved in TGF‐β1 signalling. The early and transitory myofibroblast induction seen in fetal cells is blocked by an inhibitor of p‐JNK (n = 4; p =< 0.05). In contrast, the later and more prolonged induction of myofibroblasts seen in postnatal cells was not affected by inhibition of p‐JNK. Despite the fact that fetal fibroblasts do indeed differentiate into myofibroblasts they were further shown to exhibit differential behavior to postnatal myofibroblasts with respect to TGF‐β1 stimulation of collagen expression, producing significantly less soluble collagen (approximately 50%, n = 4) after 24 or 48 hours of treatment with TGF‐β1 in serum‐free media. These results appear to indicate a role for TGF‐β1 during cutaneous wound healing in the early fetus and show that the response of fetal cells to this important profibrotic cytokine is rapid and more controlled, being short lived. It is possible that a prolonged response to this cytokine may lead to the overblown nature of postnatal wound healing, resulting in fibrosis.
Myofibroblasts in Hypertrophic Scars, Where do they come from?
Authors: Ulrich MMW , Van Den Bogeardt AJ , Verkerk M , Reijnen L , Van Zuijlen PPM , Middelkoop E
Published: January 2005

Wound healing is accompanied by the appearance of myofibroblasts (myoFB) in the wound. In skin wound healing these cells can be derived from dermal fibroblasts (dFB) by the action of TGF‐β, released from platelets and inflammatory cells entering the wound in the first phase of wound healing. MyoFB show an increased expression of components of the extracellular matrix such as collagen type I and III. In partial thickness wounds the myoFB disappear after the wound is closed probably by apoptosis. In full thickness wounds however the myoFB remain present and the healing of these wounds is accompanied by the formation of hypertrophic scars. Processes such as excessive collagen depositions, altered collagen remodeling and contraction play a key role in hypertrophic scar formation. We hypothesise that the presence of a specific myoFB subtype, which is less susceptible for apoptotic triggers, is responsible for sustained duration of these processes. In full‐thickness wounds the fibroblasts (FB) that populate the wound area are not only recruited from the surrounding dermis but also from other tissues such as the subcutaneous fat. To test the possible involvement of subcutaneous fat fibroblasts (fFB) in scar formation we studied the expression levels of proteins which are thought to play a role in hypertrophic scar formation in these cells. The expression levels of these proteins were compared with the expression levels in dFB and scar derived fibroblasts (sFB). Cultured fFB do show increased expression of α‐SMA, collagen types I and III, and TIMPs and decreased expression of MMPs in comparison with cultured dFB. Furthermore the cells show a collagen cross‐linking profile comparable with bone collagen resulting in collagen fibers which are less accessible for proteases. This kind of cross‐linking is also seen in hypertrophic scars. Taken together the differences in expression profile will lead to excessive collagen deposition. The expression pattern of fFB resembles that of sFB. This suggests that indeed the myoFB from the subcutaneous fat may play a role in hypertrophic scar formation.
The Glasbox®: A New System for Measurement of Contractile Forces Developed by Fibroblasts in Collagen Lattices
Authors: Viennet C , Armbruster V , Gharbi T , Humbert P
Published: January 2005

Cellular contraction, defined as the force cells exhibit on the extracellular matrix, plays a significant role in dermal tissue. To study the contractility of cells in vitro, we have developed a new patented device named GlaSbox®“Growing Lattice Study Box”. It consists of a cell chamber composed with 8 rectangular wells (26 × 33 mM) in which lattices develop. Two opposite silicon beams hang down into each well. The lattice is attached to this sensor through a grid directly etched on the lower part of the beams. A strain gauge is deposited at the beam surface, and connected to form a Wheastone bridge. The strain gauges signal output is amplified then converted and collected by a computer which includes an acquisition card and a specific program giving directly the forces in real time. The significant advantages of the GlaSbox® are:

the absence of any interface between the collagen lattice and the beams which maintain it stretched,

its ability to test eight lattices simultaneously,

the low volumes of cells and collagen required.

To demonstrate the applicability of this system, contractile forces of human fibroblasts from different skin conditions were measured such as chronic venous leg ulcers and striae distensae. These fibroblasts were found to produce an average force of 1.10−8 N/cell and have greater contractile capacity than normal fibroblasts (Viennet et al., J Wound Care 2004, in press). The GlaSbox® is a useful tool to study the contractility of dermis equivalent composed of various fibroblast lines and, the effect of different active principles or even growth factors (e.g., TGF‐β) on cellular contraction. In addition, it can be used to examine the relationship between cellular contraction, cell proliferation, and collagen production, which is important for the understanding of the mechanism of scar tissue formation.
Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes
Authors: Nakagaki I. , Sasaki S. , Yahata T. , Takasaki H. , Hori S.
Published: January 2005

Aim:  We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca2+ mobilizations in cold‐acclimated rats.

Methods:  A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca2+ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied.

