Fibroblasts In Terms Research Articles

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.

Orally active antioxidative copper(II) aspirinate: synthesis, structure characterization, superoxide scavenging activity, and in vitro and in vivo antioxidative evaluations

Ever since it was proposed that reactive oxygen species (ROS) are involved in the pathogeneses of various diseases, superoxide dismutase (SOD)-mimetic complexes have been intensively studied. We prepared copper(II) aspirinate [Cu2(asp)4] from Cu(II) and aspirin, which has been in use for many years as an antipyretic, an analgesic, and an anti-inflammatory agent. However, Cu2(asp)4 has been found to have additional activities, including anti-inflammatory, antiulcer, anti-ischemic/reperfusion agent, anticancer, antimutagenic, and antimicrobial activities. The activity of copper salicylate [Cu(sal)2] was also compared with that of Cu2(asp)4. The structure of the Cu2(asp)4 was determined using X-ray structure analysis. Its SOD-mimetic activity was determined using cytochrome c, electron spin resonance (ESR) spectroscopy, and ESR spin trap methods. The activity of Cu2(asp)4 was slightly greater than CuSO4 and copper acetate [Cu(ace)2] and slightly less than that of Cu(sal)2. The in vitro antioxidant activity, evaluated in human epithelial or transformed neoplastic keratinocyte cells, HaCaT, and normal dermal fibroblasts in terms of cell survival following ultraviolet B (UVB) irradiation, was significantly increased in the presence of Cu2(asp)4, Cu(sal)2, and CuSO4. Further, ROS generation following UVA irradiation in the skin of hairless mice following oral treatment with Cu2(asp)4 for three consecutive days was significantly suppressed compared to the vehicle- or Cu(ace)2-treated mice. On the basis of these results, Cu2(asp)4 was observed to be a potent antioxidative compound possessing antioxidative activity in biological systems. In conclusion, Cu2(asp)4 is a potent antioxidative agent that may be useful for future treatment of diseases resulting from ROS.

Therapeutic Potential of Growth Factors for Aging Voice

It has been reported that in aged vocal folds, dense collagen deposition takes place and hyaluronic acid decreases in the lamina propria, which are thought to contribute to the vocal problems occurring with age (presbyphonia). To restore aged vocal folds to their younger state, it seems crucial to address these age-related lamina propria changes. Intervention that might increase hyaluronic acid and decrease collagen would appear to be a potentially useful approach. The present study examined the effects of growth factors on aged fibroblasts in terms of the production of hyaluronic acid and collagen type I. In vitro study using animal model. Fibroblasts were harvested from young and aged rat vocal folds and cultured with or without hepatocyte growth factor and/or basic fibroblast growth factor at different concentrations. Subsequently, the production of hyaluronic acid and collagen type I was examined in the supernatant culture media using enzyme-linked immunosorbent assay. Aged fibroblasts produced less hyaluronic acid than younger fibroblasts. When aged and young fibroblasts were cultured with basic fibroblast growth factor, hyaluronic acid production increased and collagen type I production decreased regardless of the concentration, whereas the effects of hepatocyte growth factor appeared to vary with concentration. The basic fibroblast growth factor also was associated with stimulation of growth of aged fibroblasts. The present results suggest that growth factors, especially basic fibroblast growth factor, may have therapeutic potential in restoration of aged vocal fold.

