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PubMed1996

Expression of cytokines and their receptors by psoriatic fibroblasts. II. decreased TNF receptor expression.

Psoriatic fibroblasts produce enhanced amounts of IL-6 in vitro. This state of activation may reflect an altered expression of cytokine receptors, involved in auto/paracrine induction of IL-6. Cultures of dermal fibroblasts derived from lesional psoriatic (PP) and normal control (NN) skin were therefore analysed for their ability to bind biotinylated recombinant human cytokines using flow cytometry. PP and NN fibroblasts bound negligible amounts of IL-1 alpha and IL-1 beta, but clearly bound IL-4, IL-6 and TNF-alpha. Serum upregulated the number of NN fibroblasts which bound TNF-alpha, and to a lesser extent IL-6, but not the number of binding sites per cell. In contrast, this upregulation was significantly less in PP fibroblasts. This was not a result of differences in growth characteristics, receptor occupancy or an inability of stimulated PP fibroblasts to bind TNF-alpha. Immunocytochemistry of cells grown on slides showed that the TNF receptor type 1 (TNFR1, p55) was the predominant receptor in NN fibroblasts and was localized to the nucleus of cytoplasma. The expression of TNFR1 was clearly decreased in PP fibroblasts, which confirmed the binding studies. A slow and serum-induced shedding of TNFR1 was observed, but not of the TNFR2 (p75), in both types of fibroblasts. Confluent multi-passaged PP fibroblasts display both a decreased TNFR expression as well as an enhanced IL-6 production under serum conditions. These inherent abnormalities of PP fibroblasts imply the involvement of dermal fibroblasts in the maintenance of chronic inflammation in psoriasis.

Calcium↗
PubMed1999

Histamine effects on conjunctival fibroblasts from patients with vernal conjunctivitis.

Histamine, an important mast cell mediator in allergic disorders, may affect extracellular matrix production and cell growth in vernal keratoconjunctivitis (VKC). In the present study, the histamine reactivity of conjunctival fibroblasts derived from VKC patients was investigated in vitro. Conjunctival fibroblast cultures were derived from biopses of 8 tarsal VKC patients and 5 normal subjects. These cells were maintained in vitro and stimulated with different concentrations of histamine with and without H1 (clorpheniramine) and H2 (cimetidine) receptor antagonists. Comparisons were made to fibroblasts grown in the same media without histamine and to fibroblasts stimulated with just antihistamine. The effects of histamine were evaluated by: (1) the MTT test to assess cell proliferation; (2) an in vitro wound model for cell migration and (3) the measurement of procollagen I (PIP) and procollagen III (PIIIP) in supernatants for collagen production. Results showed: (1) While VKC-derived fibroblasts proliferated at a faster rate than normal cells in unstimulated media, after histamine stimulation, VKC and normal cells grew at a similar rate. Both H1 and H2 antagonists significantly inhibited (P<0.05) histamine-induced cell proliferation. (2) Histamine enhanced cell migration after wounding; this effect was inhibited only by H2 antagonism. (3) When stimulated with histamine, VKC fibroblasts produced significantly more PIP than those in control media. Furthermore, VKC-derived fibroblasts were more sensitive to histamine challenge, producing significantly more PIP than normal fibroblasts. H1 and H2 antagonists did not modify histamine-stimulated PIP production. The enhanced proliferative and productive capacity of VKC fibroblasts may be the result of a selective overgrowth of one or more fibroblast subpopulations in a chronically inflamed tissue. Histamine increased proliferation, migration and collagen production in both normal and VKC fibroblasts. Since H2 antagonism modulated both cell growth and migration, but not histamine-induced collagen production, the latter may be mediated by a different receptor. These results showed that histamine is at least partially responsible for fibroblast stimulation.

Cell Culture Techniques↗
PubMed1996

Differential expression of IGFBPs by normal and hypertrophic scar fibroblasts.