Results:  The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca2+ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca2+ ([Ca2+]cyto) followed by mitochondrial Ca2+ ([Ca2+]mito), the effect being transformed into an increase in [Ca2+]cyto whereas a decrease in [Ca2+]mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca2+ release from mitochondria. CCCP induced Ca2+ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca2+]cyto followed by [Ca2+]mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells.

Conclusions:  These results suggest that noradrenaline and forskolin evoked an elevation in [Ca2+]cyto followed by [Ca2+]mito, in which H+ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca2+ signalling in cold‐acclimated brown adipocytes.
Local cold acclimation of the hand impairs thermal responses of the finger without improving hand neuromuscular function
Authors: Geurts C. L. M. , Sleivert G. G. , Cheung S. S.
Published: January 2005

Aim:  To investigate the effects of cold acclimation on the thermal response and neuromuscular function of the hand.

Methods:  Ten healthy subjects [three female, seven male, age (mean ± SD): 27.9 ± 7.9 years] immersed their right hand in 8 °C water for 30 min, 5 days a week for 3 weeks. On the first and the last day, neuromuscular function of the first dorsal interosseus (FDI) muscle was tested.

Results:  There was no significant change in maximal voluntary contraction strength or evoked contractile characteristics of the FDI after cold acclimation. Minimum finger temperature decreased significantly from 10.6 ± 1.2 to 9.3 ± 0.8 °C after 3 weeks (P < 0.01), with most of the decrease occurring after a single exposure. Mean finger temperature dropped significantly from 14.2 ± 1.9 to 11.7 ± 1.4 °C following cold acclimation (P < 0.05), with 90% of this adaptation occurring after 5 days. Onset time of cold‐induced vasodilatation increased from 446 ± 171 to 736 ± 384 s (P < 0.05) and the amplitude decreased from 5.3 ± 3.2 to 2.5 ± 2.1 °C (P < 0.05). This was significantly different from the control group, who immersed their right hand on the first and last days only.

Conclusion:  These data suggest that cold acclimation does not enhance hand temperature or function but may put the hands at a greater risk of cold injury when exposed to the cold.
The acute effects of insulin on the cardiorespiratory responses to hypoxia in streptozotocin‐induced diabetic rats
Authors: Saiki C. , Seki N. , Furuya H. , Matsumoto S.
Published: January 2005

Aims:  We examined whether or not streptozotocin (STZ)‐induced diabetic rats, which have a lower heart rate (HR, beats min−1) than control rats, could maintain hypoxic ventilatory response.

Methods:  Twenty‐six Wistar rats, which had been injected with STZ (60 mg kg−1, EXP) or vehicle (0.1 m citrate buffer, CONT) intraperitoneally at 9 weeks of age, had their cardiorespiratory responses to normoxia and 12%O2 examined after 5 weeks.

Results:  Compared with CONT rats, EXP rats had a higher blood glucose [24 ± 3 vs. 5 ± 1 (mean ± SD) mmol L−1], a lower body weight (320 ± 23 vs. 432 ± 24 g), lower HR (303 ± 49 vs. 380 ± 44 in normoxia, and 343 ± 56 vs. 443 ± 60 in hypoxia) and a lower mean arterial blood pressure (MAP) (89 ± 6 vs. 102 ± 10 mmHg in hypoxia). In contrast, both groups had similar values in ventilation (), –metabolic rate (MR) ratio and arterial blood gases (ABGs). In EXP rats, with an acute insulin supplement (i.v., 0.75 U h−1 for 1.5–2 h), not only blood glucose, but also HR, and MAP were normalized as those obtained in CONT rats, and in hypoxia further increased without affecting –MR ratio and ABGs. Such acute cardiorespiratory stimulating effects of insulin could not be obtained in non‐diabetic rats (n = 7, 355 ± 24 g), in which euglycaemia (mean 6.4 mmol L−1) was maintained during the measurements.

Conclusions:  Our results suggest that, in STZ‐induced diabetic rats: (1) ventilation is hardly suppressed by hyperglycaemia, (2) cardiorespiratory responses can be acutely stimulated by short insulin injection, and (3) the effects, including those through acute blood glucose normalization, are possibly specific for the diabetic impairments.
Effects of renal nerve stimulation on intrarenal blood flow in rats with intact or inactivated NO synthases
Authors: Walkowska A. , Bądzyńska B. , Kompanowska‐Jezierska E. , Johns E. J. , Sadowski J.
Published: January 2005

Aim:  We studied a possible action of nitric oxide (NO), an intrarenal vasodilator, to buffer a decrease in renal perfusion induced by electrical stimulation of renal nerves (RNS).

Methods:  In anaesthetized rats RNS was performed (15 V, 2 ms pulse duration) for 10 s at the frequencies of 2, 3.5, 5 and 7.5 Hz. The total renal blood flow (RBF), an index of cortical perfusion, was measured using a Transonic probe on the renal artery. The outer and inner medullary blood flow (OMBF, IMBF) was measured by laser‐Doppler flowmetry. The effect of RNS on RBF, OMBF and IMBF was determined in rats which were either untreated or pre‐treated with l‐NAME (0.6 or 1.8 mg kg−1 i.v.), or S‐methyl thiocitrulline (SMTC, 20 μg kg−1 min−1 i.v.), a selective inhibitor of neuronal NO synthase (nNOS).