Cytotoxicity studies of chromium(III) complexes on human dermal fibroblasts

The cytotoxicity of certain Cr(III) complexes, such as [Cr(salen)(H 2O) 2] +, [Cr(edta)(H 2O)] −, [Cr(en) 3] 3+, [Cr(ox) 3] 3−, [Cr(pic) 3], and CrCl 3, which differ in ionic character and ligand environment in human dermal skin fibroblasts, has been studied. After 72 h of exposure to 100 μM doses of chromium(III) complexes, the order in which the complexes had an inhibitory effect on cell viability was [Cr(en) 3] 3+ > [Cr(salen)(H 2O) 2] + > [Cr(ox) 3] 3− > [Cr(edta)(H 2O)] − > [Cr(pic) 3] > CrCl 3. Based on viability studies it was confirmed that [Cr(en) 3] 3+, a triply charged cation, inhibits cell proliferation, and therefore, it was chosen to carry out further investigations. [Cr(en) 3] 3+, at a dose of 50 μM, was found to bring about surface morphological changes, evidenced by cellular blebbing and spike formation accompanied by nuclear damage. TEM analysis revealed substantial intracellular damage to fibroblasts in terms of the formation of apoptotic bodies and chromatin condensation, thus reflecting cell death. FACS analysis further revealed DNA damage by formation of a sub-G 1 peak with 84.2% DNA as aneuploid DNA and arrest of the G 2 / M phase of the cell cycle. Cellular DNA damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in DNA isolated from [Cr(en) 3] 3+-treated fibroblasts. The proposed mechanism suggests the plausible role of Cr(V), formed as a result of oxidation of Cr(III) by cellular oxidative enzymes, in the cytotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the cytotoxicity and genotoxicity of a variety of Cr(III) complexes and to understand the potential hazards of Cr(III) complexes encountered in research laboratories.

Cyclic mechanical stretching of human patellar tendon fibroblasts: activation of JNK and modulation of apoptosis.

Accelerated rehabilitation after tendon, ligament and bone injuries is widely accepted to avoid adverse effects of immobilization. However, progressive rehabilitation may also lead to an excessive inflammatory soft tissue response and often leads to structural and functional problems such as excessive scarring. The equivalent at the molecular/cellular level is in part the regulation of the sensitive homeostasis between proliferation and apoptosis. However, little attention has been paid to this aspect of tendon pathogenesis. This study investigated the response profile of human tendon fibroblasts in terms of apoptosis and anticipated alteration of Jun N-terminal kinase (JNK) activation to cyclic mechanical stretching. Human tendon fibroblasts of six patients were stretched for 15 or 60 min with 1 Hz and an elongation of 5%. Activation of stress-activated protein kinase (SAPK)/JNK was measured by western blot analysis. Apoptotic cells were determined in the stretched cells and in controls by annexin-V staining and detection by flow cytometry. Additionally DNA laddering was determined by ligation-mediated (LM) polymerase chain reaction (PCR). Application of 15 and 60 min stretch increased activation of SAPK/JNK at a maximum after 60 min. However, JNK activation after the longer stretch period 50% less than after the shorter stretch period (15 min). The apoptosis rate was correspondingly increased after short stretch application but not after longer stretch. This might be caused by an inactivation of the activated JNKs by cell protection mechanisms. The findings suggest that mechanical stretching directly activates intracellular signaling pathways, which in turn induce apoptosis. The longer stretch period resulted in a decreased apoptotic rate due to development of stress tolerance. This might be caused by heat-shock protein mediated suppression of JNK activation. This novel observation is an important issue, as defined mechanical stretching, depending on its duration, modulates apoptosis and thus affects tendon remodeling.

Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation.

Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system. The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects. Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action. On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered. Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT.

Protection of cultured gastric cells against tert-butyl hydroperoxide by glutathione isopropyl ester

Oxidants are involved in the pathogenesis of a variety of gastrointestinal disorders. Intracellular GSH protects rat gastric cells against oxidants. We examined whether GSH isopropyl ester (GSH ester) can protect against oxidants and whether a system of GSH ester uptake is present in these cells; we also investigated a possible role of GSH in inhibiting lipid peroxidation. Cytotoxicity was quantified by 51 Cr release. Lipid peroxidation was assessed by measuring malondialdehyde production. tert-Butyl hydroperoxide caused a dose-dependent increase in 51 Cr release. Treatment with GSH ester attenuated the toxicity of tert-butyl hydroperoxide. Incubation with GSH ester enhanced the cellular GSH content, which was prevented by dl-buthionine-[ S, R]-sulfoximine, an inhibitor of γ-glutamylcysteine synthetase. GSH ester prevented tert-butyl hydroperoxide-induced lipid peroxidation, corresponding with the degree of protection. Therefore, we concluded that GSH isopropyl ester protects gastric cells against oxidants through the accumulation of intracellular GSH. This protection is mediated in part by the prevention of hydroperoxide-induced lipid peroxidation. However, the gastric epithelial system of GSH ester uptake appears distinctly different from those observed in hepatocytes, lymphoid cells and fibroblasts in terms of mediation of γ-glutamylcysteine synthetase activity.