Insulin-like growth factor-I (IGF-I) is a potent fibroblast mitogen which influences wound healing. IGF action is regulated by a family of six IGF-binding proteins (IGFBPs). The purpose of this study was to determine if expression of IGFBPs is altered in hypertrophic scarring, a wound-healing condition commonly associated with deep dermal injury. Fibroblast populations from the superficial and deep dermal layers of normal human skin (SN and DN, respectively) and from superficial and deep layers of hypertrophic scars (SSc and DSc, respectively) were established and cultured in serum-free medium with or without several growth factors known to modulate wound healing, including basic fibroblast growth factor, the BB isoform of platelet-derived growth factor, IGF-I, and transforming growth factor-beta (TGF-beta). IGFBP release was analyzed by radioligand blot assays of culture media. Two main forms of IGFBPs were released, IGFBP-3 and a 24-kDa form which comigrated with serum IGFBP-4. DSc fibroblasts accumulated significantly more IGFBP-3 into serum-free culture medium than did SN, DN, or SSc fibroblasts in all conditions except TGF-beta treatment and confluence. Additionally, comparisons of IGFBP-3 release by each cell type with and without TGF-beta revealed TGF-beta stimulated IGFBP-3 accumulation by SN and DN fibroblasts but not by SSc or DSc fibroblasts. DN and DSc fibroblasts accumulated significantly more of the 24-kDa IGFBP species than SN or SSc fibroblasts in all conditions except TGF-beta or IGF-I treatment. These findings indicate that superficial and deep dermal fibroblasts are heterogeneous with respect to IGFBP release, and suggest that hypertrophic scar fibroblasts may represent a population of cells with regulatory properties distinct from those of normal dermal fibroblasts.

Cell Count↗
PubMed1996

Effects of hyaluronic acid on fibroblast behavior in peritoneal injury.

The process of intraperitoneal adhesion formation is affected by macrophages and fibroblasts which are major components of postsurgical peritoneal repair. Hyaluronic acid (HA) has been shown to affect cellular behavior. We studied the effects of HA on experimental adhesions in vivo and its in vitro effect on cultured postsurgical macrophages and fibroblasts. Experimental adhesions were facilitated by laparotomy and localized peritoneal controlled trauma in two groups of rats (A, B). Postoperatively, group A received intraperitoneal (ip) treatment by HA (1 mg/kg) for 7 days, and group B, ip saline. The rats were then reoperated upon, and adhesions scored. In vitro studies were performed on postsurgical macrophages and fibroblasts. Fibroblasts were obtained using a single-cell suspension technique by debridement of adhesions. The fibroblasts were cultured for 7 days, and their metabolic activity was assessed by the uptake of [3H]thymidine. Postoperative macrophages were obtained from the peritoneal fluid of the rats operated on, and their effect upon fibroblast [3H]thymidine uptake was studied in mixed cultures. The adhesion score of the HA-treated rats was smaller than the score of the saline-treated group. This observation suggests that ip treatment by HA may decrease adhesion formation in this rat model. [3H]Thymidine uptake by cultured postsurgical fibroblasts was significantly lower in the HA-treated group compared to that of controls. In vitro addition of HA to cultured "saline fibroblast" resulted in a significant decrease in [3H]thymidine uptake, suggesting a direct effect of HA on postsurgical fibroblast metabolism. However, [3H]thymidine uptake by fibroblasts in mixed cultures with macrophages obtained from HA-treated rats was significantly increased. These observations suggest that HA may affect the process of peritoneal healing by direct effect on fibroblast metabolic activity, and indirectly via modification of the macrophage-fibroblast interrelationship.

Animals↗
PubMed1994

Mast cells and fibroblasts: two interacting cells.

Mast cell (MC) fibroblast interactions may have a role in health and disease. We analyzed the relationships between these cells by utilizing our in vitro model in which mucosal (MMC) and connective tissue (CTMC) type MC were cocultured long-term with different fibroblasts. Mouse 3T3 fibroblasts were used to provide a normal microenvironment for MC, while fibroblasts derived from mouse with chronic graft-versus-host disease (cGVHD) provided a fibrotic one. We found that both 3T3 and cGVHD fibroblasts maintain CTMC viability, phenotype and functional activity. When MMC were cocultured with 3T3 or cGVHD fibroblasts, they changed their phenotype towards that of CTMC. On the other hand, MCs were found to affect fibroblast properties. Coculture of MMC on 3T3 monolayers was shown to increase Forsmann antigen production and collagen synthesis and stimulate fibroblast proliferation. Resting CTMC or CTMC activated by anaphylactic stimuli induced 3T3 and cGVHD fibroblasts to proliferate more. In addition, CTMC activation increased collagen production by 3T3 fibroblasts. In conclusion, fibroblasts were found to regulate MC survival and differentiation, whereas MCs were shown to affect the biochemical properties of fibroblasts, which can lead to fibrosis.

