The Study of Clinical Usefulness of Basic FGF

Fibroblast‐collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)‐stimulated collagen matrix contraction is independent of Rho kinase whereas platelet‐derived growth factor (PDGF)‐stimulated contraction is Rho kinase dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA‐ and PDGF‐stimulated fibroblast‐collagen matrix contraction. Fibroblasts whose MLC kinase was knocked down using siRNA were used to measure matrix contraction in the presence of LPA or PDGF with or without the Rho kinase inhibitor added. The extent of contraction of MLC kinase‐silenced cells was not detectably different from control cells. Other experiments were carried out to test the effects of LPA and PDGF on MLC phosphorylation with and without Rho kinase inhibitor. After 15 min of growth factor stimulation, levels of diphosphorylated MLC were highest in cells in LPA‐containing medium and lowest in PDGF containing medium. Prior addition of Rho kinase inhibitor markedly reduced phosphorylation in every case. These observations suggested that stimulation of collagen matrix contraction required neither growth factor stimulation of MLC phosphorylation nor MLC kinase.     

Enhanced flexor tendon healing through controlled delivery of PDGF‐BB

A fibrin/heparin‐based delivery system was used to provide controlled delivery of platelet derived growth factor BB (PDGF‐BB) in an animal model of intrasynovial flexor tendon repair. We hypothesized that PDGF‐BB, administered in this manner, would stimulate cell proliferation and matrix remodeling, leading to improvements in the sutured tendon's functional and structural properties. Fifty‐six flexor digitorum profundus tendons were injured and repaired in 28 dogs. Three groups were compared: (1) controlled delivery of PDGF‐BB using a fibrin/heparin‐based delivery system; (2) delivery system carrier control; and (3) repair‐ only control. The operated forelimbs were treated with controlled passive motion rehabilitation. The animals were euthanized at 7, 14, and 42 days, at which time the tendons were assessed using histologic (hyaluronic acid content, cellularity, and inflammation), biochemical (total DNA and reducible collagen crosslink levels), and biomechanical (gliding and tensile properties) assays. We found that cell activity (as determined by total DNA, collagen crosslink analyses, and hyaluronic acid content) was accelerated due to PDGF‐BB at 14 days. Proximal interphalangeal joint rotation and tendon excursion (i.e., tendon gliding properties) were significantly higher for the PDGF‐BB‐treated tendons compared to the repair‐alone tendons at 42 days. Improvements in tensile properties were not achieved, possibly due to suboptimal release kinetics or other factors. In conclusion, PDGF‐BB treatment consistently improved the functional but not the structural properties of sutured intrasynovial tendons through 42 days following repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Activation of PLC and PI 3 kinase by PDGF receptor alpha is not sufficient for mitogenesis and migration in mesangial cells

