Procollagen mRNA Levels Research Articles

Amino acid availability regulates type I procollagen accumulation in human lung fibroblasts

Fibrotic lung diseases are characterized by excessive deposition of type I collagen. Amino acid availability regulates type I collagen mRNA levels in quiescent human lung fibroblasts. In these studies, the effect of amino acid availability on type I collagen protein accumulation in quiescent human lung fibroblasts was examined. Following amino acid deprivation, alpha1(I) procollagen protein levels were not detected by Western blot analysis in either the intracellular or the extracellular compartments. Fibronectin levels and total protein levels were not affected. Amino acid deprivation resulted in a more pronounced decrease in alpha1(I) procollagen protein levels than in alpha1(I) procollagen mRNA levels, suggesting that post-transcriptional events were responsible for the further decrease inalpha1(I) procollagen protein levels. The addition of transforming growth factor-beta to amino acid deprived fibroblasts increased alpha1(I) procollagen mRNA levels without affecting alpha1(I) procollagen protein levels, confirming a post-transcriptional site for regulatory control by amino acid deprivation. In the absence of ascorbic acid, alpha1(I) procollagen protein levels increased in amino acid deprived fibroblasts, but alpha1(I) procollagen mRNA levels were not affected. The absence of ascorbic acid likely resulted in the accumulation of nonhelical procollagen in the endoplasmic reticulum, indicating that translational mechanisms for alpha1(I) procollagen were intact. The addition of chloroquine, an inhibitor of lysosomal degradation of proteins, increased alpha1(I) procollagen protein levels in amino acid deprived fibroblasts. These data suggest that following amino acid deprivation of quiescent fibroblasts, newly synthesized type I collagen was degraded intracellularly, primarily by a process that involved lysosomal proteinases.

Involvement of type VI collagen in interleukin-4-induced mineralization by human osteoblast-like cells in vitro

We recently showed that interleukin-4 (IL-4) enhanced collagen and osteocalcin accumulation and caused mineralization in human periosteal osteoblast-like (SaM-1) cells. At that time, the expression of α1(VI) collagen mRNA was induced. In the present study, the possible role of IL-4-induced type VI collagen in the in vitro mineralization in osteoblasts was investigated. Addition of IL-4 in the early stage (for the first 10 days) was essential for the mineralization. The mRNA levels of α1(VI) and α2(VI) collagen and protein level of type VI collagen were transiently increased by IL-4 treatment up to day 5, whereas the α1(I) procollagen mRNA level was greater at day 10 than at day 5. Addition of anti-type VI collagen antibody remarkably reduced the extracellular accumulations of calcium and hydroxyproline induced by IL-4. Furthermore, the transfection of antisense oligonucleotides of α1(VI) to SaM-1 cells in the presence of IL-4 partially inhibited IL-4-induced type I collagen accumulation. These results demonstrated that type VI collagen played important roles for IL-4-induced mineralization and hydroxyproline accumulation, mostly type I collagen accumulation, in human periosteal osteoblast-like cells.

Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I.

The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by high-performance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining, expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared with L‐ascorbate in human intestinal smooth muscle cells

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of l-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 μM. By contrast, l-ascorbate was required at 4–5 times the concentration for the same response. However, at 20 μM, both palmitoyl and l-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas l-ascorbate had no effect. Palmitoyl ascorbate and l-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or l-ascorbate on the activity of a procollagen α2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than l-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro. J. Cell. Biochem. 73:312–320, 1999. © 1999 Wiley-Liss, Inc.

3,4-Methylenedioxymethamphetamine (“Ecstasy”) Stimulates the Expression of α1(I) Procollagen mRNA in Hepatic Stellate Cells

3,4-Methylenedioxymethamphetamine, MDMA (“Ecstasy”), has been previously shown to produce cell necrosis and fibrosis in the liver. Our aim was to study the effect of MDMA on the type I collagen production by a cell line of hepatic stellate cells (HSC), the cell type mainly responsible for collagen synthesis in the liver. We demonstrated that MDMA increases α1(I) procollagen mRNA levels and that this increase correlates with glutathione depletion and enhanced hydrogen peroxide production by HSC. Pre-treatment with either glutathione monoethyl ester or deferoxamine prevents the MDMA-induced α1(I) procollagen mRNA expression, indicating oxidative stress to be a mediator of this effect. Lipid peroxidation was not detected in MDMA-treated cells and therefore does not seem to be involved in the pro-fibrogenic action of MDMA on HSC.