3T3 Cells↗
PubMed2001

An investigation of cell proliferation and soluble mediators induced by interleukin 1beta in human synovial fibroblasts: comparative response in osteoarthritis and rheumatoid arthritis.

OBJECTIVES AND DESIGN: The difference in cell proliferation and release of soluble factors in response to interleukin 1beta (IL-1beta) in fibroblasts obtained from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) and from normal skin has been investigated. TREATMENT: The cells were treated with recombinant IL-1beta in the presence or absence of pharmacological agents for 24 h or 48 h. METHODS: Cell proliferation was examined by WST-1 assay, and the amounts of interleukin-6 (IL-6), interleukin-8 (IL-8), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF), matrix metalloproteinase-1 (MMP-1), and prostaglandin E2 (PGE2) were measured by enzyme linked immunosorbent assay (ELISA). RESULTS: IL-1beta dose-dependently enhanced the proliferation of all fibroblasts. The proliferative response to IL-1beta in RA synovial fibroblasts was greater than that in OA synovial and skin fibroblasts. However, there was no difference in spontaneous levels of soluble factors between OA and RA fibroblasts, though medium concentrations of IL-1beta-released VEGF, MMP-1, and PGE2, but not cytokines, in RA were slightly higher than those in OA. Ability to release soluble mediators was pronouncedly increased at 3 h to 9 h after stimulating fibroblasts with IL-1beta for 1 h. The proliferative response to IL-1beta in all fibroblasts was inhibited by dexamethasone and the NF-kappaB inhibitor hymenialdisine but not the cyclooxygenase 2 (COX-2) inhibitor NS-398. But PGE2 prevented proliferation of RA fibroblasts when added to medium up to 3 h after IL-1beta stimulation. Dexamethasone also inhibited the release of IL-6, IL-8, and PGE2 induced by IL-1beta in both OA and RA fibroblasts. NS-398 exhibited an inhibition of IL-1beta-induced IL-6 production as well as PGE2 production. Hymenialdisine inhibited IL-6 production and reduced IL-8 production dependent on synovial cell strains. Methotrexate had no effect on the response to IL-1beta in synovial fibroblasts. CONCLUSION: The present results indicate that the activation of NF-kappaB plays an important role in the proliferative response to IL-1beta in human fibroblasts, and suggest that PGE2 acts as a modulator of cell proliferation in inflamed synovial tissue. It appears that the ability to produce soluble factors in RA synovial fibroblasts is not intrinsic. However, the response to IL-1beta in RA cells seems to be greater than that in OA cells.

Arthritis, Rheumatoid↗
PubMed2004

The effects of epidermal keratinocytes and dermal fibroblasts on the formation of cutaneous basement membrane in three-dimensional culture systems.

The cutaneous basement membrane (BM) plays an important role in normal and pathological conditions. However, few studies have addressed the formation of the cutaneous BM using three-dimensional culture systems. In this study, to elucidate the effects of human epidermal keratinocytes and dermal fibroblasts on the formation of the cutaneous BM, keratinocytes were cultured on several dermal substrates in the presence or absence of fibroblasts at the air-liquid interface. After 2 weeks of culture, immunohistochemical stainings for the components of the BM and electron microscopic studies of the BM zone (BMZ) were performed. In cultures of keratinocytes alone on dead reticular dermis or collagen gel without fibroblasts, beta4 integrin chain, laminin, type IV and VII collagens were all expressed. However, ultrastructurally, BMZ was not formed. In cultures of keratinocytes on fibroblast-populated collagen matrix, laminin, and type IV and VII collagens were expressed more strongly than in the absence of fibroblasts. In addition, elements of the BMZ such as hemidesmosomes, lamina lucida, lamina densa and anchoring fibrils were formed, although it was still incomplete. In the culture of keratinocytes alone on de-epidermized dermis (DED) (surface up), beta4 integrin chain, laminin, and type IV and VII collagens were strongly expressed. Also, the BMZ appeared similar to that in normal skin. In cocultures of keratinocytes and fibroblasts on DED or cultures of keratinocytes on DED combined with fibroblast-populated collagen matrix, type IV collagen was expressed more strongly than in cultures of keratinocytes alone. Ultrastructurally, similar findings to those of cultures of keratinocytes alone on DED were seen. Interestingly, when keratinocytes and fibroblasts were cocultured on DED, some fibroblasts were seen in the upper dermis as a result of migration into the dermis through partial loss of the lamina densa. These results show that keratinocytes produce most of the components of the BM such as laminin, and type IV and VII collagens. In addition, fibroblasts stimulate the expression of the components of the BM and the formation of a BMZ, suggesting that fibroblasts may produce laminin, and type IV and VII collagens or influence the effects of keratinocytes on the formation of the BM through a keratinocyte-fibroblast interaction.