BACKGROUND: Platelet-derived growth factor (PDGF) isoforms act through two distinct cell surface alpha and beta receptors. Glomerular mesangial cells express both receptors. PDGF BB and AB are potent mitogens for glomerular mesangial cells, and PDGF BB stimulates cell migration in a phosphatidylinositol 3 (PI 3) kinase-dependent manner. In this study, we investigated the effect of PDGF AA on cell migration, PI 3 kinase and phospholipase C (PLC) activation, and the role of these two enzymes in mediating biological responses in these cells in response to all three isoforms. METHODS: 3H-thymidine incorporation and modified Boyden chamber assay were used to determine DNA synthesis and directed migration, respectively, in response to all three PDGF isoforms. Differential activation of alpha and beta receptors was studied by immunecomplex tyrosine kinase assay of corresponding receptor immunoprecipitates. PLC gamma 1 activity was determined by measuring total inositol phosphates in response to different PDGF isoforms. PI 3 kinase activity was determined in antiphosphotyrosine or PDGF receptor immunoprecipitates. RESULTS: Both PDGF BB and AB resulted in stimulation of DNA synthesis and directed migration of mesangial cells. AA was neither chemotactic nor mitogenic. However, all three isoforms increased tyrosine phosphorylation of a 180 kD protein in antiphosphotyrosine immunoprecipitates, suggesting activation of respective receptors. Direct immunecomplex tyrosine kinase assay of alpha and beta receptors demonstrated significant activation of both of these receptors when cells are treated with PDGF BB or AB. PDGF AA increased tyrosine kinase activity of the alpha receptor but not the beta receptor. All three isoforms significantly stimulated the production of inositol phosphates with order of potency being BB > AB > AA. PDGF AA also dose dependently stimulated PI 3 kinase activity measured in antiphosphotyrosine immunoprecipitates of treated cells. A comparison of PI 3 kinase activity in antiphosphotyrosine immunoprecipitates from mesangial cells stimulated with three different PDGF isoforms showed significant activation of this enzyme with a decreasing order of activity: BB > AB > AA. CONCLUSION: Taken together, these data demonstrate that all three isoforms of PDGF significantly stimulate PLC gamma 1 and PI 3 kinase, two enzymes necessary for both DNA synthesis and directed migration. However, activation of alpha receptor by PDGF AA with a subsequent increase in PLC and PI 3 kinase activities is not sufficient to induce these biological responses in mesangial cells. These data indicate that the extent of activation of signal transduction pathways may be a major determinant of the biological activity of different PDGF isoforms in mesangial cells.     

Recombinant human platelet-derived growth factor and transforming growth factor-β mediated contraction of human dermal fibroblast populated lattices is inhibited by Rho/GTPase inhibitor but does not require phosphatidylinositol-3'' kinase

Matrix reorganization and tissue contraction are essential for wound healing. However, the intracellular signals that mediate these processes are largely unknown. We investigated cytokine-induced signaling and its potential role in contraction of adult human dermal fibroblast populated collagen lattices. The results document that recombinant human platelet-derived growth factor-BB and transforming growth factor-1 individually stimulate contraction of fibroblast populated collagen lattices, while a combination of the two cytokines leads to increased contraction. Although recombinant human platelet-derived growth factor-BB promoted collagen contraction, it failed to stimulate phosphatidylinositol-3' kinase in the human dermal fibroblasts. An inhibitor for phosphatidylinositol 3' kinase, wortmannin, had no effect on the cytokine-mediated collagen contraction. In addition, this failed activation of phosphatidylinositol 3' kinase is consistent with absence of tyrosine phosphorylation of the platelet-derived growth factor receptor when the cells are in a collagen matrix. In contrast, tyrosine phosphorylation of the platelet-derived growth factor receptor was readily detected in the dermal fibroblasts in monolayers. We also probed the potential role of Rho/GTPase in the cytokine-mediated contraction of fibroblast populated collagen lattices. Toxin B from Clostridium difficile at picomolar concentrations blocked both recombinant human platelet-derived growth factor and transforming growth factor-5 induced contraction. Further, this inhibition was correlated with the inhibition of cell spreading in collagen, which suggests the formation of actin fibers inside the cells is essential for cytokine-mediated contraction of fibroblast populated collagen lattices. Taken together, these results imply that Rho/GTPase signaling but not phosphoinositol-3' kinase is involved in the cytokine-mediated contraction of fibroblasts populated collagen lattice. These findings suggest a potential mechanism for these signaling components during human wound contraction.     