Increased tropoelastin and procollagen expression in the lung of nitrofen-induced diaphragmatic hernia in rats

Background/Purpose: Collagen and elastin, the predominant components of the lung connective tissue network, have been suggested to have an important influence on lung compliance and maximal expansion. Decrease in lung complicance and distensibility often is seen in human congenital diaphragmatic hernia (CDH) lung as well as in experimentally produced CDH lung. The aim of this study was to investigate mRNA levels of tropoelastin and α1 (I) procollagen, the precursors of elastin, and type I collagen, respectively, in CDH lung and to determine whether antenatal dexamethasone treatment has any effect on the production of these extracellular matrix proteins.Methods: CDH model was induced in pregnant rats after administration of 100 mg nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (0.25 mg/kg) was given on day 18.5 and 19.5. Cesarean section was performed on day 21. The fetuses were divided into three groups: group I, normal controls; group II, nitrofen-induced CDH; and group III, nitrofen-induced CDH with antenatal dexamethasone treatment. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate relative amounts of tropoelastin and α1 (I) procollagen mRNA.Results: Levels of both tropoelastin and α1 (I) procollagen mRNA were significantly increased in group II compared with group I (P < .05). Neither tropoelastin nor α1 (I) procollagen mRNA levels were significantly different between group II and III.Conclusions: The increased local synthesis of tropoelastin and type I procollagen in CDH lung may be responsible for the increased rigidity and decreased compliance observed in the CDH hypoplastic lung. Glucocorticoids have no effect on pulmonary tropoelastin and α1 (I) procollagen gene expression in CDH lungs.

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.

Expression of type I and type III collagens during the course of dimethylnitrosamine-induced hepatic fibrosis in rats.

We wished to clarify the mechanisms that account for the increase in hepatic collagen accumulation during hepatic fibrosis. The gene expression of type I and type III procollagens and matrix metalloproteinase-1 (MMP-1) was measured by Northern blot analysis; immunolocalization of both types of collagen was estimated by indirect immunohistochemical assay; and the hepatic content of collagen and malondialdehyde (MDA), a product of lipid peroxidation, were assayed in hepatic fibrosis induced in rats with a single dose of dimethylnitrosamine (DMN). During the experimental period, more type I procollagen mRNA was found than type III procollagen mRNA. The immunoreactive intensity of type I collagen was greater in necrotic areas near central veins 3 days after DMN treatment than it was on day 9, whereas the type III collagen immunodeposition for the latter period of the hepatic fibrosis was stronger than it was on day 3. As compared with controls, hepatic collagen content increased significantly after 3 days and continued, increasing gradually, as did type I and III procollagen mRNA levels. On day 14, fibrosis was greatest and both types of procollagen gene expression were at their highest, and type I and III procollagen mRNA levels and hepatic collagen content increased as the dosage of DMN was raised. MMP-1 mRNA levels increased early in hepatic fibrogenesis, and increased on day 14 when DMN dosages were low. Hepatic MDA levels increased rapidly for 3 days after DMN treatment, remaining significantly higher than control values and showing a significant increase even in response to low DMN doses on day 14. Our results suggested that fibrotic liver collagen content may make its first notable increase due in part to the balance between type I collagen and MMP-1 expression rates. Also, lipid peroxidation may be important in the mechanism of hepatofibrogenesis.

Type I procollagen synthesis is regulated by steroids and related hormones in human osteosarcoma cells

