Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down- regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine- specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells

The capacity of cells to organize and contract collagen fibrils is fundamental to processes as diverse as embryogenesis and wound healing. We analyzed different beta 1 integrins on diploid fibroblasts for their role in modifying the tertiary structure of collagen matrices. Using monoclonal antibodies that block the interaction of integrins with their ligands, evidence was obtained that alpha 2 beta 1 integrin is required for the contraction of a type I collagen matrix. Further supporting the role of alpha 2 beta 1, cell lines expressing minimal levels of this integrin uniformly failed to contract collagen matrices. In addition, transfection of a full-length alpha 2 cDNA into one such cell line led to enhanced cell surface expression of alpha 2 beta 1 and conferred the de novo capacity to contract collagen matrices.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

Integrin alphavbeta3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin alpha2beta1

The interplay between the collagen-binding integrin, alpha2beta1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type alpha2beta1 (alpha2beta1A) or alpha2beta1 with a Y783/795F mutation in the cytoplasmic tail of the beta1 subunit (alpha2beta1Amut) in the beta1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the alpha1 and alpha2 integrin subunits and do not adhere to collagen even after transfection with beta1A. Cells expressing alpha2beta1Amut contracted three-dimensional collagen lattices less efficiently than those expressing alpha2beta1A. PDGF-BB significantly stimulated lattice contraction by GD25-alpha2beta1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130(Cas) were phosphorylated when GD25-alpha2beta1A cells, but not GD25-alpha2beta1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130(Cas) only in GD25-alpha2beta1A cells. However, when cultured within collagen lattices, FAK and p130(Cas) phosphorylation were stimulated in both alpha2beta1A- and alpha2beta1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-alpha2beta1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-alpha2beta1Amut cells were mediated by the alphavbeta3 integrin. Phosphorylation of p130(Cas), but not FAK, in GD25-alpha2beta1Amut cells seeded in collagen lattices also depended on alphavbeta3. Our results show that PDGF-BB stimulation of fibroblast-collagen interactions is mediated by the alphavbeta3 integrin when beta1 integrin function is impaired.     

Mechanical behavior of fibroblasts included in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin. Excessive steroid activity, genetic and mechanical factors and inherited defects of connective tissues are the most frequent causes of this disease. Fibroblasts derived from women presenting striae distensae lesions were included into collagen gels to study their mechanical behavior: capacity to contract free-floating lattices and to produce isometric force in tense lattices. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. An isometric force system was used to study quantitatively the forces developed during lattice contraction. alpha 2 beta 1 integrins expression (transmembrane receptors) was evaluated by flux cytometry. Striae distensae fibroblasts contract collagen gels slower than normal human fibroblasts but the final contraction is similar. They produce a greater isometric force which is associated with enhanced alpha 2 beta 1 integrins expression. By their mechanical properties, striae distensae fibroblasts appear as a different population from normal fibroblasts.     

Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling

Cultured human foreskin keratinocytes (HFKs) adhere to and grow on nonfibrous collagen via integrin alpha2beta1. During incubation, the receptors used for adhesion are changed to integrins alpha3beta1 and alpha6beta4 and those receptors bind to laminin 5 which is deposited by keratinocytes themselves. In this report, we examined the behaviors of HFKs and transformed keratinocytes on collagen fibril gels. These cells adhered to and spread on collagen gels using integrin alpha2beta1. After several hours on collagen gels, however, cells became round and apoptosis occurred. The behavior of keratinocytes contrasted to that of fibroblasts that grew well even on collagen gel. At the point of apoptosis, integrins alpha2beta1 and alpha3beta1 were not found in the contact region of HFKs. Also, deposition of laminin 5 on collagen gel was not found despite the synthesis of mRNA for laminin 5 and laminin 10/11, while soluble laminin 5 protein is readily detectable. Phosporylation of Akt, which is known as a survival signal, was detected in HFKs cultured on coated collagen; however, the protein level and signals of Akt were dramatically decreased on collagen gel after 1 day of culture. These results indicate that collagen gel has different effects than nonfibrous collagen on HFKs and transformed keratinocytes and the interactions of integrin alpha3beta1 and laminin 5/10/11 are indispensable for maintenance of keratinocyte adhesion and survival.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Role of the α1β1 integrin complex in collagen gel contraction in vitro by fibroblasts

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the β1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-β, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the α2β1 and, to a lesser extent the α1β1 integrin complexes. The present studies were undertaken to determine the role of the α1 integrin chain, a collagen/laminin receptor, in collagen gel contration by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the α1β1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the α1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the α1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the α1β integrin complex plays a significant role in cellular interactions with interstital collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing. © 1995 Wiley-Liss, Inc.     