The Glasbox®: A New System for Measurement of Contractile Forces Developed by Fibroblasts in Collagen Lattices

Fibrosis is often seen in tissues following vessel hyperpermeability, interstitial fluid flow, and edema; examples include secondary lymphedema in skin and pulmonary edema. To determine whether the biophysical changes that interstitial flow induces can influence fibroblast behavior, we developed a tissue culture model of interstitial flow that can be chronically applied to 3‐D cultures. We use it to demonstrate that even very low interstitial fluid flows (of 1–10 microns/sec) can induce human dermal and lung fibroblasts embedded in type I collagen to align perpendicular to the direction of flow and align the collagen extracellular matrix fibers. We saw that attachment to collagen is necessary for alignment, since fibroblasts in fibrin gels do not align, and those grown in fibrin‐collagen mixtures align only weakly. Furthermore, fibroblast alignment corresponds to increased expression of alpha‐smooth muscle actin, signaling that the cells are undergoing differentiation to a fibrotic myofibroblast phenotype. Collagen fiber alignment is demonstrated using confocal reflectance microscopy and FFT analysis. Our results indicate that interstitial flow, which is increased in vivo during wound healing and many pulmonary disorders such as edema, may contribute to interstitial fibrosis. The results also suggest that interstitial flow can be used to develop in vitro models of interstitial fibrosis for studies of directed matrix remodeling and cell orientation.     

Mammalian Diaphanous (mDia) may be involved in the signal transduction of sphingosine-1-phosphate on developing actin stress fiber of human fibroblasts

Sphingosine 1-phosphate (S1P) is a biologically active lipid mediator with many pivotal roles in the regulation of cell growth, migration, differentiation, and apoptosis. However, signal transduction mediated by S1P in human fibroblasts is still unclear. We investigated signal transduction by S1P in human fibroblasts using collagen matrix contraction in order to explore whether or not S1P could be applied for the treatment of skin wound healing. We found that S1P promoted floating collagen matrices' contraction, for the in vitro model of initial phase wound contraction, in which some kinds of G protein, such as Giα, Rac 1, and Rho, were involved. However, Rho-associated coiled-coil forming kinase (ROCK) was partially involved in S1P-promoting floating collagen matrices contraction. Mammalian Diaphanous (mDia) as well as ROCK have been identified to be putative downstream target molecules of Rho. In mDia-silenced cells, the ROCK inhibitor suppressed actin stress fiber formation regardless of the presence or absence of S1P. Our results indicate that mDia as well as ROCK may be situated downstream of Giα, Rac1, and Rho to induce actin stress fiber development by human fibroblasts stimulated with SIP.     

Persistent Ischemia Impairs Myofibroblast Development in Wound Granulation Tissue a New Model of Delayed Wound Healing

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.     

PDGF‐BB inhibits intervertebral disc cell apoptosis in vitro

Degeneration of the intervertebral disc (IVD) results in deterioration of the spinal motion segment and can lead to debilitating back pain. Given the established mitotic and anti‐apoptotic effects of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) in a variety of cell types we postulated that rhPDGF‐BB might delay disc cell degeneration through inhibition of apoptosis. To address this hypothesis, we treated human IVD cells isolated from five independent patients with rhPDGF‐BB in monolayer and 3D pellet cultures. The anti‐apoptotic potential, cell proliferative capacity, morphology/pellet differentiation, and gene expression of PDGF‐treated IVD cells were evaluated via flow cytometry/immunohistochemistry, MTT assays, histology, and quantitative RT‐PCR, respectively. We found that rhPDGF‐BB treatment significantly inhibited cell apoptosis, increased cell proliferation and matrix production, and maintained mRNA expression of critical extracellular matrix genes. This study suggests two possible mechanisms for the anti‐degenerative effects of rhPDGF‐BB on human IVD cells. First, PDGF treatment strongly inhibited IVD cell apoptosis in 3D cultures. Second, rhPDGF‐BB acts as an anabolic agent, promoting maintenance of IVD cell phenotype in 3D culture, based on the molecular and protein expression analysis. We speculate that rhPDGF‐BB may be used as a biologic treatment to target early degenerative IVD disease in the future. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1181–1188, 2014.     