Change in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post-transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C-terminal propeptide (PICP) and the levels of alpha 1(I) procollagen mRNA in cultured human MG-63 and SaOs-2 osteoblast-like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto-oncogenes, c-fos and c-jun, in MG-63 cells. In MG-63 cells, calcitriol stimulated the synthesis and secretion of PICP. The alpha 1(I) procollagen mRNA level was elevated with no effect on message stability, indicating a transcriptional mechanism of regulation. In contrast, dexamethasone treatment was accompanied by an accelerated rate of alpha 1(I) procollagen mRNA turnover, observed as decreased amounts of the message and the secreted PICP, implying a posttranscriptional regulation. Retinoic acid, in turn, decreased the levels of alpha 1(I) procollagen mRNA and secreted PICP by slowing down transcription of the COL1A1 gene without any effect on message stability. The ability of these hormones to regulate the alpha 1(I) transcripts was sensitive to puromycin treatment, suggesting an involvement of an induced mediator protein in the action of the hormones on the COL1A1 gene. Both dexamethasone and calcitriol rapidly but transiently increased the expression of the c-fos and c-jun proto-oncogenes. Neither proto-oncogene responded to retinoic acid treatment with significant changes in mRNA levels. Estradiol treatment was found to have no influence on type I procollagen synthesis. In SaOs-2 cells, which are not as well differentiated as the MG-63 cells, calcitriol and dexamethasone did not influence type I procollagen synthesis. Retinoic acid as well as estradiol reduced collagen gene expression in these cells. These findings suggest that hormonal effects on type I procollagen synthesis may depend on the maturational state of the osteoblastic cells that express different regulatory factors and receptors, resulting in, in each case, a finely adjusted rate of gene expression.

Type I procollagen synthesis is regulated by steroids and related hormones in human osteosarcoma cells

Changes in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post-transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C-terminal propeptide (PICP) and the levels of α1(I) procollagen mRNA in cultured human MG-63 and SaOs-2 osteoblast-like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto-oncogenes, c-fos and c-jun, in MG-63 cells. In MG-63 cells, calcitriol stimulated the synthesis and secretion of PICP. The α1(I) procollagen mRNA level was elevated with no effect on message stability, indicating a transcriptional mechanism of regulation. In contrast, dexamethasone treatment was accompanied by an accelerated rate of α1(I) procollagen mRNA turnover, observed as decreased amounts of the message and the secreted PICP, implying a posttranscriptional regulation. Retinoic acid, in turn, decreased the levels of α1(I) procollagen mRNA and secreted PICP by slowing down transcription of the COL1A1 gene without any effect on message stability. The ability of these hormones to regulate the α1(I) transcripts was sensitive to puromycin treatment, suggesting an involvement of an induced mediator protein in the action of the hormones on the COL1A1 gene. Both dexamethasone and calcitriol rapidly but transiently increased the expression of the c-fos and c-jun proto-oncogenes. Neither proto-oncogene responded to retinoic acid treatment with significant changes in mRNA levels. Estradiol treatment was found to have no influence on type I procollagen synthesis. In SaOs-2 cells, which are not as well differentiated as the MG-63 cells, calcitriol and dexamethasone did not influence type I procollagen synthesis. Retinoic acid as well as estradiol reduced collagen gene expression in these cells. These findings suggest that hormonal effects on type I procollagen synthesis may depend on the maturational state of the osteoblastic cells that express different regulatory factors and receptors, resulting in, in each case, a finely adjusted rate of gene expression. J. Cell. Biochem. 68:151–163, 1998. © 1998 Wiley-Liss, Inc.

Thrombin Stimulates Smooth Muscle Cell Procollagen Synthesis and mRNA Levels via a PAR-1 Mediated Mechanism

Thrombin is a serine protease involved in haemostasis which exerts a number of cellular effects, including stimulating mesenchymal cell migration, proliferation, and has been implicated both in normal wound healing and pathological conditions associated with hyperproliferation of smooth muscle cells such as atherosclerosis and restenosis. We hypothesize that thrombin, in addition to its proliferative effects, may also influence the deposition of matrix proteins at sites of vascular injury by directly stimulating smooth muscle cell procollagen production. 10 nM thrombin significantly stimulated rat aortic smooth muscle cell procollagen production by 34 +/- 3% compared to media control cells over a 48 h incubation period, and increased steady state alpha1(I) procollagen mRNA levels by up to 104 +/- 22%. These effects are mediated via interaction of thrombin with the PAR-1 receptor since TRAP (Thrombin Receptor Activating Peptide) stimulated procollagen production by 23 +/- 0.5%. In addition, conditioned medium from thrombin-treated cells stimulated procollagen production by 30 +/- 3% suggesting that thrombin is acting via the production and/or release of an autocrine mediator. These data suggest a novel role for thrombin in vascular wound healing and the development of pathological conditions associated with increased connective tissue deposition.