Results:  In untreated rats, RNS decreased IMBF significantly less than RBF and OMBF. High‐dose l‐NAME treatment significantly enhanced the RNS induced decrease of RBF but not of OMBF or IMBF. SMTC treatment significantly enhanced the decrease of IMBF, without affecting the response of RBF or OMBF.

Conclusion:  At intact NO synthesis the inner medullary circulation is not controlled by renal nerves to the extent observed for the outer medulla or cortex. NO generated by all NOS isoforms present in the kidney buffers partly the intrarenal vasoconstriction triggered by electrical RNS. The NO derived from nNOS seems of particular importance in the control of inner medullary perfusion, interacting with NO generated by endothelial NOS and renal nerves.
Mechanisms regulating GLUT4 glucose transporter expression and glucose transport in skeletal muscle
Authors: Zorzano A. , Palacín M. , Gumà A.
Published: January 2005

Skeletal muscle is a major glucose‐utilizing tissue in the absorptive state and the major glucose transporter expressed in muscle in adulthood is GLUT4. GLUT4 expression is exquisitely regulated in muscle and this seems important in the regulation of insulin‐stimulated glucose uptake by this tissues. Thus, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes. Recent information indicates that glut4 gene transcription is regulated by a number of factors in skeletal muscle that include MEF2, MyoD myogenic proteins, thyroid hormone receptors, Krüppel‐like factor KLF15, NF1, Olf‐1/Early B cell factor and GEF/HDBP1. In addition, studies in vivo indicate that under normal conditions the activity of the muscle‐specific GLUT4 enhancer is low in adult skeletal muscle compared with the maximal potential activity that it can attain at high levels of the MRF transcription factors, MEF2, and TRα1. This finding indicates that glut4 transcription may be greatly up‐regulated via activation of this enhancer through an increase in the levels of expression or activity of these transcription factors. Understanding the molecular basis of the expression of glut4 will be useful for the appropriate therapeutic design of treatments for insulin‐resistant states. The nature of the intracellular signals that mediate the stimulation of glucose transport in response to insulin or exercise is also reviewed.
Lipid metabolism in human skeletal muscle cells: effects of palmitate and chronic hyperglycaemia
Authors: Aas V. , Rokling‐Andersen M. , Wensaas A. J. , Thoresen G. H. , Kase E. T. , Rustan A. C.
Published: January 2005

This review focuses on the effect of exogenous factors known to be of importance for the development of insulin resistance in differentiated human myotubes. Recent data from our laboratory on the effects of fatty acid pre‐treatment and chronic glucose oversupply on fatty acid and glucose metabolism, without and with acute insulin are presented, and discussed in the context of other recent publications in the field. Pre‐treatment of myotubes with palmitate, chronic hyperglycaemia, and acute high concentrations of insulin changed fatty acid metabolism in favour of accumulation of intracellular lipids. Acute insulin exposure increased 14C‐oleate uptake and levels of free fatty acids (FFA) and triacylglycerol (TAG). Palmitate pre‐treatment further increased oleate uptake, both under basal conditions and in the presence of insulin, with a marked increase in the phospholipid (PL) fraction, with a concomitant reduction in oleate oxidation. Chronic hyperglycaemia also promoted increased lipogenesis and elevated levels of cellular lipids. Changes in fatty acid metabolism in human muscle, in particular fatty acid oxidation, are probably crucial for the molecular mechanism behind skeletal muscle insulin resistance and impaired glucose metabolism. Differentiated human skeletal muscle cells may be an ideal system to further explore the mechanisms regulating lipid metabolism.
Mechanisms regulating phosphoinositide 3‐kinase signalling in insulin‐sensitive tissues
Authors: Shepherd P. R.
Published: January 2005

A great deal of evidence has accumulated indicating that the activity of PI 3‐kinase is necessary, and in some cases sufficient, for a wide range of insulin's actions in the cell. Most biochemical, genetic and pharmacological studies have focused on identifying potential roles for the class‐Ia PI 3‐kinases which are rapidly activated following insulin stimulation. However, recent evidence indicates the alpha isoform of class‐II PI 3‐kinase (PI3K‐C2α) may also play a role as insulin causes a very rapid activation of this as well. The basic mechanisms by which insulin activates the various members of the PI 3‐kinase family are increasingly well understood and these studies reveal multiple mechanisms for modulating the activity and functionality of PI 3‐kinase and for down regulating the signals they generate. These include inhibitory phosphorylation events, lipid phosphatases such as PTEN and SHIP2 and inhibitor proteins of the suppressors of cytokine signalling (SOCS) family. The current review will focus on these mechanisms and how defects in these might contribute to the development of insulin resistance.