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PubMedDate not supplied

Activated caspase expression and apoptosis increase in keloids: cytochrome c release and caspase-9 activation during the apoptosis of keloid fibroblast lines.

To characterize apoptosis in keloids and the mechanisms responsible for this process, the expression of activated caspase-9 and -3 in fibroblasts obtained from keloids was analyzed. Immunohistochemistry revealed that the number of fibroblasts positive for terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) or activated caspase-9 or -3 was low but was significantly higher in keloid tissues than in normal scar tissues. Significant relationships between the number of caspase-positive fibroblasts and TUNEL-positive fibroblasts suggested that the activation of caspase-9 and -3 induces apoptosis in a subpopulation of keloid fibroblasts. All keloid fibroblast cell lines established in this study showed activation of caspase-9 and -3 after serum deprivation for 3 or 4 hours, as shown using Western blotting. Furthermore, serum deprivation-induced apoptosis in a keloid fibroblast line was blocked by a caspase-9 inhibitor (acetyl-Leu-Glu-His-Asp-al), indicating that activation of caspase-9 was necessary for the process of apoptosis in keloid fibroblasts. Although serum deprivation did not significantly change the level of apoptosis protease activating factor-1 in any of the lines, cytochrome c release was detected in cytosolic fractions of the lines after serum deprivation for 3 or 4 hours. These results strongly suggest that keloid fibroblasts are predisposed to apoptosis and cytochrome c release and that caspase-9 activation may underlie regulation of apoptosis in keloid fibroblasts in vivo.

Adolescent↗
PubMed1994

Extracellular matrix derived from hair and skin fibroblasts stimulates human skin melanocyte tyrosinase activity.

There is indirect evidence that both skin and hair melanocytes are regulated by the activity of adjacent cells. In hair, the specialized fibroblasts (dermal papilla cells) appear to play a role in the regulation of hair growth. Hair pigmentation may relate to hair growth. In skin, melanocytes are located adjacent to the basement membrane zone. As far as we are aware, direct interactions of fibroblasts with melanocytes have not previously been investigated. Accordingly, the objective of this study was to develop co-culture conditions in which to investigate whether dermal fibroblasts from skin or hair could influence melanocyte differentiation. The influence of fibroblast-conditioned media, co-culture with fibroblasts, and fibroblast-derived extracellular matrix (ECM) on normal human skin melanocyte tyrosinase activity was examined. Fibroblasts from both skin and hair were capable of altering melanocyte morphology and significantly increasing tyrosinase activity when melanocytes were cultured in the absence, but not the presence, of the major proliferative drives. Although stimulation of tyrosinase activity was detectable with conditioned medium and co-culture with fibroblasts, the most striking result was obtained with the fibroblast-produced ECM which, on average, produced a four-fold increase in tyrosinase activity within 6 days. Thus, the study describes co-culture conditions in which the stimulatory effect of the fibroblast on melanocyte differentiation can be examined.

Cell Differentiation↗
PubMed2004

Tumour necrosis factor-alpha but not interferon-gamma is the main inducer of inducible protein-10 in skin fibroblasts from patients with atopic dermatitis.

BACKGROUND: Inducible protein (IP)-10 belongs to the CXC chemokine subfamily and acts through the CXCR3 receptors that attract T lymphocytes. Keratinocytes are thought to be the main cell source of this chemokine in the skin, but other sources need to be elucidated. OBJECTIVES: To determine whether skin fibroblasts, besides keratinocytes, are able to produce IP-10 and the possible involvement of these cells in pathogenesis of atopic dermatitis (AD). METHODS: We studied the production pattern of IP-10 in dermal fibroblasts obtained from healthy donors, AD patients and in the HaCaT cell line (normal human keratinocytes) used as control. We stimulated fibroblasts after the sixth and seventh passage with tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and by means of reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay analysis detected the production pattern of IP-10. To determine whether a different pattern of production of IP-10 by fibroblasts corresponds to the level of this chemokine in the plasma of patients with AD, we also checked the plasma IP-10 levels in 33 AD patients and 10 healthy donors. RESULTS: The pattern of chemokine production between dermal fibroblasts and HaCaT cells was different. The main inducer of IP-10 in fibroblasts was TNF-alpha, whereas IFN-gamma was the main inducer of IP-10 in HaCaT cells. We demonstrate that fibroblasts from AD patients have higher IP-10 expression and are more sensitive to TNF-alpha stimulation compared with healthy controls. Consequently, IP-10 levels in plasma of AD patients were higher than in healthy donors. CONCLUSIONS: Skin fibroblasts could be an important source of IP-10. TNF-alpha is the main inducer of IP-10 by skin fibroblasts, but not IFN-gamma or IL-4. The increased level of IP-10 in the plasma of patients with AD could be connected with increased activity of skin fibroblasts.

