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At least 937 records · Page 52Linked to original sources
PubMed2004

Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction.

OBJECTIVES: Myocardial infarction leads to extensive changes in the organization of cardiac myocytes and fibroblasts, and changes in gap junction protein expression. In the immediate period following ischemia, reperfusion causes hypercontraction, spreading the necrotic lesion. Further progressive infarction continues over several weeks. In reperfusion injury, the nonspecific gap junction channel uncoupler heptanol limits necrosis. We hypothesize that gap junction coupling and fibroblast invasion provide a substrate for progressive infarction via a gap junction mediated bystander effect. METHODS: A sheep coronary occlusion infarct model was used with samples collected at 12, 24 and 48 h, and 6, 12 and 30 d (days) post-infarction. Immunohistochemical labelling of gap junction connexins Cx40, Cx43, and Cx45 was combined with cell-specific markers for fibroblasts (anti-vimentin) and myocytes (anti-myomesin). Double and triple immunolabelling and confocal microscopy were used to follow changes in cardiac myocyte morphology, fibroblast content and gap junction expression after myocardial infarction. Gap junction protein levels and fibroblast numbers were quantified. RESULTS: Within 12 h of ischemia, myocyte viability is impaired within small islands in the ischemic region. These islands spread and fuse into larger infarct zones until 12 d post-infarction. Thereafter, surviving myocytes within the infarct and in the border-zone appear to become stabilized. Distant from the infarct, continuing myocyte disruption is regularly observed, even after 30 d. Cx43 becomes redistributed from intercalated discs to the lateral surface of structurally compromised myocytes within 12 d. Cx45 expressing fibroblasts infiltrate the damaged region within 24 h, becoming most numerous at 6-12 d post-infarction, with peak Cx45 levels at 6 d. Later, Cx43 expressing fibroblasts are observed, and the related Cx43 label increases over the 30 d observation period, even though fibroblast numbers decline after 12 d. Cx40 was only seen in vascular endothelium. CONCLUSIONS: Progressive infarction, identified by myocyte sarcomere disruption and subsequent cell loss, occurs in parallel with fibroblast invasion and gap junction remodeling. Two fibroblast phenotypes occur within infarcts, expressing either Cx43 or Cx45. Coupled fibroblasts may play a number of roles in tissue remodeling following myocardial infarction, including provision of a possible substrate for progressive infarction via a gap junction mediated bystander effect.

Animals↗
PubMed2005

Regulation of fibroblasts by activated and non-activated immune cells.

BACKGROUND: Rejection of transplanted tissue is characterized by cell infiltration and interstitial edema. Graft fibroblasts and fibroblast products are partly involved in the regulation of both these phenomena. Knowledge about the mechanisms behind fibroblast activation may lead to new strategies to prevent rejection. This study investigated whether cells of the immune system have the capacity to regulate fibroblast activation. METHODS: Fibroblasts isolated from rejecting heart transplants or from normal heart tissue were cultured in the presence of supernatants of stimulated or non-stimulated immune cells. The immune cells were challenged either in vitro (incubation with phytohemagglutinin) or in vivo (organ transplantation). Fibroblast proliferation and hyaluronan production were measured. RESULTS: Normal, sub-confluent heart fibroblasts showed an increased proliferation rate in the presence of supernatants of activated immunocompetent cells, irrespective of if these cells had been stimulated in vitro or in vivo. As expected, proliferation rate and hyaluronan production were upregulated in fibroblasts isolated from rejecting tissue. However, supernatants of biopsy specimens obtained from non-rejecting organs (syngeneic transplants or normal hearts) had an inhibitory effect on the growth rate of confluent fibroblasts isolated from rejecting tissue. CONCLUSIONS: We conclude that graft-infiltrating cells and immune cells activated in vitro have the capacity to stimulate fibroblasts, most probably as a result of the production and secretion of fibroblast-stimulating factors.

Animals↗
PubMed2005

The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts.

BACKGROUND: Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung. OBJECTIVE: To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling. METHODS: CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), alpha-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-alpha1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-beta(1) release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments. RESULTS: CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-beta(1) release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3-neutralizing antibodies. CONCLUSION: These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.

Cell Differentiation↗
PubMed2005

Electrical coupling of fibroblasts and myocytes: relevance for cardiac propagation.

