Chylomicron and apoB48 metabolism in the JCR:LA corpulent rat, a model for the metabolic syndrome.

Postprandial (PP) lipaemia is a significant contributor to the development of dyslipidaemia and cardiovascular disease (CVD). It is also evident that PP lipaemia is prevalent during conditions of obesity and insulin resistance (IR) and may contribute to increased progression of CVD. Our group has assessed the potential of the obese JCR:LA-cp rat as a model of PP lipaemia in order to explore CM (chylomicron) metabolism during the onset and development of IR in the metabolic syndrome. Studies confirm that both fasting plasma and PP apoB48 (apolipoprotein B48) area under the curve are significantly elevated in the obese JCR:LA-cp phenotype as compared with lean controls. Mechanistic studies have also shown that the concentration of lymphatic CM apoB48 and CM size are significantly increased in this model. Furthermore, PP dyslipidaemia in the obese rat can be improved acutely with supplementation of n-3 polyunsaturated fatty acids. Using a different approach, we have subsequently hypothesized that the vascular remodelling that accompanies IR may explain accelerated entrapment of apoB48-containing particles. Small leucine-rich proteoglycans (including biglycan and decorin) have been observed to co-localize with apoB in human tissue. However, the potential impact of IR on vascular remodelling, particularly in the presence of obesity, remains unclear. Preliminary observations from the JCR:LA-cp model indicate that biglycan protein core content increases with age and is exacerbated by IR, suggestive of pro-atherogenic remodelling. The focus of this review is to contribute to the perspective of PP lipaemia in CVD risk associated with the metabolic syndrome through the use of animal models.

Healing of burn wounds in transgenic mice overexpressing transforming growth factor-beta 1 in the epidermis.

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Myofibroblasts and apoptosis in human hypertrophic scars: the effect of interferon-alpha2b.

BACKGROUND: Hypertrophic scars (HSc) are a dermal fibroproliferative disorder that leads to considerable morbidity. Preliminary evidence suggests that interferon (IFN) may improve HSc clinically. The aims of this study were (1) to compare the cell density in HSc and in wounds that heal without the development of HSc (normotrophic scars), (2) to examine the presence of myofibroblasts and apoptosis in normotrophic and HSc scars over time, and (3) to determine if the systemic administration of IFN-alpha2b can induce apoptosis. METHODS: Two groups of patients underwent serial tissue biopsies. Six burn patients were studied prospectively by obtaining biopsy specimens from wound granulation tissue, normal skin, post-burn HSc, and normotrophic scars (healed donor sites). A second patient group with HSc was treated with systemic IFN-alpha2b and had biopsy material taken before, during, and after IFN therapy. The tissue was analyzed by immunohistochemical staining for alpha-smooth muscle actin (alpha-SMA) and in situ DNA fragmentation terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis. RESULTS: The total numbers of fibroblasts in HSc were found to be similar to granulation tissue and twice that of normal skin and normotrophic scar. Over time the numbers of cells in HSc tissue decreased toward normal skin levels. There was a significantly higher percentage of fibroblasts staining for alpha-SMA in HSc as compared with normotrophic scar or normal skin obtained from the same patient (P >.05). Serial biopsy specimens of resolving HSc tissue obtained from the patients who received systemic IFN-alpha2b showed a general reduction in total number of fibroblasts and myofibroblasts associated with a significant increase in the percentage of apoptotic cells compared with normal dermis from the same patient. CONCLUSIONS: HSc tissues have greater numbers of fibroblasts and myofibroblasts than normal skin and normotrophic scars. As HSc remodels, the numbers of fibroblasts and myofibroblasts reduces, possibly by the induction of apoptosis. Systemic IFN-alpha2b may contribute to the resolution of HSc in part by the enhanced induction of apoptosis.

Proteomic analysis of human skin fibroblasts grown on titanium: novel approach to study molecular biocompatibility.

Despite the growing use of titanium as an implant material, there is a lack of consensus on what constitutes the molecular basis of its biocompatibility. In the present study we compared the response of skin fibroblasts to two different growth supporting surfaces: commercially pure titanium (cpTi) and tissue culture polystyrene (TCPS). Proteins from extracts of whole cells and adsorbed serum were separated and identified by 2-dimensional gel electrophoresis. In all, 40 proteins (46 spots) were identified by matrix-assisted laser desorption/ionization and mass spectrometry, database searching, immunoblotting, running a standard, or a combination of these techniques. Many of the proteins collected from the two surfaces were found to derive from the serum used in the culture medium. The surface properties of titanium appeared to promote the formation of a more concentrated carpet of serum proteins. Several proteins from bovine or human serum, such as albumin, alpha2-HS-glycoprotein, alpha-fetoprotein, plasminogen, thrombospondin 1, and serotransferrin, along with a few unidentified serum components, were found to adsorb onto cpTi in comparatively high concentrations. The adsorption of serum proteins did not appear to be selective on either substrate. We found that among the major cellular proteins, fibronectin and a cytoskeletal protein (non-muscle myosin heavy chain type A) were expressed at lower levels by fibroblasts grown on cpTi compared to TCPS. By analyzing the changes in the entire proteome of cells in response to different growth substrates, we may gain a better understanding of the molecular basis of biocompatibility.

Differential morphometric and ultrastructural remodelling in the left atrium and left ventricle in rapid ventricular pacing-induced heart failure.