Differential regulation of free-floating collagen gel contraction by human fetal and adult dermal fibroblasts in response to prostaglandin E2 mediated by an EP2/cAMP-dependent mechanism

In contrast to fetal wound healing, dermal adult wound healing results in imperfect repair and scar formation. Fibroblasts are responsible for the contraction and remodeling of the wound matrix, which is influenced by inflammatory mediators including prostaglandin E2 (PGE2). This study addresses the mechanism by which PGE2 regulates contraction of collagen gels by human fetal and adult dermal fibroblasts. We hypothesized that the intrinsic phenotypic properties of the two types of fibroblasts and their responses to PGE2 alter their contraction properties and contribute to different wound healing outcomes. Contraction was evaluated using free-floating fibroblast-populated collagen gels that contract by migratory forces. PGE2 was found to differentially inhibit collagen gel contraction by fetal and adult fibroblasts. This effect was mimicked by a specific PGE2 receptor agonist as well as by two pharmacological agents, indicating a cyclic adenosine monophosphate-dependent signaling pathway mediated through the EP2 receptor. Our results indicate that fetal fibroblast contraction is maintained by a more stable actin cytoskeleton. Therefore, the migratory phenotype may be sufficient for physical remodeling of the wound matrix leading to regenerative repair. Maintenance of this phenotype in the later stages of wound healing could potentially be achieved by targeting cyclic adenosine monophosphate via the EP2 receptor.     

014Macrophage Suspensions Treatment of Infected Sternal Wounds Following Cabg Surgery

Detachment of an attached (stressed) fibroblast‐populated collagen matrix (FPCM) induces fibroblast apoptosis; this may be secondary to increased p53 after detachment. We have noted, however, that the p53 response after detachment of an attached FPCM (i.e., stress‐release) varies among cells strains. We hypothesized that modulation of p53 after FPCM stress‐release may be related to the fibroblast population density at the time of release. Human foreskin fibroblasts (initially seeded at 0.5 × 10⁶/ml in 0.2 ml) were cultured for 48 hr in attached collagen matrices prior to detachment for 0–6 hr; p53 levels and lysate DNA concentration (used as a measure of fibroblast population density) were determined with immunoblot densitometry and a spectrophotometer, respectively. The experiment was performed with 15 different strains of fibroblasts. The p53 level increased (defined as ≥100% increase in baseline p53 level) during the 6 hr detachment period in 6/15 (40%) of fibroblast strains (responders), and downregulated or unchanged in the remaining strains (nonresponders). The mean DNA concentration in the responders vs . nonresponders was 1.56 μg/ml (median 1.63, range 0.89–2.25) vs . 2.57 (median 1.94, range 1.38–5.09), respectively (p < 0.05, Wilcoxon rank sum test). Stress‐release of the FPCM resulted in a variable p53 response which appeared to have a relationship with the fibroblast population density; a lower density was associated with p53 upregulation after detachment. This variable p53 response may in turn be related to varying proliferative capacity in the collagen matrix, as there was a broad range (nearly an order of magnitude) of DNA concentration among the 15 cell strains after 48 hr of attached FPCM cuture.     

Actin-related protein 2/3 complex is required for actin ring formation.