Cloning and characterization of hcKrox, a transcriptional regulator of extracellular matrix gene expression

cKrox is a novel zinc finger-containing transcription factor that binds to the α1(I) and α2(I) collagen gene promoters. The gene coding for cKrox is a new member of a family of early growth response genes, that play important roles in development. In the mouse, cKrox is expressed, beginning at 9.5 days of gestation and at 10.5 days in regions destined to become skin. In adult animals, expression is predominantly in skin, one of the two major organs where type I collagen is expressed. We have isolated cDNA clones for human cKrox. Theoretical translation of the nucleic acid sequence reveals 90% conservation of amino acids between the mouse and human proteins; however, the human gene product contains a 117-amino-acid N-terminal extension. The amino acid sequences of the zinc-finger DNA binding domains of mouse and human cKrox are identical. RT-PCR analysis of human fibroblasts indicates constitutive low-level expression of cKrox which can be transiently elevated by treatment with retinoic acid. Transient transfection assays indicate that hcKrox represses transcription of the α1(I) procollagen promoter, and electrophoretic mobility shift assays demonstrate that hcKrox binds to both the human and murine promoter DNA. Deletion derivatives of hcKrox demonstrate transcription-activating potential that is promoter-dependent. NIH3T3 cells permanently expressing hcKrox demonstrate a threefold and 10-fold decrease in α1(I) procollagen and fibronectin mRNA levels, respectively, compared to control cells. Consistent with this finding, a fibronectin promoter reporter construct is repressed more than 80% by hcKrox. These data suggest that hcKrox represses collagen transcription directly, and it may function as a repressor of fibronectin and possibly other matrix genes.

High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma.

Remodeling of pulmonary capillaries occurs after chronic increases in capillary pressure (e.g., mitral stenosis). Also, remodeling of pulmonary arteries begins within 4 h of increased wall stress and is endothelium dependent. We have previously shown that high lung inflation increases wall stress in pulmonary capillaries. This study was designed to determine whether high lung inflation induces remodeling of the extracellular matrix (ECM) in lung parenchyma. Open-chest rabbits were ventilated for 4 h with 9-cmH2O positive end-expiratory pressure (PEEP) on one lung and 1-cmH2O PEEP on the other (High-PEEP group), or with 2-cmH2O PEEP on both lungs (Low-PEEP group). An additional untreated control group was also included. We found increased levels of mRNA in both lungs of High-PEEP rabbits (compared with both the Low-PEEP and untreated groups) for alpha1(III) and alpha2(IV) procollagen, fibronectin, basic fibroblast growth factor, and transforming growth factor-beta1. In contrast, alpha2(I) procollagen and vascular endothelial growth factor mRNA levels were not changed. We conclude that high lung inflation for 4 h increases mRNA levels of ECM components and growth factors in lung parenchyma.

Apoptosis and proliferation of fibroblasts during postnatal skin development and scleroderma in the tight-skin mouse.

Tight-skin (Tsk) is a dominant gene mutation that causes a fibrotic skin disease in mice, similar to human scleroderma. Both conditions are characterized by increased numbers of dermal fibroblasts containing high levels of procollagen mRNA. Whether this fibroblast population arises from fibroblast growth or fibroblast transcriptional activation is debated. Proliferation and apoptosis of fibroblasts of normal and Tsk mice were studied in skin sections before, at onset, and in established fibrosis. Tissues sections were immunostained with proliferating cell nuclear antigen (PCNA) as proliferation marker. Apoptosis was investigated by in situ end-labeling of fragmented DNA and nuclear staining with propidium iodide. The expression of the apoptosis inhibitor Bcl-2 was investigated by immunohistochemistry. We demonstrate differences in fibroblast proliferation and apoptosis related to postnatal skin growth and development. Neonatal skin exhibits the highest levels of proliferation and apoptosis in fibroblasts. In contrast, low proliferation and absence of apoptosis characterizes adult fibroblasts. Skin fibroblasts express Bcl-2 only in newborns, and at other ages Bcl-2 was restricted to epithelial cells. Our results also suggest that neither increased fibroblast proliferation nor defective apoptosis accounts for the fibrotic phenotype of Tsk. Therefore, transcriptional activation of extracellular matrix genes appears more relevant in the pathogenesis of Tsk fibrosis.