Myocytes, while giving rise to the bulk volume of normal cardiac muscle, form a "minority cell population" in the heart compared with nonmyocytes, chiefly fibroblasts. The heterogeneous cell types show very intimate spatial interrelation in situ, with virtually every myocyte in the mammalian heart bordering to 1 or more fibroblasts. Nonetheless, gap junction coupling in the heart is traditionally assumed to occur exclusively between myocytes. Yet, both freshly isolated cells and cell cultures have unambiguously shown functional heterogeneous myocyte-fibroblast coupling (mainly via connexin 43). Such coupling is sufficient, in vitro, to synchronize spontaneous beating in distant myocytes, connected over distances of up to 300 microm by fibroblasts only. More recently, functional myocyte-fibroblast coupling (via connexin 45) has been demonstrated in situ for sinoatrial node pacemaker tissue, and preliminary immunohistochemical data suggest that myocyte-fibroblast coupling may be present in postinfarct scar tissue. The functional relevance of such heterogeneous coupling for cardiac electrophysiology is only starting to emerge and has thus far mainly been assessed in theoretical studies. According to this research, fibroblasts may affect the origin and spread of excitation in several ways above and beyond formation of "passive" barriers that obstruct electrical conduction. Thus, fibroblasts may act as current sinks, contributing to the formation of unidirectional block or to the delay in atrioventricular conduction. Via short-range interaction, fibroblasts may help to smooth out propagating wave fronts, in particular in the sinoatrial node and in the cross-sheet direction of healthy ventricular myocardium, 2 tissues that might otherwise be expected to show fragmented conduction patterns. As long-distance communication lines, fibroblasts may bridge posttransplantation or ischemic scar tissue, with beneficial or detrimental effects on organ function (depending on the relation to normal conduction patterns), and explain the recruitment of myocyte islands embedded in fibrotic scar tissue. The inherent mechanosensitivity of cardiac fibroblasts could, furthermore, allow them to play a sensory role and to affect cardiac electrophysiology via mechanoelectric feedback. This article reviews the currently available experimental and theoretical evidence on the previous scenarios, and highlights areas for further research.

Animals↗
PubMed2006

Alterations in cultured myocardial fibroblast function following the development of left ventricular failure.

A structural event in the progression of left ventricular (LV) failure is myocardial extracellular matrix (ECM) remodeling. The myocardial fibroblast is a major cell type influencing the ECM, but whether and to what degree specific phenotypic differences in myocardial fibroblasts can be demonstrated to occur in culture with the development of LV failure remains unclear. Adult pigs (25 kg) were used for control myocardial fibroblast preparations (N=5) or following pacing-induced LV failure (N=5; 240 bpm, 3 weeks). LV remodeling occurred with pacing as evidenced by increased LV end diastolic volume (132+/-11 vs. 60+/-4 mL for control; P<0.05). Functional parameters including migration, adhesion, collagen and matrix metalloproteinase release were assessed in fibroblast cultures from passages 1-4. The following findings were consistent with each passage and the results were analyzed with control values set to 100%. Migration of LV failure fibroblasts increased by over 170% (P<0.05). Adhesion to collagen I, laminin and fibronectin was increased by over 160% in LV failure fibroblasts (P<0.05). beta(1) integrin density decreased by 50% in LV failure fibroblasts (P<0.05). Fibrillar collagen release increased by over 130% and matrix metalloproteinase-2 increased by 140% in LV failure fibroblasts (P<0.05). The unique findings of this study are two-fold. First, after a pathological stimulus in-vivo, adult myocardial fibroblasts maintain a consistent phenotype through early passages in-vivo. Second, a differential release of, and response to ECM components occurred in LV failure fibroblasts. Thus, a phenotypic transformation of the myocardial fibroblast occurs with the development of LV failure, which in turn may contribute to matrix remodeling and presents as a potential cellular therapeutic target.

Animals↗
PubMed2003

Increased plasminogen activator inhibitor-1 in keloid fibroblasts may account for their elevated collagen accumulation in fibrin gel cultures.

Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts.

Adolescent↗
PubMed2000

Selectins (CD62L, CD62P) and megakaryocytic glycoproteins (CD41a, CD42b) mediate megakaryocyte-fibroblast interactions in human bone marrow.