Actin rings are vital for osteoclastic bone resorption, and actin-related protein 2/3 complex is a pivotal regulator of actin polymerization. Actin-related protein 2/3 complex was found in the podosomes of actin rings. A short interfering RNA knocked down expression of actin-related protein 2 in osteoclasts and disrupted actin rings, suggesting that the complex is crucial to actin ring formation. INTRODUCTION: To resorb bone, osteoclasts form an extracellular acidic compartment segregated by a sealing zone. This is dependent on an actin ring that is composed of filamentous actin organized into dynamic structures called podosomes. The actin-related protein 2/3 (Arp2/3) complex is a vital regulator of actin polymerization. We tested whether the Arp2/3 complex is a component of actin rings and is important for actin ring formation. MATERIALS AND METHODS: Western blot analysis was used to determine levels of Arp2 and Arp3, two components of the Arp2/3 complex in osteoclast-like cells. Confocal microscopy studies using antibodies for immunocytochemistry demonstrated localization of Arp2/3 complex in osteoclasts. Short interfering RNA oligonucleotides (siRNAs) were made against Arp2 and used to knock down its expression. RESULTS: A 3-fold increase in Arp2 and Arp3 was detected during RANKL-induced differentiation of RAW 264.7 cells into osteoclast-like cells. Arp2/3 complex was concentrated in actin rings and enriched near the sealing zone. Arp2/3 complex co-localized with cortactin, a component of podosomes, but not vinculin, which surrounds podosomes. siRNA against Arp2, transfected into RAW 264.7 cells 5 days after stimulation with RANKL, reduced Arp2 protein levels 70% compared with cells transfected with ineffective siRNAs. Cytochemical characterization of RAW 264.7 osteoclast-like cells and marrow osteoclasts in which Arp2 was knocked down revealed fewer podosomes and no actin rings, although many cells remained well spread. CONCLUSIONS: These data show that Arp2/3 complex is a component of actin rings and that the presence of Arp2/3 complex is vital to the formation of actin rings. In addition, the results show the use of siRNAs for the study of RAW 264.7 osteoclast-like cells.     

Cytoskeletal rearrangement and signal transduction in TGF-beta1-stimulated mesangial cell collagen accumulation

TGF-beta1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-beta1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-beta1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle alpha-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-beta1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-beta1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-beta1-stimulated alpha1(I) collagen mRNA but did not inhibit TGF-beta-stimulated alpha1(IV) mRNA expression. A similar decrease in alpha1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-beta1-stimulated alpha1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-beta receptor number, or receptor affinity. Thus, TGF-beta1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.     

Early interstitial accumulation of collagen type I discriminates chronic rejection from chronic cyclosporine nephrotoxicity

Little is known regarding the composition of the interstitial extracellular matrix of kidney allografts with deteriorating function. Collagen I, III, and IV, the collagen IV alpha3 chain, and the laminin beta2 chain were investigated in biopsies of allografted kidneys with chronic cyclosporine A nephrotoxicity (CsAT) (n = 17), chronic rejection (CR) (n = 12), or chronic allograft nephropathy (CAN) (n = 19). alpha-Smooth muscle actin expression was also examined. Normal native kidneys were used as control samples (n = 11). Biopsy samples were studied with routine light microscopy and immunostaining. The mean interstitial fibrosis scores were significantly higher for the CR and CAN groups, compared with the chronic CsAT group. The cortical tubulointerstitial areas of the CR and CAN groups, but not the chronic CsAT group, contained more collagen I than did normal control samples. Differences were noted even in biopsies with mild fibrosis. Accumulation of collagen III, IV, and IV alpha3 was increased in all patient groups. Collagen III accumulation was greater in the CR and CAN groups than in the chronic CsAT group. Receiver-operating characteristic curve analysis demonstrated that collagen I staining had the best discriminatory value in differentiating CR from chronic CsAT, with a sensitivity of 63% and a specificity of 94% at a cutoff value of 19%. Laminin beta2 staining did not differentiate CR from CsAT. Increased alpha-smooth muscle actin staining did not differ among the three groups. It was concluded that, during chronic CsAT, collagen III and IV were preferentially accumulated in the tubulointerstitium. Early increases in the deposition of collagen I, with collagen III and IV, were more specific for CR. CR seems to elicit a more pronounced fibrotic response than does chronic CsAT.     

PDGF Receptor β Is a Potent Regulator of Mesenchymal Stromal Cell Function

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet‐derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)β gene by Cre‐loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF‐A, ‐B, ‐C, and ‐D as well as PDGFRα and β was detected. Depletion of PDGFRβ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT‐PCR. PDGF‐BB, but not PDGF‐AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF‐BB were eliminated by depletion of PDGFRβ in MSCs except that PDGF‐BB still suppressed osterix expression in PDGFRβ‐depleted MSCs. Depletion of PDGFRβ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRβ gene deletion, we showed that PDGFRβ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRα on the osteogenic differentiation were very subtle. PDGFRβ could represent an important target for guided tissue regeneration or tissue engineering of bone.     