Lack of influence of cyclosporin A on levels of gingival procollagen types I and III mRNAs in rats of different ages

Previous studies showed that gingival overgrowth following cyclosporin A (CsA) administration is not associated with an increase in interstitial collagen. It also was shown that CsA causes a significant decrease in collagen content within the gingival stroma. In order to determine whether this decrease is caused by down-regulation of collagen mRNA, the procollagen mRNA level in gingiva of young and old rats was measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labelled cDNA probes for procollagen types I and III with total RNA extracted from the molar gingiva showed that administration of CsA did not change the steady-state levels of mRNAs for both procollagens in the gingiva of either young or old rats. The ratio of gingival interstitial collagen to DNA was significantly reduced in the CsA-treated animals (4.2 ± 0.85) relative to the controls (7.8 ± 1.6). It is concluded that the reduction in interstitial collagen following CsA treatment is not age-related, and is most probably caused by increased degradation rather by decreased biosynthesis.

TGF-β3 Stimulates and Regulates Collagen Synthesis Through TGF-β1-Dependent and Independent Mechanisms

In contrast to the TGF-beta1 and beta2 isoforms, TGF-beta3 has shown the ability to downregulate scarring and fibrosis in vivo under certain experimental conditions. In this study, we determined the direct effects of TGF-beta3 on cultures of human dermal fibroblasts. TGF-beta3 (0.1 to 100 pg per ml) increased DNA synthesis up to 50% (p < 0.01, r = 0.970), collagen protein synthesis up to 200% (dose range of 0.1 to 5 ng per ml, p < 0.001, r = 0.990), and increased alpha1(I) procollagen mRNA levels (r = 0.999), with maximal effects (200% of control) observed by 24 h. Collagen lattice contraction was increased by more than 50% in response to TGF-beta3 (p < 0.001), and to a similar extent as the TGF-beta1 isoform. Stimulation of collagen synthesis and of alpha1(I) procollagen mRNA levels in response to TGF-beta3 was partially blocked by a TGF-beta1-specific anti-sense oligonucleotide but was still detectable (35% greater than baseline) when TGF-beta3 was added to dermal fibroblasts from TGF-beta1 knock-out mice. In contrast with these stimulatory effects, however, downregulation of alpha1(I) procollagen, alpha1(III) procollagen, and TGF-beta1 mRNA levels toward baseline occurred when TGF-beta3 (0.1 to 5 ng per ml) was added simultaneously and in combination with TGF-beta1. We conclude that stimulation of collagen synthesis by TGF-beta3 occurs through TGF-beta1-dependent and independent pathways. By downregulating the response to TGF-beta1 and by shifting from one pathway to the other, TGF-beta3 can dampen and provide fine-tuning to the overall TGF-beta's induced program of collagen deposition.

Selective modulation of collagen gene expression by different isoforms of platelet-derived growth factor in experimental wound healing.

Subcutaneous sponges in rat were used as a wound model to study the changes in collagen gene expression induced by different isoforms of platelet-derived growth factor (PDGF). The steady state levels of proalpha1(I) and proalpha1(III) collagens rose markedly in response to PDGF-AB treatment. In marked contrast to PDGF-AB, PDGF-BB exerted a down-regulatory effect on the expression of type I and III procollagen mRNA levels. In situ hybridisation signal for proalpha1(I) collagen mRNA was most prominent in the periphery of the sponges in response to PDGF-AB. In PDGF-AA and PDGF-BB treated sponges, however, the hybridisation signal for proalpha1(I) collagen mRNA was evenly distributed. Immunolabellings demonstrated the presence of type I and III collagen epitopes and thus revealed the deposition of these proteins into developing granulation tissue. The volume of ingrown granulation tissue was estimated by measuring the cross-sectional area of the samples. PDGF-AB and PDGF-BB were both effective resulting in a significant increase in the amount of granulation tissue. However, only a limited enhancement of granulation tissue ingrowth was observed in response to PDGF-AA treatment. To conclude, PDGF-AB and PDGF-BB induced significantly the ingrowth of repair tissue in wounds. PDGF-AB distinctively upregulated collagen gene expression, while PDGF-BB primarily seemed to be mitogenic.