Adult↗
PubMed1982

Inhibition of protein glycosylation and selective cytotoxicity toward virally transformed fibroblasts caused by B3-tunicamycin.

The biological effect of B3-tunicamycin, the only known homologue of tunicamycin which contains a saturated fatty-acid side chain, was examined using chick embryo fibroblasts, a mouse fibroblastic line (3T3) and a virally transformed mouse fibroblastic line (SV40-3T3). This homologue inhibited the transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine to dolichyl phosphate, catalyzed by microsomes from chick liver or from cultured mouse fibroblasts. B3-tunicamycin also inhibited the incorporation of mannose into glycoproteins synthesized by chick or mouse fibroblasts. Incorporation of the amino acids proline and tyrosine was inhibited by B3-tunicamycin to a lesser extent than the incorporation of mannose. The mannose incorporation into glycoproteins synthesized by virally transformed cells was inhibited by B3-tunicamycin to a higher degree than what was achieved in the nontransformed lines or in the chick primary fibroblasts. When the activity of B3-tunicamycin as an inhibitor of protein glycosylation was compared to other homologues of tunicamycin, it was found to be the most active. This homologue caused complete (more than 95%) inhibition of protein glycosylation at a concentration of 50 ng/ml in chick and in mouse fibroblasts and at a concentration of 10 ng/ml in transformed mouse fibroblasts. When the cytotoxic activities of tunicamycin homologues were examined on nontransformed and virally transformed 3T3 cells, it was found that B3-tunicamycin displayed the highest selective cytotoxicity toward the transformed cells. When transformed fibroblasts (10(5) cells/plate) were treated with B3-tunicamycin (100 ng/ml) for 48 h, complete cell death was observed. The viability and the proliferative activity of the nontransformed fibroblast were normal even when treated with concentrations up to 500 ng/ml of B3-tunicamycin. This suggests that B3-tunicamycin may be a suitable candidate for studies of tumor growth in animals.

Animals↗
PubMed1987

Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts.

The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase.

Cell Line↗
PubMedDate not supplied

A simple in vitro model for investigating epithelial/mesenchymal interactions: keratinocyte inhibition of fibroblast proliferation and fibronectin synthesis.

Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Cell Proliferation↗
PubMedDate not supplied

Growth characteristics of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies.

OBJECTIVE: To determine if there is a difference in in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. To determine the effects of a corticosteroid and monokine on in vitro growth of fibroblasts isolated from the trunk and distal aspect of the limb of horses and ponies. STUDY DESIGN: Growth of fibroblasts from tissues harvested from the trunk and limb were compared from horse and pony samples grown in control media and control media with triamcinolone or monokine added. ANIMALS OR SAMPLE POPULATION: Dermal and subcutaneous tissue from 22 horses and 17 ponies of various ages and breeds. METHODS: Fibroblast growth was assessed by tritiated thymidine uptake using standard cell culture techniques. The effect of a monokine or triamcinolone plus control media were compared with control media for fibroblast growth. RESULTS: Fibroblast growth from tissues isolated from the horse limb was significantly less than growth from the horse trunk and the limb and trunk of ponies. Monokine was more effective than triamcinolone in suppressing fibroblast growth from tissues isolated from the trunk and limb in both horses and ponies. CONCLUSIONS: There are growth differences in fibroblasts isolated from the limb of horses compared with those isolated from the trunk and from the limb and trunk of ponies. CLINICAL RELEVANCE: The difference in fibroblast growth from tissues isolated from the trunk and limb of horses and ponies may provide evidence for the difference reported in the healing characteristics of limb wounds in horses and ponies. Influencing fibroblast growth may provide a key to controlling the development of exuberant granulation tissue in horses and ponies.