Basement Membrane↗
PubMed2005

Contractile forces generated by striae distensae fibroblasts embedded in collagen lattices.

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin that are reddish at first and finally white. They are due to stretching of the skin, as in rapid weight gain, or mechanical stress, as in weight lifting. The pathogenesis of striae distensae is unknown but probably relates to changes in the fibroblast phenotype. In order to characterize striae distensae fibroblasts, alpha-smooth muscle actin expression and contractile forces were studied. Five healthy women with early erythematous striae and five healthy women with older striae were selected. Paired biopsies were taken from the center of lesional striae and adjacent normal skin. Fibroblasts were obtained by an explant technique and expanded in vitro in Dulbecco's modified Eagle's medium. Contractile forces generated by fibroblasts in collagen lattices were measured with the Glasbox device developed in our laboratory. Alpha-smooth muscle actin expression was studied by immunofluorescence labeling of cells and by flow cytometry. Fibroblasts from early striae distensae were the richest cells in alpha-smooth muscle actin filaments and generated the highest contractile forces. Their peak contractile force was 26% greater than normal fibroblasts. There was a 150% higher level of alpha-smooth muscle actin content in fibroblasts from early striae distensae compared with fibroblasts from normal skin. In contrast, there was no significant difference in force generation between old striae fibroblasts and normal fibroblasts with cells expressing no alpha-smooth muscle actin. The contractile properties of fibroblasts from striae distensae varies depending on the stage of the disease. In early striae distensae, fibroblasts acquire a more contractile phenotype, corresponding to that of myofibroblasts.

Actins↗
PubMed2002

Crucial role of fibroblasts in regulating epidermal morphogenesis.

Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.

Cell Division↗
PubMed1990

Mast cells enhance migration and proliferation of fibroblasts into an in vitro wound.

The effects of mast cells (MC) in an in vitro wound model were studied. The model consisted of rat peritoneal MC cultured on confluent monolayers of 3T3 fibroblasts (MC/3T3). A linear wound was performed by cutting along the midline and scraping one half of the monolayer. After 42 h fibroblasts were counted in the scraped area of the wound. In the MC/3T3 cocultures 27.6 +/- 2.1 fibroblasts were found compared to 16.6 +/- 0.9 in the 3T3 cultures. The most significant increase in the number of fibroblasts was obtained upon activation of the MC with anti-IgE antibodies immediately after wound production (39.9 +/- 2.1). Stimulation with compound 48/80 had a weaker effect (32.7 +/- 1.5). Incubation of 3T3 wounded monolayers with supernatants of anti-IgE- or compound 48/80-activated MC, or with sonicated MC, induced an increase in fibroblast number similar to that found in unactivated MC/3T3. [3H]Thymidine incorporation followed by autoradiography was performed to assess fibroblast mitosis. The highest number of labeled fibroblasts beyond the wound line was found in immunologically activated MC/3T3 (29.7 +/- 4.4), followed by compound 48/80-activated MC/3T3 (18.4 +/- 1.5), MC/3T3 (15.1 +/- 3.6), and 3T3 (10.6 +/- 2.6). After addition of aphidicolin, which inhibited fibroblast mitosis, MC were still effective in enhancing fibroblast migration. In all the cocultures MC were observed to have migrated alongside fibroblasts. Thus merely the presence of MC adhering to wounded fibroblast monolayers significantly enhanced migration and proliferation of the fibroblasts. A further increase was achieved by immunological activation of the MC. We therefore suggest that MC have a facilitating role in this in vitro wound model.