Previous in vitro studies are in keeping with the finding that isolated and enriched megakaryocytes attach to bone marrow fibroblasts and generate an increased growth of these cells. This process was assumed to depend on a close spatial relationship between both cell types which supports the paracrine effect of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta1. Moreover, adhesion molecules including beta1 integrin receptors and fucosylated structures were determined to play an important role in these complex interactions. However, up to now the influence of megakaryocyte expressed glycoproteins CD41a and CD42b in these processes was not investigated. In addition, the role of megakaryocytic CD62P and also of CD62L, both adhesion molecules of the selectin group, could also be of interest. Following isolation and enrichment of bone marrow megakaryocytes and fibroblasts, both cell populations were characterized regarding their expression of these factors by applying immunocytochemical techniques. Additionally, their influence on adhesion of megakaryocytes to fibroblasts as well as fibroblast growth was evaluated by comparative megakaryocyte-fibroblast co-cultures and inhibition studies using specific monoclonal antibodies (mabs). Fibroblast monocultures served as controls. In these experiments, selectin-specific antibodies significantly reduced megakaryocyte attachment to fibroblast feeder layers and fibroblast growth in the co-cultures. The effect of CD41a and CD42b specific antibodies was limited to megakaryocyte-dependent fibroblast growth. These results elucidate the involvement of the selectins CD62P and CD62L in the basal activation of megakaryocytes inducing their attachment to bone marrow fibroblasts. In contrast, the megakaryocyte glycoproteins CD41a and CD42b exert their effect on the megakaryocyte dependent fibroblast growth. Altogether, it is tempting to speculate that the various interactions of these mediators reflect certain steps in the complex pathomechanisms causing the evolution of (reactive) myelofibrosis in hematopoietic neoplasias accompanied by megakaryocytic proliferation.

Bone Marrow↗
PubMed1997

G protein subunit levels in fibroblasts from familial Alzheimer's disease patients: lower levels of high molecular weight Gs alpha isoform in patients with decreased beta-adrenergic receptor stimulated cAMP formation.

Abnormalities in G protein linked signal transduction pathways have been detected in fibroblasts from individuals with familial and sporadic Alzheimer's disease. The present study used Gs alpha, Gi alpha, Gq alpha and Go alpha G protein subunit antisera, immunoblotting and densitometry to quantify levels of these proteins in control fibroblasts and in fibroblasts from individuals with familial Alzheimer's disease (FAD). The FAD fibroblasts were from individuals with the APPK670N,M671L mutation, different presenilin 1 (PS1) mutations and one fibroblast cell line from an individual with FAD of unknown genetic aetiology. Results revealed a significant reduction in the large Gs alpha subunit in fibroblasts with the PS1 mutations and in the fibroblast cell line of unknown genetic aetiology, when compared to control levels. This decrease was not apparent in the APPK670N,M671L FAD fibroblasts. Immunoreactivity for Go alpha was not detected in any of the fibroblast cell lines. No differences were observed in Gi alpha or Gq alpha levels when comparing any of the control and Alzheimer's disease fibroblast groups. WE conclude that with the exception of decreased levels of the large Gs alpha subunit, gross alterations in the levels of the Gi alpha, Gq alpha and Go alpha are not associated with the G protein-coupled signal transduction disturbances described previously for some of these FAD fibroblasts.

Alzheimer Disease↗
PubMed1997

Elevated basic fibroblast growth factor levels in stroke-prone spontaneously hypertensive rats.

Basic fibroblast growth factor is a biologically active polypeptide with mitogenic, angiogenic and neurotrophic properties. In the present study, the temporal and spatial expressions of basic fibroblast growth factor in stroke-prone spontaneously hypertensive rats were compared to two related strains of rat: spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Higher levels of total RNA concentration were found in cerebral cortex of four-week-old stroke-prone rats compared to spontaneously hypertensive rats and Wistar Kyoto rats. Northern blot analysis showed no changes in levels of basic fibroblast growth factor messenger RNA with increasing age in cerebral cortex of Wistar Kyoto and spontaneously hypertensive rats. However, significant increases were found in 26- and 38-week-old stroke-prone rats compared to four-week-old stroke-prone rats. Although messenger RNA increases were also found in subcortical and cerebellar regions, a significant difference in levels of basic fibroblast growth factor messenger RNA was observed only in cerebral cortices among these three strains. This age-related increase in basic fibroblast growth factor messenger RNA correlated with the increase incidence of stroke in stroke-prone rats. Immunohistochemical study further revealed a dramatic increase in levels of basic fibroblast growth factor immunoreactivity in cerebral cortex of 30-week-old stroke-prone rats as compared to young stroke-prone rats, as well as age-matched Wistar Kyoto and spontaneously hypertensive rats. This increase in basic fibroblast growth factor immunoreactivity therefore appears very specific to aged stroke-prone rats. However, immunoreactivity decreased once severe tissue damages were observed in the cerebral cortex. Basic fibroblast growth factor-positive cells were diffusely expressed in cerebral cortex; double staining with glial fibrillary acidic protein showed the majority of these basic fibroblast growth factor-positive cells to be astrocytes. In summary, although young stroke-prone spontaneously hypertensive rats showed significantly higher RNA concentration, significant increases in levels of basic fibroblast growth factor, including both messenger RNA and protein expression, were observed in aged stroke-prone rats with a high incidence of stroke. These findings suggest the possibility that basic fibroblast growth factor may play a role in the developmental sequelae of cerebral lesions in stroke-prone spontaneously hypertensive rats.

