Fibroblast Procollagen Research Articles

Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator

The deposition of fibrin is an integral part of the tissue repair process, but its persistence is also associated with a number of fibrotic conditions. This study addressed the hypothesis that reduced fibrinolysis and fibrin persistence are associated with an enhanced accumulation of collagen and the development of skin fibrosis. Decreased fibrinolysis was confirmed in fibrin gel cultures that contained human dermal fibroblasts plus the specific plasmin inhibitor alpha(2)-antiplasmin or dermal fibroblasts isolated from plasminogen activator (PA)-deficient mice. Collagen accumulation was significantly increased in the presence of inhibitor and in tPA-deficient, but not uPA-deficient, fibroblasts compared with controls. These findings were also confirmed using a skin fibrosis model in which multiple injections of fibrin were given subcutaneously to PA-deficient mice. Injection sites from tPA-deficient mice displayed significantly increased collagen levels compared with uPA-deficient mice and wild-type controls. Up-regulation of fibroblast procollagen gene expression and reduced activation of pro-MMP-1 appeared to mediate the increase in collagen by human dermal fibroblasts in the presence of alpha2-antiplasmin. These findings suggest that persistent fibrin is associated with enhanced collagen accumulation that may result in the development of fibrotic skin disorders in which reduced fibrinolysis is a feature.

Airway fibroblasts exhibit a synthetic phenotype in severe asthma

Platelet-derived growth factor (PDGF) may have a significant role in airway remodeling in asthma, because it is a powerful inductor of many airway fibroblast activities such as collagen synthesis. To determine whether PDGF is a significant contributor to airway remodeling in patients with asthma by enhancing airway fibroblast procollagen I expression. Six normal controls without asthma, 10 subjects with mild to moderate asthma, and 5 subjects with severe asthma underwent bronchoscopy with endobronchial biopsy. Biopsies were placed in Dulbecco modified Eagle medium and fibroblasts cultured in the presence and absence of PDGF isoforms -AA, -BB, and -AB (1, 5, 10, 100 ng/mL) and insulin-like growth factor 1 (100 ng/mL). Fibroblast procollagen I and PDGF receptors (PDGFRs) alpha and beta expression were determined by ELISA. Platelet-derived growth factor BB significantly enhanced fibroblast procollagen I expression in patients with severe asthma compared with patients with mild/moderate asthma and normal controls. Furthermore, the baseline fibroblast expression of PDGFR-beta was significantly greater in patients with severe asthma compared with the other groups. This pilot study suggests that airway fibroblasts from patients with severe asthma exhibit a synthetic phenotype, which may be driven by the overexpression of PDGFR-beta.

Angiotensin II and the fibroproliferative response to acute lung injury.

Angiotensin II (ANG II), generated by activation of local renin-angiotensin systems, is believed to play an important role in tissue repair and remodeling, in part via transforming growth factor-beta (TGF-beta). Angiotensin-converting enzyme (ACE) inhibitors have been shown to abrogate experimental lung injury via a number of potential mechanisms; however, the potentially fibroproliferative role for ANG II in the lung has not been characterized. We hypothesized that, after lung injury, ANG II would stimulate fibroblast procollagen synthesis and promote lung collagen deposition in rats. In vitro, ANG II was a potent inducer of procollagen production in human lung fibroblasts via activation of the type 1 receptor and, at least in part, via the autocrine action of TGF-beta. After bleomycin-induced lung injury, an increase in lung ANG II concentration was observed by day 3 that preceded increases in lung collagen and was maintained until death at day 21. Administration of an ACE inhibitor (ramipril) reduced ACE activity, ANG II concentration, TGF-beta expression, and collagen deposition. Losartan (an ANG II type 1 receptor antagonist) also attenuated the increase in TGF-beta expression and lung collagen deposition. These observations suggest that ANG II, possibly generated locally within the lung, may play an important role in the fibrotic response to acute lung injury, at least in part via the action of TGF-beta. ACE inhibitors and receptor antagonists, already widely used clinically, should be assessed as potential new therapies for fibrotic lung disease.