Animals↗
PubMed1995

Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan.

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5 x 10(5) cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-3H]--lysine labelled purified chick--embryo aorta elastin substrate. After incubation for 10 h at 37 degrees C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84 +/- 1.15 mg/ml) and lysyl oxidase activity (3558.6 +/- 345.5 cpm/10(6) cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35 +/- 0.96 mg/ml and 2436.0 +/- 352.6 cpm/10(6) cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.

Adolescent↗
PubMed1993

Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells.

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival epithelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular matrix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibited enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular matrix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.

Animals↗
PubMed2007

Differential chemokine response of fibroblast subtypes to complement C1q.

BACKGROUND AND OBJECTIVE: The pathogenesis of periodontitis includes an inappropriate activation of the classical complement cascade (C') with accumulation of inflammatory C' products in fluids and tissues. Our hypothesis is that in vivo the C' product, C1q, may act as a regulatory component of the innate immune response of distinct matrix fibroblasts to the inflammatory environment. This study analyzed the C1q induction of pro-inflammatory cytokine secretion in fibroblast subtypes derived from distinct periodontal tissues, and identified a mechanism of the cell response. MATERIAL AND METHODS: Primary human gingival fibroblast, periodontal ligament fibroblast, and granulation tissue fibroblast cultures were treated for 24 h with C1q. Protein arrays assessed the secretory profile of constitutive and C1q-inducible pro-inflammatory cytokines, and enzyme-linked immunosorbent assays were used to quantify the kinetics of each inducible cytokine. RESULTS: Granulation tissue fibroblast cultures were unresponsive to C1q challenge. In contrast, periodontal ligament fibroblasts responded with a release of monocyte chemoattractant protein (MCP)-1, interleukin-6, interleukin-8, and macrophage inflammatory protein (MIP)-1beta higher than the basal level by 8.2-, 7.0-, 3.8-, and 7.2-fold, respectively. Human gingival fibroblast cultures increased secretion of these chemokines by 5.2-, 4.5-, 3.0-, and 9.8-fold, respectively. Inhibitor studies revealed that C1q-inducible release of chemokines by the human gingival fibroblast and periodontal ligament cultures was contingent upon p38 mitogen-activated protein kinase activity. CONCLUSION: The ability of C1q to stimulate secretion of pro-inflammatory chemokines depends upon which specific fibroblast subtype is involved. Targeting C1q-activated intracellular signaling pathways may be an effective means to inhibit the production of chemokines that promote inflammatory cell infiltration into gingival and periodontal ligament tissues.

Cells, Cultured↗
PubMed1986

Gamma-interferon-induced inhibition of the growth of Rickettsia prowazekii in fibroblasts cannot be explained by the degradation of tryptophan or other amino acids.

We examined the role of amino acid deprivation in gamma-interferon-induced (IFN-gamma) suppression of the growth of Rickettsia prowazekii in mouse L929 cells and human fibroblasts by measuring the amino acid pools in untreated and IFN-gamma-treated cells. In recombinant IFN-gamma-treated cultures of human fibroblasts, tryptophan was undetectable in both the intracellular pool and the extracellular medium. In contrast, tryptophan was not depleted from the intracellular pool or the extracellular medium of L929 cells treated with recombinant IFN-gamma or crude mouse lymphokines. None of the other amino acids measured was severely depleted in IFN-gamma-treated L929 cells and human fibroblasts. Extracts prepared from IFN-gamma-treated human fibroblasts exhibited indoleamine 2,3-dioxygenase activity, converting tryptophan into products that cochromatographed with N-formylkynurenine and kynurenine; however, extracts prepared from untreated human fibroblasts, untreated L929 cells, recombinant IFN-gamma-treated L929 cells, and mouse lymphokine-treated L929 cells did not degrade tryptophan. Human HeLa cells resembled the human fibroblasts in that they degraded tryptophan after IFN-gamma treatment. Similarly, mouse 3T3-A31 cells and mouse embryo fibroblasts resembled mouse L929 cells in that they did not degrade tryptophan. Supplementation of the extracellular medium with additional tryptophan reconstituted the tryptophan pool in mock-infected and R. prowazekii-infected, X-irradiated, IFN-gamma-treated human fibroblasts to values greater than those observed in untreated control cultures. However, reconstitution of the tryptophan pool did not relieve IFN-gamma-induced inhibition of rickettsial growth. Addition of kynurenine or N-formylkynurenine to rickettsia-infected human fibroblasts at concentrations four times the usual tryptophan concentration did not inhibit growth of R. prowazekii. We conclude that neither tryptophan depletion nor depletion of the other amino acids studied explains the inhibitory effect of IFN-gamma on rickettsial growth in mouse L929 cells. In IFN-gamma-treated human fibroblasts, either tryptophan depletion is not involved in the inhibition of rickettsial growth or tryptophan depletion and some other mechanism(s) together contribute to the inhibition of rickettsial growth.