Animals↗
PubMed1996

Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase.

This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 microM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 microM and 50 microM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 microM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4 beta-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachidonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A2 not phospholipase D activation.

Animals↗
PubMed1999

Differences between fibroblasts cultured from oral mucosa and normal skin: implication to wound healing.

It is generally agreed that oral mucosa heals faster with less scar than skin does, and hypertrophic scar or keloid is very rare in the oral cavity. Fibroblasts are thought to play an important role in wound healing and scar formation, whose control is mediated by growth factors. We have studied whether there are any differences in the cellular behavior of fibroblasts between oral mucosa and skin, and in their response to growth factors. Oral mucosal fibroblasts proliferated slightly more than dermal fibroblasts on average. Dermal fibroblasts in collagen gel possessed greater contraction potency than oral mucosa fibroblasts, irrespective of the presence of growth factors; however, oral mucosa fibroblasts showed an earlier collagen gel contraction with or without TGF-beta1. There were no differences in basal collagen synthetic rate between dermal and oral mucosal fibroblasts, while the latter synthesized more collagen than dermal fibroblasts when they were stimulated with TGF-beta1. Our study showed that oral mucosal fibroblasts and dermal fibroblasts had selective differences in cellular behavior and in their responses to growth factors, which seems to contribute to the differences in wound healing.

Cell Division↗
PubMed1989

Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging.

Acute local gamma-irradiation of porcine skin induces, as in human skin, extensive mutilating sclerosis, characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of extracellular matrix macromolecules. Collagen synthesis, content, and types were studied by measuring the incorporation of the radiolabeled precursor [3H]-proline into confluent primary cultures and subcultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. A significant increase in collagen biosynthesis and deposition, with a preferential enhancement of type III collagen, was observed in primary cultures of fibrotic fibroblasts when compared to those of normal dermis. However, the percentage (36%) of neosynthesized collagen in relation to the total neosynthesized non-collagenous and collagenous proteins remained unchanged. In subcultures of normal cells, collagen synthesis and content remained constant until about the 10th passage and then progressively declined until death of the fibroblasts, at around the 15th passage. During the senescence phase, normal fibroblasts acquired the capacity to synthesize higher levels of non-collagenous proteins. On the contrary, collagen synthesis and content diminished in subcultures of fibrosis-induced fibroblasts from the second passage, and then progressively declined as a function of aging; particularly the ratio of type III to type I collagen returned to normal values from the second passage. Moreover, these fibroblasts did not become senescent, persisted in synthesizing low levels of collagen, and acquired later (around the 40th passage) a higher capacity to synthesize non-collagenous proteins. These results demonstrate that primary cultures of porcine fibroblasts obtained from normal or radiation-induced fibrotic dermis reproduce the in vivo situation. The results strongly suggest that: 1) the modification of collagen synthesis, content, and types observed in primary cultures of fibroblasts might be due to factors causing long-lasting changes in phenotypic expression and/in stimulation of the expansion of some fibroblast clones engaged in the accelerated synthesis of extracellular matrix macromolecules, such as collagen or non-collagenous proteins. 2) the rapid decrease in this active capacity to synthesize and accumulate collagen, observed in subcultures of fibrosis-induced fibroblasts, might be related to the loss of some activation factors or to the dedifferentiation of the cells. 3) the switch from collagen to non-collagenous protein synthesis during later subcultures of fibroblasts obtained from radiation-induced fibrosis, might be due to molecular modification at the transcriptional or DNA level.

Animals↗
PubMed2003

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2.

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.

Adenoviridae↗
PubMed1998

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing.

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.

Adult↗
PubMed1995

Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is a paracrine growth factor for cultured fibrosis-derived kidney fibroblasts.