Animals↗
PubMed1992

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts.

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.

Animals↗
PubMed1988

Incorporation of thymidine by fibroblasts: evidence for complex regulation by postsurgical macrophages.

Macrophages and fibroblasts are major components of the postsurgical repair process. In order to understand more fully the interaction between these two cell types, we studied the modulation by macrophages of the incorporation of [3H]thymidine into postsurgical fibroblasts recovered from the site of peritoneal injury. Peritoneal exudate cells (PEC):(greater than 95% macrophages) were collected from rabbits 4 and 7 days after resection and reanastomosis of the small intestine. PEC were suspended in Medium 199 (M-199) with 3% fetal calf serum (FCS) and incubated for 48 hr. Fibroblasts were obtained from rabbits that underwent abrasion of the parietal peritoneum 7 days previously, and were cultured for 7 days in M-199 with 3% FCS. Fibroblasts were then replated and incubated with macrophage-spent medium. Incorporation of [3H]thymidine into fibroblasts was significantly suppressed after 24 hr of incubation with macrophage-spent media compared to the incorporation by fibroblasts incubated with fresh medium (control). This suppression was most profound when fibroblasts were incubated with resident (nonsurgical) macrophage-spent medium. The incorporation of thymidine by macrophage-spent media groups then increased rapidly and reached control levels at 48 hr of incubation. After 54 hr of incubation, the incorporation of thymidine by fibroblasts incubated with media from postsurgical macrophages was significantly higher than that of control. Morphological changes in fibroblasts also appeared as the culture with macrophage-spent media progressed. Initially, fibroblasts were shaped like pine needles, but after 7 days of culture, fibroblasts assumed a spherical shape. Round-shaped fibroblasts returned to the original morphology (pine needle shape) after incubation for 48 hr with macrophage-spent medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Generation of contractile force by cultured Dupuytren's disease and normal palmar fibroblasts.

Contractile fibroblasts are believed to be responsible for palmar fascia contracture in Dupuytren's Disease. An in vitro collagen lattice model was used to examine the contractile properties of Dupuytren's fibroblasts from 10 patients undergoing partial fasciectomy, and palmar fascia fibroblasts from 6 patients undergoing carpel tunnel release. Dupuytren's and palmar fascia fibroblasts cultured within a stabilized collagen lattice acquired morphological characteristics similar to those of 'myofibroblasts' in Dupuytren's diseased fascia. Both types of fibroblasts generated contractile forces that resulted in rapid collagen lattice contraction after release of the lattice from points of stabilization. Generation of contractile force by the fibroblasts was inhibited by disruption of the actin cytoskeleton, lack of cells, or serum removal. Afferent neuropeptides (substance P, galanin and neurokinin A) did not promote lattice contraction. These results demonstrate that normal palmar fascia fibroblasts can modulate into Dupuytren's-like fibroblasts and that cultured fibroblasts, from either Dupuytren's diseased or normal palmar fascia, can generate contractile forces that are transmitted to extracellular matrix. In addition, fibroblast contraction is an actin based process which requires specific factor(s) present in serum. It is suggested that in Dupuytren's disease extracellular cues trigger the modulation of fibroblasts to Dupuytren's fibroblasts and the promotion of contractile forces responsible for palmar fascia contrature.

Actins↗
PubMed1994

Species-specific binding of IL-1, but not the IL-1 receptor antagonist, by fibroblasts.