Inhibition of wound contraction by papaverine: in vitro analysis with a submucosal tissue model.

We studied the effect of topical application of papaverine, a smooth-muscle relaxant, on the contraction of open, dorsal skin wounds in rats. Wound contraction was inhibited significantly in papaverine-applied wounds compared with saline-dressed control wounds. The in vitro effect of papaverine on wound contraction was studied by using human oral fibroblasts cultured three-dimensionally in the hydrated collagen gels containing papaverine. Papaverine inhibited collagen gel contraction in a dose-dependent manner. Any change in actin filament organization in fibroblasts cultured three-dimensionally in the collagen gels was observed by staining the filaments with fluorescent dye-conjugated phalloidin. In the control cultures, well-organized stress fibers were formed in the cell projections; in papaverine-treated fibroblasts, during the early stages of the treatment, the stress fibers were disrupted and actin filament aggregation was observed. In addition, papaverine induced a delay in the formation of the processes in fibroblasts cultured in collagen gels. These results indicate that wound contraction inhibition by papaverine is mediated by its effects on the organization of actin filaments of fibroblasts.     

Altered cultured mesangial cell phenotypes from RF/J mice: a spontaneous immune complex mediated glomerulonephritis with progressive glomerulosclerosis.

BACKGROUND/AIM: RF/J mice are a model of spontaneous immune complex mediated glomerulonephritis showing massive extracellular matrix accumulation and progressive glomerulosclerosis. The aim of this study was to investigate whether there is an altered cultured mesangial cell (MC) phenotype in RF/J mice associated with these glomerular changes. METHODS: The nature of cultured MCs from RF/J mice in the proliferative response to platelet-derived growth factor (PDGF) BB was compared with that of normal mice (BALB/c) by 3H-thymidine incorporation. The binding of PDGF-BB was examined with Scatchard analysis, and the messenger RNAs (mRNAs) of PDGF beta-receptor, collagen I, collagen IV, and fibronectin were detected using Northern blot analysis in the MCs of each mouse. RESULTS: The 3H-thymidine incorporation of MCs from RF/J mice showed significantly lower responses to PDGF-BB stimulations with concentrations ranging from 0.5 to 10.0 ng/ml in comparison with those of BALB/c mice which exhibited a proportional dose- dependent increase of the incorporation (p < 0.05 for 0.5 ng/ml PDGF-BB, p < 0.01 for 1.0-10.0 ng/ml). According to the Scatchard analysis, MCs from BALB/c mice showed aKD of 105 pM of PDGF-BB binding to its receptors, and the density of receptors was 5.82 fmol/10(5) cells. However, no binding PDGF-BB site on the surface of MCs from RF/J mice was noted. Northern blot analysis of MCs from RF/J mice indicated negative expression of detectable PDGF-beta receptor mRNA. As for matrix protein messages, MCs from RF/J mice did not express mRNA of type I collagen, but did express a higher amount of type IV collagen and fibronectin in comparison with MCs from normal BALB/c mice. CONCLUSIONS: An altered phenotype in MCs of RF/J mice was demonstrated, possibly contributing to the characteristic pathological glomerular changes. However, the precise association remains to be clarified.     