Amino Acids↗
PubMed1994

Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis.

OBJECTIVES: To determine whether fibroblasts from patients with systemic sclerosis (SSc) produce excessive amounts of endothelin-1 (ET-1), which is recognised as having vasoconstrictive properties and as having a potent mitogenic effect on fibroblasts. METHODS: Dermal fibroblasts were removed from 11 patients with SSc and from five normal controls (NC). The assay of ET-1 protein was measured by an ELISA that used two anti-ET-1 antibodies. The gene expression of prepro ET-1 mRNA was evaluated by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. RESULTS: Levels of ET-1 protein were significantly higher in SSc fibroblast cultures than in those of normal fibroblasts (p < 0.01). The expression of prepro ET-1 mRNA was also higher in SSc fibroblasts than in normal fibroblasts. The addition of interleukin-1 beta (IL-1 beta) increased the production of ET-1 by fibroblasts. CONCLUSION: The findings indicate that the overproduction of ET-1 is a novel abnormal function in SSc fibroblasts, and that ET-1 induced by fibroblasts may play a role in the fibrosis and Raynaud's phenomenon of SSc.

Adult↗
PubMed2006

Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21.

BACKGROUND: T cell-mediated immunity plays a central part in the pathogenesis of tissue damage in inflammatory bowel disease (IBD). The mechanism by which T cells mediate tissue damage during IBD remains unclear, but evidence indicates that T cell-derived cytokines stimulate fibroblasts to synthesise matrix metalloproteinases (MMPs), which then mediate mucosal degradation. We have previously shown that, in IBD, there is high production of interleukin (IL) 21, a T cell-derived cytokine, which enhances Th1 activity. AIM: To investigate whether IL21 controls MMP production by intestinal fibroblasts. METHODS: IL21 receptor (IL21R) was evaluated in intestinal fibroblasts by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Fibroblasts were stimulated with IL21 and MMPs were evaluated by RT-PCR and western blotting. The effect of a neutralising IL21R fusion protein (IL21R/Fc) on the induction of MMPs in fibroblasts stimulated with IBD lamina propria mononuclear cell (LPMC) supernatants was also evaluated. RESULTS: Intestinal fibroblasts constitutively express both IL21R and the common gamma chain receptor, which are necessary for IL21-driven signalling. IL21 enhances fibroblast production of MMP-1, MMP-2, MMP-3 and MMP-9, but not tissue inhibitors of MMP-1 and MMP-2. Moreover, IL21 synergises with tumour necrosis factor alpha to increase synthesis of MMP synthesis. IL21 enhances MMP secretion without affecting gene transcription and protein synthesis. IBD LPMC supernatants stimulate MMP secretion by intestinal fibroblasts, and this effect is partly inhibited by IL21R/Fc. CONCLUSIONS: These results suggest that fibroblasts are a potential target of IL21 in the gut and that IL21 controls MMP secretion by fibroblasts.

Cells, Cultured↗
PubMed1992

Differences in gap junction channels between cardiac myocytes, fibroblasts, and heterologous pairs.

Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.

Animals↗
PubMed2002

Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia.