Our laboratory previously reported that bovine thymocytes and fibroblasts proliferated only in response to bovine IL-1, whereas proliferation of murine thymocytes was augmented equally well by murine, human, and bovine IL-1. In this study, we used direct and competitive receptor binding assays to determine whether differential binding of homologous versus heterologous IL-1 accounts for the species-specific response of bovine fibroblasts. Our results demonstrated that bovine and human fibroblasts bound homologous IL-1 with high affinity and heterologous IL-1 with lower affinity. In contrast, murine fibroblasts bound both homologous and heterologous IL-1 with high affinity. Because IL-1 and the human IL-1 receptor antagonist (IL-1ra) both bind to type 1 IL-1 receptors, we also determined whether the IL-1ra demonstrated receptor binding properties similar to human IL-1 for bovine, human, and murine fibroblasts. To our surprise, the human IL-1ra bound equally well to bovine, human and murine fibroblasts. We used this characteristic of the IL-ra to perform affinity cross-linking analysis of the IL-1 receptors on bovine, human and murine fibroblasts. These comparisons demonstrated the IL-1 receptors on bovine and human fibroblasts have similar molecular sizes (M(r) 73 kDa and M(r) kDa, respectively), whereas, IL-1 receptors on murine fibroblasts have an estimated molecular size of M(r) 88 kDa. These data demonstrate that IL-1 receptors on bovine fibroblasts preferentially bind homologous IL-1, but bovine fibroblasts do not discriminate binding by the human IL-1ra. In contrast, murine fibroblast IL-1 receptors bind heterologous IL-1 with high affinity.

3T3 Cells↗
PubMed2005

Hypoxia upregulates cyclooxygenase-2 and prostaglandin E(2) levels in human peritoneal fibroblasts.

OBJECTIVE: To determine the levels of COX-1, COX-2, and prostaglandin (PG) E(2) in human fibroblasts isolated from normal peritoneal and adhesion tissues. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Fibroblast cultures from both peritoneum and adhesion tissues of five patients. INTERVENTION(S): Treatment of the primary cultured fibroblasts with NS398. MAIN OUTCOME MEASURE(S): We used Western blot to determine the effects of hypoxia on COX-1 and COX-2 levels from lysates of normal peritoneal and adhesion fibroblasts before and after hypoxia. We also used the ELISA techniques to determine PGE(2) levels in media collected from these fibroblasts before and after hypoxia and with and without NS398, a COX-2-specific inhibitor. RESULT(S): There was no difference in COX-1 levels between normal and adhesion fibroblasts with and without hypoxia. Basal COX-2 and PGE(2) levels were significantly higher in adhesion than normal fibroblasts. Hypoxia gradually increased COX-2 and PGE(2) levels in normal peritoneal fibroblasts over time, reaching a peak at 24 hours but had no effect on adhesion fibroblasts. Inhibition of COX-2 by NS398 significantly reduced PGE(2) levels in both normal and adhesion fibroblasts. CONCLUSION(S): The presence of higher levels of COX-2 in adhesion fibroblasts and the induction of COX-2 in normal peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development.

Cell Survival↗
PubMed2005

Wound healing morbidity in STS patients treated with preoperative radiotherapy in relation to in vitro skin fibroblast radiosensitivity, proliferative capacity and TGF-beta activity.

BACKGROUND AND PURPOSE: In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-beta) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-beta activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms. PATIENTS AND METHODS: Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: approximately 0.02 Gy/min) and TGF-beta assays (high dose-rate: approximately 1.06 Gy/min) following gamma-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF(2.4)) and binucleation index (BNI), respectively. Active and total TGF-beta levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined. RESULTS: Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after irradiation compared with those from individuals in the treatment group with normal wound healing, consistent with the results of our previous study. No link was observed between fibroblast radiosensitivity and WHC. Neither active nor total TGF-beta levels in cultures were significantly affected by irradiation. Fibroblast proliferation in unirradiated and irradiated cultures, as well as radiosensitivity, was not influenced by TGF-beta content. TGF-beta expression in fibroblast cultures did not reflect wound healing morbidity. CONCLUSIONS: These data are consistent with our previous study and combined the results suggest that in vitro fibroblast proliferation after irradiation may be a useful predictor of wound healing morbidity in STS patients treated with preoperative radiotherapy. TGF-beta levels in culture do not predict WHC, suggesting that the role of TGF-beta in wound healing is likely controlled by other in vivo factors.

Adolescent↗
PubMed2003

In vivo fluorescent labeling of corneal wound healing fibroblasts.