Effects of bone associated growth factors on DNA, collagen and osteocalcin synthesis in cultured fetal rat calvariae

Studies in bone and bone cell cultures have shown that osteocalcin synthesis is dependent on the maturity of the osteoblast and the presence of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3. The bone matrix is a rich source of growth factors that play a role in bone formation, but their effects on osteocalcin synthesis and their interactions with 1,25(OH)2D3 have not been examined. Insulin-like growth factor I (IGF I), basic and acidic fibroblast growth factor (bFGF and aFGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta), are growth factors associated with the bone matrix. These factors were shown to stimulate [3H]thymidine incorporation into DNA in 24 h cultures of fetal rat calvariae, and their effect was not modified by 1,25(OH)2D3. IGF I and TGF beta stimulated [3H]proline incorporation into calvarial collagen while the other growth factors studied did not; 1,25(OH)2D3 inhibited collagen synthesis in control as well as in IGF I and TGF beta treated calvariae. IGF I, bFGF and aFGF stimulated osteocalcin synthesis 1.5 to 2.5 fold but only IGF I was synergistic with the stimulatory effect of 1,25(OH)2D3. PDGF and TGF beta had no effect on osteocalcin synthesis. In conclusion, bone matrix-associated factors have important mitogenic effects in bone cultures, but only IGF I and FGFs stimulate osteocalcin synthesis, an effect that is of small magnitude when compared to that of 1,25(OH)2D3.     

PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis

BACKGROUND: Platelet-derived growth factor (PDGF) has been consistently implicated in the cell proliferation and extracellular matrix accumulation, which characterize progressive glomerular disease. In the present study, the effects of a potent and selective inhibitor of PDGF receptor tyrosine kinase, STI 571, were examined in vitro and in vivo. METHODS: Cultured mesangial cells were incubated with PDGF (50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL) and treated with STI 571 (0.13 to 2.0 micromol/L). Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 antibody with rats randomized to receive either STI 571 (50 mg/kg intraperitoneally daily) or vehicle. Animals were examined six days later. RESULTS: In vitro, both PDGF and FGF-2 induced a threefold increase in mesangial cell 3H-thymidine incorporation. STI 571 reduced PDGF but not FGF-2-stimulated mesangial cell proliferation in a dose-dependent manner, with complete abolition at 0.4 micromol/L. In animals with Thy-1.1 glomerulonephritis, PDGF receptor tyrosine kinase blockade was associated with significant reductions in mesangial cell proliferation (P < 0.001), the number of activated (alpha-smooth muscle positive) mesangial cells, and glomerular type IV collagen deposition (P < 0.001). CONCLUSION: The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by blockade of PDGF receptor activity suggests the potential clinical utility of this approach as a therapeutic strategy in glomerular disease.     

In vitro alterations in cytoskeletal tensional homeostasis control gene expression in tendon cells.

An in vitro collagen gel system was used to determine the effect of alterations in cytoskeletal tensional homeostasis on gene expression in tendon cells. Collagen gel matrices, seeded with rat tail tendon cells, underwent cytochalasin D and gel contraction treatments designed to alter the internal cytoskeletal homeostasis of the cells. Gels were examined for cytoskeletal organization using a rhodamine phalloidin stain for actin. The effect of altered cytoskeletal organization on mRNA expression of a catabolic (interstitial collagenase) and anabolic (alpha1(I) collagen) gene was examined using northern blot analysis. Tendon cells in adhered gels demonstrated a highly organized cytoskeleton and showed evidence of alpha1(I) collagen mRNA expression but no evidence of collagenase mRNA expression. Treatment of the attached gel with cytochalasin D disrupted the cytoskeletal organization and resulted in the up-regulation of collagenase mRNA and the inhibition of alpha1(I) collagen mRNA expression. Release of the gels resulted in a cell mediated gel contraction, an immediate loss of cytoskeletal organization, and an mRNA expression pattern similar to that seen with cytochalasin D treatment. Isometric contraction of the gel on itself or around a 3-point traction device resulted in an mRNA expression pattern similar to the adhered gel. Gene expression in the contracted gels could be reversed through chemical cytoskeletal disruption or removal of the traction device which permitted further gel contraction. The results of the study suggest that tendon cells can establish an internal cytoskeletal tension through interactions with their local extracellular environment. Alterations in this tension appear to control the expression of both catabolic and anabolic genes in a reciprocal manner.