P53 transactivation and protein accumulation are independently regulated by UV light in different phases of the cell cycle.

DNA damage-induced activation of the p53 tumor suppressor gene is suggested to be central in the cellular damage response pathway. In this study, we analyzed the responses of p53 to UVC radiation in synchronized mouse fibroblasts in terms of p53 accumulation, transcriptional activation, and sequence-specific DNA-binding activity. UVC was found to induce accumulation of p53 cell cycle dependently in G1/S- and S-phase cells but not in G0 or G1 cells. In contrast, p53 transcriptional activity and its target genes, p21 and GADD45, were stimulated by UVC in G0 and G1 cells in the absence of detectable p53 protein. The accumulation of p53 and increased p21 and GADD45 expression were replication dependent in S-phase cells. Interestingly, sequence-specific p53 DNA-binding activity was stimulated also replication independently in S phase, though the effect was not conveyed to stimulation of p53 target genes, suggesting that additional events are required for p53-stimulated gene expression. The results show that opposed to the cell cycle dependence of p53 accumulation, the UVC-mediated transactivation by p53 is independent of the cell cycle phase and protein stabilization.

Growth inhibition of human pancreatic cancer cells by sphingosylphosphorylcholine and influence of culture conditions.

Sphingosylphosphorylcholine (SPC) has been shown to be a potent mitogen for Swiss 3T3 fibroblasts and also to be an inhibitor of cell growth of some cancer cells, suggesting cell-selective action of the lipid. We examined the effects of SPC, and its structurally-related sphingosine (SP), sphingosine 1-phosphate (S1-P) and membrane-permeable derivatives of ceramides on cell growth of four strains of human pancreatic cancer cells, MLA PaCa-2, PANC-1, PK-1 and PK-9. Under the reported conditions for SPC-induced stimulation of 3T3 fibroblasts, where cells were grown to confluency in the presence of 10% fetal bovine serum (FBS) in culture prior to experiments and insulin was supplemented in experimental culture, none of the agents tested stimulated DNA synthesis in MIA PaCa-2 cells and ceramide at high concentration even inhibited it. On the other hand, in reduced FBS concentration in preculture and in the absence of insulin in experimental culture, SP, S1-P and ceramides suppressed cell growth of all the cells tested including Swiss 3T3 fibroblasts. However, under these conditions, SPC inhibited three out of four species of pancreatic cancer cells but stimulated Swiss 3T3 fibroblasts in terms of both DNA synthesis and cell proliferation. Cell cycle analysis showed that SPC stimulated cell cycle progress from the G1 to the S phase in Swiss 3T3 fibroblasts but inhibited it in PANC-1 cells in reduced FBS concentrations. We suggest that extracellular SPC can inhibit cell growth of human pancreatic cancer cells through regulation of the cell cycle process depending upon both the cell species and environmental conditions.

Molecular markers of senescence in fibroblast-like cultures.

The loss of replicative capacity in vitro of normal human diploid fibroblasts is a model for studying molecular changes that accompany both regulated growth control and cellular senescence. We describe the molecular phenotype of senescent fibroblasts in terms of markers that are altered with proliferative decline. We describe these markers by analyzing pathways and associated mechanisms related to the responsiveness of proliferatively competent and senescent cells to growth signals including changes in the extracellular environment, growth factors, growth factor receptors, secondary messengers, cell-cycle progression, transcription factors, and the fidelity of DNA synthesis. There is an abundance of molecular markers for senescence in culture at every level of information transfer. Although it seems clear that some alterations in gene expression with senescence are the result of specific changes in upstream events, more global dysregulation of coordinated growth control point to as yet undefined mechanisms.