Numerous studies have shown that fibroblasts play an important role in corneal wound healing, however, the dynamic cellular events underlying wound tissue organization and contraction remain unclear. The purpose of this study was to develop a system to enable live cell imaging of corneal wound healing fibroblasts in situ. To this end, concentrated preparations of an RD114 pseudotyped MLV-based vector expressing the enhanced green fluorescent protein (EGFP) were evaluated in vitro for gene transfer efficiency using cultured rabbit corneal keratocytes. Primary rabbit keratocytes were efficiently labeled in vitro (up to 50% EGFP(+)) at a low multiplicity of infection (MOI=10). To evaluate this gene transfer vector in vivo, rabbit corneal fibroblasts were transduced by direct application of vector supernatant to injured corneas following lamellar keratectomy. Fluorescent fibroblasts were then visualized in situ using epifluorescence microscopy and multiphoton confocal microscopy of excised fresh tissue at multiple time points from 14 days to four months following gene transfer. Fourteen days post-transduction, labeled fibroblasts expressing EGFP were readily detectable by fluorescence microscopy. Detectable fluorescence was noted up to eight weeks post-transduction. Labeled fibroblasts were detected in clusters located predominantly along the margin circumscribing the wound and to a lesser extent within the wound area. Cell growth in clusters was suggestive of the expansion of individual transduced clones. High-resolution imaging showed fluorescent fibroblasts to have a broad, flattened, dendritic morphology, distinct from the spindle shape of cultured fibroblasts. Utilizing multiphoton confocal microscopy, three-dimensional imaging of viable, labeled cells showed wound healing fibroblasts to be extensively interconnected and multi-layered within the corneal wound. These results demonstrate that rabbit corneal fibroblasts can be efficiently transduced in vitro and in vivo using RD114 pseudotyped MLV-based vectors and that these vectors direct long-term transgene expression without apparent toxicity, pathogenesis or perturbation of native fibroblast morphology. Our data further suggest that, in vivo, wound-healing fibroblasts have a defined life span within the wound.

Animals↗
PubMed1998

Acute intermittent nicotine treatment produces regional increases of basic fibroblast growth factor messenger RNA and protein in the tel- and diencephalon of the rat.

Several findings show a neuroprotective effect of nicotine treatment in different experimental models, and a negative correlation has been observed between cigarette smoking and the incidence of Parkinson's disease. It seems possible that nicotine may in part exert its neuroprotective actions by favouring the synthesis of neurotrophic factors. The aim of this study was to determine whether the nicotine treatment could be associated with the induction of a neurotrophic factor in brain regions with nicotinic receptors. Thus, we analysed by in situ hybridization and RNAse protection assay the effects of (-)nicotine on basic fibroblast growth factor messenger RNA and by immunocytochemistry fibroblast growth factor-2 protein in the tel- and diencephalon of rats following single or acute intermittent (-)nicotine treatment. The present results showed that acute intermittent (-)nicotine treatment (four i.p. injections at intervals of 30 min), but not single injections, lead to a substantial and dose-related (0.1-2 mg/kg) up-regulation of fibroblast growth factor-2 messenger RNA levels in the cerebral cortex, in the hippocampus, in the striatum and ventral midbrain. This induction of fibroblast growth factor-2 expression peaked 4 h after the first injection and returned to normal levels within 24 h. The change of fibroblast growth factor-2 messenger RNA levels was associated with increased fibroblast growth factor-2 immunoreactivity mainly localized to nerve cells. The treatment was effective also when repeated in the same animals three or five days after the first injection. The pre-treatment with the non-competitive (-)nicotine receptor antagonist mecamylamine blocked the (-)nicotine effects on fibroblast growth factor-2 messenger RNA levels. In the above areas, no changes were observed in the fibroblast growth factor-1, 2 and 3 receptor messenger RNA levels nor in brain-derived neurotrophic factor messenger RNA levels. The present data indicate an ability of intermittent (-)nicotine to increase fibroblast growth factor-2 in many tel- and diencephalic areas. In view of the trophic function of fibroblast growth factor-2, the previously observed neuroprotective effects of (-)nicotine may at least in part involve an activation of the neuronal fibroblast growth factor-2 signalling, and open up new avenues for treatment of Parkinson's disease and Alzheimer's disease based on the existence of nicotinic receptor subtypes enhancing fibroblast growth factor-2 signalling in many regions of the tel- and diencephalon.

Animals↗