Proliferation of fibroblasts contributes to the adventitial thickening observed during the development of hypoxia-induced pulmonary hypertension. However, whether all or only specific subpopulations of fibroblasts proliferate during this process is unknown. Because lung, skin, and gingiva contain multiple fibroblast subpopulations, we hypothesized that the pulmonary artery (PA) adventitia of neonatal calves is composed of multiple fibroblast subpopulations and that only selective subpopulations expand under chronic hypoxic conditions. Fibroblast subpopulations were isolated from PA adventitia of control calves using limited dilution cloning techniques. These subpopulations exhibited marked differences in morphology, actin expression, and serum-stimulated growth. Only select fibroblast subpopulations demonstrated the ability to proliferate in response to hypoxia. Fibroblast subpopulations were similarly isolated from calves exposed to hypoxia (14 days). With regard to morphology, actin expression, and serum-stimulated growth of subpopulations, there were no obvious differences in fibroblast subpopulations between the hypoxic and the control calves. However, the number of fibroblast subpopulations with about a twofold increase in hypoxia-induced DNA synthesis was significantly greater in the hypoxic calves (26%) compared with control calves (10%). We conclude that the bovine PA adventitia comprises numerous phenotypically and biochemically distinct fibroblast subpopulations and that select subpopulations expand in response to chronic hypoxia.

Animals↗
PubMed1991

Cytokine signaling in lung: transforming growth factor-beta secretion by lung fibroblasts.

Control of growth and phenotypic expression of interstitial fibroblasts is a critical determinant of lung architecture and physiology during processes of growth and remodeling. We examined the ability of lung fibroblasts to produce transforming growth factor-beta (TGF-beta), a cytokine that is known to modulate proliferation and phenotypic expression of mesenchymal cells. Cultures of fibroblasts isolated from rat lungs spontaneously secrete TGF-beta as measured in the standard bioassay of anchorage-independent growth of normal rat kidney (NRK) cells in soft agar. Rat lung fibroblasts secrete TGF-beta in an inactive precursor form. Fibroblasts cultured from adult and fetal rat lungs produced comparable amounts of TGF-beta. The ability of lung fibroblast supernatant fluids to induce colony formation in soft agar could be completely neutralized by preincubation of samples with anti-TGF-beta immunoglobulin (Ig). Anti-platelet-derived growth factor IgG had no effect on anchorage-independent growth of NRK cells driven by rat fibroblast culture supernatant samples. These results indicate that TGF-beta does not require the presence of and interaction with secondary cytokines for its activity. In contrast to the results obtained with rat cells, neither human fetal nor adult lung fibroblasts secreted detectable amount of active TGF-beta or its inactive precursor. This was not due to the presence of TGF-beta inhibitors in fibroblast culture media, because the addition of purified porcine TGF-beta to conditioned medium from human lung fibroblast cultures yielded the expected increase in NRK cell growth in soft agar. These results point to differing cytokine control patterns in the lungs of the two species.

Animals↗
PubMed2000

Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2.

Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.

Adult↗
PubMed1999

Lovastatin induces fibroblast apoptosis in vitro and in vivo. A possible therapy for fibroproliferative disorders.

Diseases associated with pathological fibroproliferation represent a major cause of morbidity and mortality. Despite the importance of this class of disorders, current therapy is of limited value, and no therapy is available to reduce the fibroblast population size within existing fibrotic lesions. In this regard, constitutive expression of growth-promoting genes can sensitize cells to undergo apoptosis. Studies in our laboratory have demonstrated that lovastatin potently induces apoptosis in fibroblasts constitutively expressing Myc, and that lung fibroblasts isolated from fibrotic lesions constitutively express growth-promoting genes. In this study, we sought to determine if nontransformed lung fibroblasts would manifest susceptibility to lovastatin-induced apoptosis similar to that observed in fibroblasts ectopically expressing Myc. Here we show that clinically achievable concentrations of lovastatin induce apoptosis in normal and fibrotic lung fibroblasts in vitro, as evidenced by acridine orange staining, terminal transferase nick end translation (TUNEL), and DNA laddering. Apoptosis of human lung fibroblasts was dose- and time-dependent, and blocked by exogenous mevalonic acid. Furthermore, apoptosis was associated with decreased levels of mature Ras, a molecule directly implicated in fibroblast rescue from apoptosis. The ability of lovastatin to induce fibroblast apoptosis in vivo was examined using a guinea pig wound chamber model. Lovastatin (5 microM, 8 d) reduced granulation tissue formation in the wound chambers by 64.7%, with associated ultrastructural evidence of fibroblast apoptosis. These findings support further study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors as potential therapy for patients with fibroproliferative disorders.

Animals↗