Interleukin-1 beta and phenytoin reduce alpha 1 (I) procollagen mRNA expression in human gingival fibroblasts.

Effects of and interactions between interleukin-1 beta (IL-1 beta) and phenytoin (PHT) on alpha 1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E2 (PGE2) formation were studied. IL-1 beta (300 pg/ ml) reduced the steady-state level of alpha 1 (I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 micrograms/ml) reduced the level of alpha 1 (I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1 beta, PHT potentiated the inhibitory effect of IL-1 beta on alpha 1 (I) procollagen mRNA level that was accompanied by an increased PGE2 formation. Preincubation with indomethacin (10(-6) M) partially reduced the inhibitory effect of IL-1 beta as well as of IL-1 beta in combination with PHT on the mRNA level of alpha 1 (I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE2 (> or = 10 nM) dose-dependently reduced steady-state level of alpha 1 (I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of alpha 1 (I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces alpha 1 (I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.

Keratinocyte growth factor reduces lung damage due to acid instillation in rats.

Acid aspiration is a serious complication of anesthesia and other forms of unconsciousness that can result in the adult respiratory distress syndrome (ARDS), which continues to have a very high mortality despite our current therapeutic interventions. This type of injury damages the alveolar epithelium, principally alveolar type I cells, and requires proliferation of alveolar type II cells to restore gas exchange units. Since keratinocyte growth factor (KGF) has been shown to be a potent mitogen for alveolar type II cells, we evaluated whether intrabronchial administration of KGF would minimize lung injury due to the unilateral instillation of 0.1 N hydrochloric acid (HCl). Rats were pretreated or post-treated by intrabronchial instillation of KGF (5 mg/kg) into the left lung before HCl instillation. All rats receiving KGF at 48 or 72 h before HCl instillation survived for the 7-day observation period, whereas the mortality rate for those receiving HCl alone or saline followed by HCl was 31% and 33%, respectively. Pretreatment with KGF at 72 h but not at 24 or 48 h considerably ameliorated morphologic damage produced by HCl. Inflammatory cells in bronchoalveolar lavage were markedly decreased 3 and 7 days after HCl instillation by the 72-h KGF pretreatment. Pretreatment with KGF at 72 h also attenuated the reduction of total lung capacity, decreased the alpha 1(I) procollagen mRNA levels, and diminished hydroxyproline accumulation due to HCl instillation. Saline pretreatment at 72 h had no significant effect on the HCl injury and subsequent physiologic abnormalities. Our attempts to improve outcome with post-treatment instillation of KGF were unsuccessful. We conclude that KGF pretreatment reduces lung injury due to acid instillation and can prevent subsequent pulmonary fibrosis.

Site-specific responses to monocrotaline-induced vascular injury: evidence for two distinct mechanisms of remodeling.

Monocrotaline (MCT)-induced pulmonary vascular injury was used to begin studying the mechanism(s) of vascular remodeling in Fischer 344 rats, using extracellular matrix (ECM) gene expression to define areas of remodeling. By day 28 after injection, pulmonary artery pressures were increased and right ventricular hypertrophy had developed compared with normal controls. Tropoelastin, fibronectin, and alpha 1(I) procollagen mRNA levels increased at least 2-fold by day 28. In situ hybridization demonstrated tropoelastin gene expression by cells adjacent to the lumen and procollagen gene expression at the medial-adventitial border in both small muscular and large elastic pulmonary arteries. This pattern of gene expression was observed as early as 1 wk after MCT injury. These observations indicated two distinct areas of remodeling, one along the vascular lumen at the site of monocrotaline-induced injury and the other at a second distinct site. To determine whether other differences may be involved at these two sites, the presence of transforming growth factor-beta (TGF-beta) was studied. Total TGF-beta protein was 4-fold higher in remodeling lungs compared with normal lungs. Gene expression for all three isoforms of TGF-beta colocalized with tropoelastin gene expression along the vascular lumen but not with alpha 1(I) procollagen gene expression. These results suggest a complex site-specific response to injury mediated by two distinct pathways in this model of pulmonary vascular remodeling.