Effects of a factor derived from polymorphonuclear leukocytes on the growth and collagen metabolism in normal and scleroderma skin fibroblast cultures

Effects of a factor (15 kDa) derived from polymorphonuclear leukocytes (PMNs) on the growth and collagen metabolism of normal and scleroderma skin fibroblasts in culture were investigated. Skin fibroblasts from papillary, reticular or whole dermis of 5 patients with scleroderma and 7 normal controls were cultured in the presence or absence of PMN factor. The factor affected skin fibroblasts, (i) to stimulate cell proliferation, (ii) to stimulate collagenase production as observed by its functional activity, but (iii) to suppress collagen production with little effect on collagen types at a stage before the translational level. Scleroderma fibroblasts were less susceptible to the factor than normal fibroblasts in terms of cell proliferation and protein synthesis including collagen. Stimulation of cell proliferation by the factor was higher with reticular cells than papillary cells, while stimulation of collagenase production was inversely high in papillary cells and little stimulation was noted with reticular cells. Suppression of collagen production was also eminent in papillary cells, compared with that in reticular cells. These results suggest that PMNs function at the inflammatory stage in shifting tissue metabolism to a catabolic phase by releasing a factor which stimulates connective tissue cells, and that decreased susceptibility of scleroderma cells and reticular cells to PMN factor may provide for a positive factor in the accumulation of collagen in the reticular dermis of scleroderma skin.

Testing garlic for possible anti-ageing effects on long-term growth characteristics, morphology and macromolecular synthesis of human fibroblasts in culture

The beneficial effects claimed for the use of garlic as a nutritional supplement include detoxification, antioxidation, antifungal activity, antibacterial activity, tumour suppression and, possibly, anti-ageing and rejuvenating effects. We have used the Hayflick system of cellular ageing in culture in order to test garlic for its anti-ageing effects on long-term growth characteristics, morphology and macromolecular synthesis of human skin fibroblasts. Our results show that an addition of garlic extract into the normal cell culture medium can support serial subculturing for over more than 55 population doublings in 475 days, and that this treatment has some youth-preserving, anti-ageing and beneficial effects on human fibroblasts in terms of maximum proliferative capacity and morphological characteristics. In comparison, similar or lesser doses of garlic extracts are growth inhibitory for cancerous cells that could not be grown over longer periods in the presence of garlic. To our knowledge, this is the first report of the effects of garlic on the long-term growth characteristics and macromolecular synthesis of normal human skin cells, the results of which have applications for both anti-ageing and anti-cancer research.

Human recombinant interleukin-4 induces proliferation and interleukin-6 production by cultured human skin fibroblasts

The effect of human recombinant interleukin-4 (hrIL-4) on normal human adult dermal fibroblasts in terms of proliferation and IL-6 production was studied. Fibroblasts were exposed to different concentrations of IL-4 for various periods of time. Proliferation was measured using a [ 3H]thymidine incorporation assay. IL-6 production was measured at the transcriptional, protein, and functional levels by Northern blot analysis, radioimmunoassay, and B9 bioassay, respectively. Our results show that hrIL-4 significantly stimulated (two- to fivefold) fibroblasts to increase the incorporation of [ 3H]thymidine in a dose- and time-dependent manner. However, hrIL-1, hrIL-2, hrIL-5, or hrTNFα, at the same concentration (100 U/ml) and for the same time period (4 days), did not. In addition, IL-4 significantly induced (four- to eightfold) the production of immunoreactive and biologically functional IL-6. However, IL-4 was not as potent an inducer of IL-6 as IL-1. The IL-4-induced IL-6 production was dose and time dependent and was due, at least in part, to a dramatic increase in the steady-state levels of IL-6 mRNA. This is the first report describing the ability of IL-4 to activate human dermal fibroblasts in terms of proliferation and IL-6 production.

Change in phospholipid composition of mouse bone marrow-derived mast cells during cultivation with fibroblasts