We studied the role of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) in the proliferative response of transformed human renal interstitial fibroblast cell lines established from either a kidney with glomerulonephritis and interstitial fibrosis or a normal kidney in comparison to primary human foreskin fibroblasts. Growth of fibrosis-derived renal fibroblasts was inhibited in the presence of IL-1 receptor antagonist (IL-1Ra) by 35% (P < 0.005), suggesting that these cells produce IL-1 and possess IL-1 receptors as part of paracrine growth. In contrast, spontaneous proliferation of fibroblasts derived from a normal kidney or normal skin were not inhibited by IL-1Ra. In fibrosis-derived but not in normal renal cells, fibronectin synthesis was increased 2.2-fold (P < 0.01) in the presence of IL-1Ra. Addition of exogenous IL-1 beta or bFGF stimulated proliferation of skin fibroblasts. In contrast, growth of fibrosis-derived renal fibroblasts was stimulated by IL-1 beta and unchanged by bFGF. Growth of normal kidney fibroblasts was unaffected by bFGF and inhibited by IL-1 beta. We conclude that compared to normal fibroblasts, fibrosis-derived renal fibroblasts have a different cytokine-response profile, are IL-1-dependent, produce IL-1 as a paracrine growth factor and do not proliferate to bFGF, a classical fibroblast growth factor.

Cell Count↗
PubMed1999

Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin-1alpha.

1. Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts. 2. Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL-1alpha-dependent receptor induction was blocked by cycloheximide. 3. BK-stimulated cytosolic Ca2+ elevation was higher in confluent than in non-confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL-1-alpha-induced up-regulation of BK receptors. 4. Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6-oxo-PGF1alpha 6-7 fold. 5. Preincubation with IL-1alpha had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6-oxo-PGF1alpha 7-10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL-1alpha induced rise in BK-stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo-oxygenase 2. 6. IL-1alpha sensitized BK-stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL-1alpha effects on prostanoid release were not related to BK receptor numbers nor to the BK-stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation-associated kinins and cytokines.

6-Ketoprostaglandin F1 alpha↗
PubMed2003

Smads regulate collagen gel contraction by human dermal fibroblasts.

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction that is implicated in efficient wound healing. The Smad family of proteins mediates signal transduction of the TGF-beta superfamily. However, its role in fibroblast contraction remains unclear. OBJECTIVES: To determine whether Smad proteins regulate fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with human dermal fibroblasts infected with adenoviruses carrying Smads. RESULTS: Overexpression of Smad3, a major signal transducer in the Smad family, enhanced collagen gel contraction by fibroblasts when compared with fibroblasts overexpressing a control lacZ. Addition of a very low concentration of TGF-beta1 that did not affect the collagen gel contraction by itself enhanced the contraction by fibroblasts overexpressing Smad3. In contrast, TGF-beta1-mediated collagen gel contraction was suppressed by overexpression of Smad7, a major inhibitory regulator in the Smad family, in fibroblasts. In addition, inhibitors of the Erk and p38 pathways, PD98059 and SB203580, did not affect TGF-beta1-mediated collagen gel contraction by dermal fibroblasts. CONCLUSIONS: Modulation of Smad3 or Smad7 expression in dermal fibroblasts affected their contraction of collagen gels possibly by regulating TGF-beta signalling in fibroblasts.

Blotting, Western↗
PubMed1996

Expression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.

In chronic inflammatory conditions, mononuclear cells infiltrate the involved connective tissue and may persist, perhaps because of binding to adhesion molecules on connective tissue cells, as well as extracellular matrix. Here we investigated whether vascular cell adhesion molecule-1 (VCAM-1) may be induced on human dermal fibroblasts by proinflammatory cytokines. Expression of VCAM-1 was determined by Northern blotting, cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Only trace amounts of VCAM-1 mRNA or protein were constitutively expressed on dermal fibroblasts but both were rapidly (within 4 hr) upregulated by tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) and somewhat slower (20 hr) by interferon-gamma (IFN-gamma). The combination of TNF-alpha and IFN-gamma had at least an additive effect. The adhesion function of the expressed VCAM-1 was examined by studying the adhesion of the Jurkat T lymphocytes to fibroblasts. Adhesion of Jurkat cells to dermal fibroblasts was markedly increased by stimulation of fibroblasts with TNF-alpha and IFN-gamma (22% of cells adhered versus 9% on unstimulated fibroblasts). The increased adhesion was inhibited to that on unstimulated fibroblasts by monoclonal antibody (mAb) to domain 1 of VCAM-1, but not by mAb to domain 4. MAb to very late antigen-4 (VLA-4), the integrin counter-receptor on lymphocytes for VCAM-1, completely inhibited the increase in Jurkat cell adhesion to activated fibroblasts and partly also inhibited basal adhesion to unstimulated fibroblasts. These results suggest that VCAM-1 expression by dermal fibroblasts is inducible at the mRNA, protein and functional levels by proinflammatory cytokines. The VLA-4/VCAM-1 pathway maybe involved in adhesive interactions between T lymphocytes and activated fibroblasts in the skin during chronic dermal inflammatory conditions.

Blotting, Northern↗