Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

Interferon-alpha and interferon-gamma reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture

The effects of interferon-alpha and interferon-gamma on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-gamma treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-gamma reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-alpha on procollagen mRNA levels was somewhat weaker than that of interferon-gamma. The results suggest potential use of interferon-gamma in treatment and prevention of human fibrotic conditions.     

The Effects of Age and the Expression of SPARC on Extracellular Matrix Production by Cardiac Fibroblasts in 3-D Cultures

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.     

Endothelin-1 expression in vascular adventitial fibroblasts

Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was accompanied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II.     

Interferon-α and interferon-γ reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture

The effects of interferon-α and interferon-γ on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-γ treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-γ reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-α on procollagen mRNA levels was somewhat weaker than that of interferon-γ. The results suggest potential use of interferon-γ in treatment and prevention of human fibrotic conditions.     

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-beta) enhanced procollagen synthesis by 45 +/- 3% (P < 0.001), and 38 +/- 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 +/- 8% (P < 0.01) and 72% +/- 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-beta-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.     

Role of feeder cells in spreading and cytoskeleton organization of newborn rat keratinocytes.

We studied the effect of feeder cells (fibroblasts) and a mixture of the extracellular matrix components, Matrigel, on spreading and cytoskeleton organization of newborn rat keratinocytes (REK). REK formed lamellipodia on being plated together with feeder cells and on the Matrigel as a substrate whereas the same REK plated alone on a plastic surface formed filopodia. REK lamellipodia formation in co-cultures depended on the fibroblast addition time. Although conditioned medium from fibroblast cultures was not enough to induce lamellipodia, the extracellular matrix left after fibroblast removal was as effective as Matrigel. Our results indicate that lamellipodia formation seems to depend on the factor(s) secreted by fibroblasts and associated with the extracellular matrix.     

Co-ordinate increase in the expression of type I and type III collagen genes in progressive systemic sclerosis fibroblasts.

Progressive systemic sclerosis (PSS), is a connective tissue disease characterized by excessive accumulation of collagen in the skin and various internal organs which is due, at least in part, to increased collagen production by PSS fibroblasts. In order to examine the molecular mechanisms responsible for this abnormality, we compared the kinetics of collagen biosynthesis, the intracellular degradation of collagen and the expression of Types I and III procollagen genes between normal and PSS dermal fibroblasts in culture. Two age- and sex-matched normal and PSS dermal fibroblast cell lines were studied. The results showed that the PSS cultures produced higher amounts of collagen than did normal fibroblasts and displayed an abnormal kinetic pattern. Furthermore, the PSS cells showed a slight but statistically significant increase in the fraction of collagen degraded intracellularly when compared with normal cells (23% against 18% respectively). The levels of mRNA for procollagen Types I and III were determined by Northern and dot-blot hybridization with specific cloned cDNA probes for alpha 1(I), alpha 2(I) and alpha 1(III) and it was found that they were 2-3-fold higher for each of the three chains in the PSS cell lines compared with the controls. These findings indicate, therefore, that the overproduction of collagen characteristic of PSS fibroblasts can be largely accounted for by the increased levels of collagen mRNA.     

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-beta and triggers apoptosis in dermal fibroblasts.

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-beta1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-beta measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-beta, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-beta and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring.     

Insulin-like growth factor I receptor is downregulated after alveolarization in an apoptotic fibroblast subset

After alveolar formation, >20% of interstitial lung fibroblasts undergo apoptosis, a process that is of critical importance for normal lung maturation. The immature lung contains two morphologically distinct fibroblast populations, lipid-filled interstitial fibroblasts (LIF) and non-LIF (NLIF), which differ with respect to contractile protein content, proliferative capacity, and expression of mRNAs for fibronectin and types I and III collagen, but not tropoelastin. After alveolarization, apoptosis occurs in only one fibroblast population, the LIF. Using flow cytometry to analyze fibroblasts stained with a lipophilic, fluorescent dye, we identified a subset, designated LIF(-), that contained fewer lipid droplets. Unlike LIF that retain lipid, LIF(+), the LIF(-) do not undergo apoptosis after alveolarization. In LIF(+), apoptosis was correlated with downregulation of insulin-like growth factor I receptor (IGF-IR) mRNA and cell surface protein expression. Treatment with anti-IGF-IR decreased total lung fibroblast survival (P = 0.05) as did treatment with the phosphatidylinositol 3-kinase inhibitor LY-294002 and the ras-raf-mitogen-activated protein kinase inhibitor PD-98059 (P < 0.002), which block IGF-I/insulin receptor survival pathways. These observations implicate downregulation of IGF-IR expression in fibroblast apoptosis after alveolar formation.     

Induction of procollagen processing in fibroblast cultures by neutral polymers

In cultures of dermal fibroblasts, procollagen and the intermediates pC- and pN-collagen accumulated in the culture medium with little further processing to collagen. When polyethylene glycol (PEG) or other neutral polymers were added to fibroblast culture medium, no collagen or procollagen was found in the medium, but all the collagen was associated with the cell layer. The type I procollagen was fully processed to collagen with an initial transient accumulation of pN-collagen, and the processed collagen formed covalently cross-linked dimers. The presence of pepsin-sensitive COOH-terminal telopeptides and the accumulation of pN-collagen in PEG-treated cultures of dermatosparactic fibroblasts indicated that it was likely that processing occurred via the correct in vivo propeptidase activities. At the levels used in this study, PEG did not have any toxic effect during the incubation period on the fibroblasts in culture, since the amount of total protein synthesis was not altered by addition of PEG to cultures. However, the level of collagen production was reduced to about half, indicating that there was increased degradation or that the released collagen propeptides or the accumulation of processed collagen in association with the cells exerted a feedback regulation on collagen synthesis. Addition of neutral polymers to the culture medium provided a simple means of achieving complete and accurate processing of procollagen which more closely resembled the in vivo process.     

Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-β-induced myofibroblast formation. FnE forms a stable complex with α_Vβ₃ integrin, and the integrin β₃ subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-β-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis.     

Different regulation of collagen I gene transcription in three-dimensional lattice cultures.

Human skin fibroblasts were cultivated in confluent monolayers, retracting collagen lattices, retracting fibrin lattices and non-retracting fibrin lattices and the expression of messenger RNA specific for the alpha 1 chain of type I procollagen comparatively studied by Northern blot and dot blot hybridization. Two factors contribute to the lower level of procollagen messenger RNA in collagen lattices: the retraction and the nature of the fibrillar protein that constitutes the lattices. Fibrin lattices, when they do not retract, make as much collagen and procollagen mRNA as monolayer confluent cells.