A mouse bone marrow-derived mast cell (BMMC) clone, designated as MC-MKM, was established. MC-MKM cells released histamine and generated lipid chemical mediators such as prostaglandin D 2 or platelet-activating factor upon stimulation with IgE and antigen. It is known that BMMCs aquire the characteristics of connective tissue mast cells (CTMCs) during coculture with fibroblasts. In the present study, we found that cellular phospholipid composition changed drastically when MC-MKM cells were cultured for 2 weeks in the presence of mouse 3T3 fibroblasts. In MC-MKM cells, phosphatidylcholine was the predominant phospholipid class, followed by phosphatidylethanolamine. During coculture with fibroblasts for 2 weeks, total phospholipid content in MC-MKM cells increased 2.5-fold. Among major phospholipid classes, a percentage of phosphatidylethanolamine increased most dramatically in accordance with a decrease in that of phosphatidylcholine, while no appreciable change in composition of other phospholipids was observed. The phospholipid composition of cocultured MC-MKM cells was closer to that of rat peritoneal CTMCs than that of the starting MC-MKM cells. The present findings that BMMCs converted their phenotype into CTMC-like cells by the interaction with fibroblasts in terms of phospholipid composition are not contradictory to the previous observations that BMMCs changed into CTMC-like cells.

Pseudohomozygous Type II Hyperlipoproteinemia

Nodular xanthomas on both elbows and a streak-like xanthoma on the intergluteal area developed in a 4-year-old girl with type IIa hyperlipoproteinemia. She had no disease associated with secondary hypercholesterolemia and no family history of hypercholesterolemia. Her xanthomas regressed under fat restriction diet and cholestyramine therapy. She was diagnosed as having pseudohomozygous type II hyperlipoproteinemia. The low-density lipoprotein (LDL) receptor activities of her cultured fibroblasts in terms of binding, internalization and degradation rate of LDL were normal. These results are consistent with a new syndrome of pseudohomozygous type II hyperlipoproteinemia and suggest that the mechanism of hypercholesterolemia, which induced xanthoma, differs from familial hypercholesterolemia.

Peroxisomes in fibroblasts from skin of Refsum's disease patients.

Skin fibroblasts were cultured from young adult patients with Refsum's disease, an inherited metabolic disorder characterized by a deficiency in oxidation of phytanic acid and by increased serum and tissue concentrations of this fatty acid. These cultures were compared to cultures of normal fibroblasts in terms of the number and distribution of peroxisomes demonstrable cytochemically in preparations incubated for catalase activity. Refsum's fibroblasts were found to contain 1-10 peroxisome profiles per 100 micron 2 of cytoplasm; the controls contained 1-2 profiles per 100 micron 2. The peroxisomes in normal fibroblasts were found in all regions of the cytoplasm. In the Refsum's material they were relatively scarce in the perinuclear region, where many of the cells showed numerous large inclusions containing lipid-like material and myelin figures. Our findings indicate that in the adult form of Refsum's disease, which is the more thoroughly studied variety, peroxisomes in fibroblasts are not diminished in number. This contrasts with a recent report concerning a case of what is thought to be an infantile form of the disorder, in which no peroxisomes were detected in a liver biopsy. If phytanic acid accumulations in the adult form are a consequence of peroxisomal defects, the defects presumably are at the level of specific enzymatic deficiencies and do not involve a generalized absence of peroxisomes.

Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: Preliminary assessment of their osteogenicity

Confluent cellular layers are reproducibly obtained (from 21 of 24 specimens) by outgrowth from composite pieces of human trabecular bone and marrow. The cells resemble fibroblasts in terms of morphology, esterase profile, and production of collagen type 1. However, the cells displayed some osteoblastlike features. Both the primary outgrowths and passaged cultures had high alkaline phosphatase activities (37 nmols min −1.μg DNA −1) in the range displayed by embryonic osteoblastlike cells. The cellular alkaline phosphatase activity, which showed similarity to the bone isoenzyme on kinetic criteria, was stimulated by 1,25-di-hydroxyvitamin D 3 but decreased by PTH (1–34). In addition, the cell preparations were shown to increase osteocalcin (bone Gla protein) production in response to 1,25-dihydroxyvitamin D 3. The osteogenic potential of the bone and marrow-derived cells has been assessed in an in vivo diffusion chamber assay in which congenially athymic (nude) mice were used as hosts. None of the 25 chambers examined showed evidence of osteogenesis, although the cells remained viable and fibroblastlike. The alkaline phosphatase activities decreased to < 1 % of the original, high in vitro values. The findings question the hypothesis that bone and marrow-derived cells are osteobiasts or osteoblastlike cells, rather than a mixture of cell lines of the bone and marrow stromal system.