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.     

The interaction of epidermal keratinocytes and fibroblasts during the formation of a live skin equivalent]

The effect of human fetal fibroblasts and adult keratinocytes on collagen contraction was studied. Keratinocytes embedded in collagen lattices did not spread and produced only a slight contraction. When keratinocytes were seeded on the surface of tht gel, the contraction began within 24 h and correlated with the formation of epithelial colonies. Transplantation of multilayered epithelial sheets on the gel significantly accelerated the onset of contraction. Keratinocytes seeded on and fibroblasts grown in collagen lattices cooperatively contracted the gel, and keratinocytes were able to stimulate gel contraction even when they had no contact with the collagen roughly populated with fibroblasts. Swiss 3T3 cells remained spherical in collagen lattices and did not contract the gel but when cultivated with keratinocytes they stimulated gel contraction. In their turn, keratinocytes influenced the behaviour of Swiss 3T3 cells which elongated and produced processes. We suggest that both keratinocytes and mesenchymal cells can affect gel contraction 1) by a direct contact with collagen lattices, and 2) through potentiation of the ability of another cell type to contract the gel.     

Smad3 mediates the TGF-beta-induced contraction of type I collagen gels by mouse embryo fibroblasts

TGF-beta signals through TGF-beta receptors and Smad proteins. TGF-beta also augments fibroblast-mediated collagen gel contraction, an in vitro model of connective tissue remodeling. To investigate the importance of Smad2 or Smad3 in this augmentation process, embryo-derived fibroblasts from mice lacking expression of Smad2 or Smad3 genes were cast into native type I collagen gels. Fibroblast-populated gels were then released into 0.2% FCS-DMEM alone or with recombinant human TGF-beta1, beta2, beta3, or recombinant rat PDGF-BB. Gel contraction was determined using an image analyzer. All three isoforms of TGF-beta significantly augmented contraction of collagen gels mediated by fibroblasts with genotypes of Smad2 knockout (S2KO), Smad2 wildtype (S2WT), and Smad3 wildtype (S3WT), but not Smad3 knockout (S3KO) mice. PDGF-BB augmented collagen gel contraction by all fibroblast types. These results suggest that expression of Smad3 but not Smad2 may be critical in TGF-beta augmentation of fibroblast-mediated collagen gel contraction. Thus, the Smad3 gene could be a target for blocking contraction of fibrotic tissue induced by TGF-beta.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

Gamma radiation inhibits fibroblast-mediated collagen gel retraction

Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.     

Differences in the mechanism for high- versus moderate-density fibroblast-populated collagen lattice contraction

The free-floating fibroblast-populated collagen lattice (FPCL) model introduced by Bell contains 0.5 x 10(5) cell/ml and here is defined as a moderate-density FPCL (MD-FPCL). One modification of the model is to increase the cell density by a factor of 10, where 5 x 10(5) cells/ml defines a high-density FPCL (HD-FPCL). The initial detection of HD-FPCL contraction is 2 h, whereas MD-FPCL is later, 6 h. A contracted HD-FPCL has a doughnut-like appearance, due to the high density of cells accumulating at the periphery. A contracted MD-FPCL is a flattened disc. The compacted collagen of MD-FPCL lattice exhibits a strong birefringence pattern due to organized collagen fiber bundles. In contracted HD-FPCL, a minimal birefringence develops, indicating minimal organization of collagen fiber bundles. MD-FPCL contraction was reduced with less than 10% serum; the disruption of microtubules, uncoupling of gap junctions, inhibition of tyrosine kinases, and addition of a blocking antibody to alpha2beta1 collagen integrin. Making HD-FPCL with only 1% serum or including the inhibitory agents had only minimal affect on lattice contraction. On the other hand, platelet-derived growth factor stimulated HD-FPCL contraction but had no influence on MD-FPCL contraction. It is suggested that the mechanism for HD-FPCL contraction is limited to the process of cells spreading. HD-FPCL contraction is independent of collagen organization, microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. MD-FPCL contraction involves collagen organization and is optimized by the involvement of microtubules, gap junctions, alpha2beta1 integrin, and tyrosine phosphorylation. When studying cell physiology in a collagen matrix, cell-density influences need to be considered.