Activation of Fibroblast Procollagen α1(I) Transcription by Mechanical Strain Is Transforming Growth Factor-β-dependent and Involves Increased Binding of CCAAT-binding Factor (CBF/NF-Y) at the Proximal Promoter

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen alpha 1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2- and 4-fold by continuous cyclic mechanical strain. This was accompanied by an approximately 3-fold increase in the levels of total active transforming growth factor-beta (TGF-beta) released into the medium. Inclusion of a pan-specific TGF-beta neutralizing antibody inhibited strain-induced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-beta1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-beta-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-beta response element in the rat COL1A1 gene.

Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways.

Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.

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-β) 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-β-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.

Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts.

Chronic inhalation of cadmium at the workplace or in cigarette smoke is associated with emphysema, a disease characterized by extensive disruption of lung connective tissue. We have previously shown that cadmium, at noncytotoxic doses, inhibits fibroblast procollagen production in vitro, with maximal inhibitory effects of 69 +/- 6% (P < 0.01) at 30 &microM cadmium chloride (CdCl2). In this paper we show that at similar doses, cadmium also inhibits proteoglycan synthesis, with values reduced by between 36 +/- 4% (P < 0.01) and 42 +/- 6% (P < 0.01) for proteoglycans secreted into the culture media and associated with the cell layer, respectively. The greatest inhibition was obtained for the major matrix-associated proteoglycans, versican, decorin, and the large heparan sulfate proteoglycans, with synthesis values reduced by between 60 and 70%. Biglycan and other heparan sulfate proteoglycans were also affected, with synthesis values reduced by between 25 and 45%. In contrast, total protein synthesis was unaffected. Furthermore, effects of cadmium at the protein level were mirrored by reduction in messenger RNA levels for alpha1(I) procollagen, versican, and decorin. These data support the hypothesis that cadmium may play an important role in the pathogenesis of emphysema associated with chronic inhalation of cadmium fumes by inhibiting the production of connective tissue proteins.

Mechanical Load Enhances the Stimulatory Effect of PDGF on Pulmonary Artery Fibroblast Procollagen Synthesis

The mechanical environment is a key regulator of pulmonary artery wall remodeling in normal growth and during the development of hypertension. Here we determined the direct effect of mechanical load and platelet-derived growth factor (PDGF) on adventitial pulmonary artery fibroblast replication and procollagen metabolism—two processes stimulated during the development of pulmonary hypertension.

Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production.

A preliminary study of the effects of laser radiation on collagen metabolism in cell culture.

A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells. Energy fluencies varied from 0-1 J/cm2 over a period of 1-4 days. Fibroblast procollagen production was monitored by the synthesis of [3H] hydroxyproline, and DNA replication was assessed by [3H] thymidine incorporation. Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation. Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals. Laser doses between 0.099 and 0.522 J/cm2 had the most significant stimulatory effects on fibroblast function. Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.

Misoprostol Partially Inhibits the Renal Scarring of Chronic Cyclosporine Nephrotoxicity.

Chronic cyclosporine (CY) nephrotoxicity is a well-known complication of this immunosuppressive agent, which may, in part, be attributed to abnormalities in renal prostaglandin content. We utilized misoprostol (M), a prostaglandin E(1) analog, in a rat model of chronic CY toxicity at 7 and 28 days to determine effectiveness in prevention of the renal damage. After 7 days, there were no differences in weight change, creatinine clearance, or renal scarring in rats treated with vehicle (V), CY, or CY + M; however, renal procollagen alpha 1(I) mRNA levels were increased in CY versus V rats (p < 0.05). In contrast, after 28 days CY rats had significant reductions in weight gain, glomerular filtration rate, and renal blood flow with increases in renal scarring and procollagen alpha 1 (IV) mRNA levels (all p < 0.05 versus V). Addition of M resulted in partial but significant improvement in GFR, RBF, and procollagen alpha 1(IV) mRNA levels, with lessening of the renal scarring. Skin fibroblasts also were incubated with CY and M to assess impact on procollagen MRNA levels. CY augmented fibroblast procollagen mRNA levels which enhanced by a large dose of M, but inhibited with a moderate dose. These data suggest that M improves renal scarring induced by CY by hemodynamic and/or direct effects.

Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein

Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.

Clinical Improvement Following Dermabrasion of Photoaged Skin Correlates With Synthesis of Collagen I

The ability of superficial dermabrasion to improve clinical features of photoaged skin is well known, but the specific biological mechanisms involved are poorly understood. The so-called repair zone, as visualized by routine histologic examination, has been attributed to new collagen formation within the papillary dermis and may be responsible for clinical improvement following dermabrasion. We investigated molecular and histologic events occurring in dermabraded skin and correlated them with clinical improvement. Ten photoaged patients (mean age, 59 years) underwent facial dermabrasion to the level of the papillary dermis. Clinical severity of photoaging was graded in a blinded manner at baseline and 12 weeks after dermabrasion. Biopsy specimens obtained at baseline and 3 and 12 weeks after dermabrasion were analyzed histologically and by in situ hybridization for fibroblast procollagen I mRNA, immunohistologically and by Western blotting with a monoclonal antibody specific for the aminoterminal cleavage site of procollagen I. Masson's trichrome staining demonstrated an increase in collagen from baseline (as an upper dermal band in the dermabrasion "repair zone") at 3 and 12 weeks' postdermabrasion. Immunohistologic examination demonstrated papillary dermal fibroblast staining for procollagen I at baseline that increased by threefold at 3 weeks' postdermabrasion and by 1.5-fold at 12 weeks' postdermabrasion. Western blotting demonstrated an average-fold increase in pN collagen I of 4.2 +/- 1.5 at 3 weeks and of 2.7 +/- 0.7 at 12 weeks. By in situ hybridization, baseline levels of procollagen I mRNA in papillary dermal fibroblasts increased sixfold at weeks 3 and 12 postdermabrasion. Increase in procollagen I mRNA correlated with clinical improvement, ie, reduction in wrinkling. Superficial dermabrasion clinically improves photoaged skin, and this improvement correlates strongly with increased collagen I gene expression.

Cadmium selectively inhibits fibroblast procollagen production and proliferation.

Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of platelet-derived growth factor isoforms on human lung fibroblast proliferation and procollagen gene expression.

Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts, is a potentially important cytokine in the pathogenesis of fibroproliferative disorders of lung. Different isoforms of PDGF include a heterodimer composed of A and B chains and homodimers composed of A or B chains. The biological significance of the different isoforms is unknown, but they have been shown to differ in their mitogenic potency in some systems. Their effects on other fibroblast functions have not been fully examined. We undertook this study to determine the effect of PDGF isoforms on human lung fibroblast proliferation and procollagen synthesis. Cultured lung fibroblasts (IMR-90, WI-38, GeNA) were incubated in the presence of varying concentrations of highly purified PDGF-AB obtained from platelets or recombinant PDGF-AA or -BB homodimers. Incorporation of [3H]thymidine was determined as a measure of mitogenic activity. Fetal lung fibroblasts (IMR-90, WI-38) and an adult fibroblast strain (GeNA) responded similarly to the different isoforms, with maximum mitogenic activity observed at 5-10 ng/mL. Cell cycle analysis using three additional normal adult lung fibroblast strains indicated that all PDGF isoforms stimulated similar proportions of cells to cycle over a 7-day period. Fibroblast procollagen synthesis, measured after pulse labeling with [3H]proline, was not increased even at concentrations of the PDGF isoforms that were maximally mitogenic. Moreover, steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNA levels, determined by northern analysis and dot blot hybridization, were not changed after exposure to any of the PDGF isoforms. While all PDGF isoforms are potent mitogens for fetal and adult lung fibroblasts, it was concluded that they do not directly stimulate procollagen gene expression or procollagen synthesis in vitro. The results suggest that PDGF isoforms are potentially important mitogens for lung fibroblasts, but other factors are likely to be involved in the stimulation of fibroblast procollagen synthesis that is observed in fibroproliferative disorders of lung.

Fibroblast procollagen production rates in vitro based on [ 3H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [ 3H]proline and measuring the subsequent formation of [ 3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [ 3H]proline, and [ 3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [ 14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [ 3H]proline and approximately 10 6 cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.