BACKGROUND: Heart failure induced by rapid ventricular pacing (RVP) is associated with left atrial (LA) but not left ventricular (LV) hypertrophy. OBJECTIVE: To determine whether differences in wall tension correlate with the differential ultrastructural remodelling in the LA and LV chambers, changes in ultrastructure, systolic function and wall tension (an index of wall stress) were compared in dogs after RVP (n=7) and with no RVP (n=9). RESULTS: Compared with dogs with no RVP (controls), dogs with RVP had increased collagen volume fraction (5.3% versus 8.3%), myocyte cross-sectional area (245 versus 366 microm(2)) and hydroxyproline (222 versus 323 microg/mg protein) in the LA (all P<0.05), but not in the LV. The increase in systolic wall tension produced by RVP was greater in the LA (five versus 43 units, P<0.0004) than in the LV (227 versus 290 units, P<0.01) chambers and correlated closely with the collagen volume fraction (r=0.87), which in turn correlated with myocyte cross-sectional area (r=0.98). In the left atrium, wall tension correlated with wall stress (r=0.99). CONCLUSIONS: The results suggest that differential wall tension may provide the stimulus for differential ultrastructural remodelling (with more hypertrophy and collagen) between LA and LV chambers in RVP-induced cardiomyopathy.

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-beta and triggers apoptosis in dermal fibroblasts.

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-beta1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-beta measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-beta, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-beta and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring.

Quantifying the adherence of fibroblasts to titanium and its enhancement by substrate-attached material.

Normal human skin fibroblasts were cultured on tissue culture polystyrene and on commercially pure titanium. In addition, substrate-attached material that remained on the surfaces after detachment of fibroblasts with a chelating agent was examined. The force required to detach 50% of the fibroblasts from each substrate was assessed by centrifugation. The results showed a time-dependent decrease in the force required to detach fibroblasts from titanium not seen on tissue culture polystyrene. Nearly all cells detached from the titanium surfaces at 7.85 x 10(x3) dynes/cell after 3 or 5 days in culture, whereas few cells detached from tissue culture polystyrene. Cells freshly seeded onto titanium substrates that had been coated with substrate-attached material by prior culture of fibroblasts for 3 or 5 days showed an approximately sixfold increased adherence. The results of immunofluorescence staining for fibronectin and its receptor suggest that the nature of the interaction between this extracellular matrix ligand and the substrate may be important in determining cellular stiffness at the cell-extracellular matrix interface.

Liposome associated interferon-alpha-2b functions as an anti-fibrogenic factor in dermal wounds in the guinea pig.

We have previously reported that interferon-alpha-2b (IFN-alpha-2b) can be encapsulated in liposomes without compromising its anti-fibrogenic effects on dermal fibroblasts in vitro. This study was conducted to determine whether this preparation applied topically to guinea pig wounds can affect their healing. The rationale for this approach is that systemic administration of IFN-alpha-2b by injection for treatment of dermal fibrosis is uncomfortable, requires a large quantity of the cytokine and cannot be easily used in children. Liposomes are potentially useful vehicles for the topical delivery of drugs. Empty sonicated liposome vesicles were mixed with various concentrations of IFN-alpha-2b and then dried and rehydrated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the efficiency of encapsulation and the stability of the preparation under experimental conditions. A total of 36 full thickness skin wounds (6/animal, 3 on each side) were made with an 8 mm disposable punch. Each wound on the right side received cream (100 mg/wound) containing 3000 units of liposome-encapsulated IFN-alpha-2b, while wounds on the left side received cream containing empty liposomes. There was a significant reduction in rate of contraction of wounds treated with IFN-alpha-2b as early as 5 days after wounding. This reduction remained significant up to 10 days. Northern analysis, used to evaluate the expression of mRNAs for type I and type III collagens in response to IFN-alpha-2b showed a marked reduction in abundance of the transcripts for the pro-alpha1(I) chain of type 1 collagen on days 11 and 14 after wounding. Similarly, the level of mRNA for type III procollagen was markedly reduced as early as day 7 and remained depressed up to day 14. These findings were consistent with results obtained for the total collagen content in tissue samples. Cellularity of the IFN-alpha-2b-treated wounds, assessed by vimentin content, was also markedly reduced at day 7 and remained depressed up to day 14. Liposome associated IFN-alpha-2b applied 5 days after completion of epithelialization reduced mRNA for the pro-alpha1(I) chain of type 1 collagen, confirming its transepidermal penetration and effectiveness. The activity of liposome-associated IFN-alpha-2b in vivo supports the concept of the topical use of this anti-fibrogenic agent for treatment of fibroproliferative disorders.

Molecular and cellular aspects of fibrosis following thermal injury.

The pathogenesis of hypertrophic scars following thermal injury remains a complex and incompletely understood process but recent investigations into the composition of the tissue itself, the activities of the scar fibroblasts, and the effects of various cytokines and growth factors, have all contributed to the emergence of an increasingly clear picture. Although it may be considered just one example of a broad range of fibroproliferative disorders that afflict many different organs, often in response to diverse environmental insults, the nature of the burn injury and the special properties of skin probably play important roles in promoting the development of this especially troublesome variety of excessive connective tissue. This knowledge has provided the rationale for a number of experimental therapies that, individually or in some combination, may augment or one day supplant the more commonly employed surgical or physical treatments.

Control of wound contraction. Basic and clinical features.

Although a substantial amount of molecular and cellular data have been generated in an effort to understand the process of wound contraction and scar contracture formation, questions remain. What seems apparent is that the myofibroblast is not the only cell that generates contractile forces within wounds, but it does appear to be intrinsically linked to the development of hypertrophic scars. The supposition that the formation of scar contractures is solely the result of a continuation of wound contraction is an oversimplification. Figure 4 provides a model of the possible evolution of contractile forces during the wound healing process and their role in the development of scar contractures. Migration of fibroblasts into and through the extracellular matrix during the initial phase of wound healing, prior to the expression of alpha-SMA, appears to be a fundamental component of wound contraction. During this migration, the pulling of collagen fibrils into a streamlined pattern in their wake, and the associated production of collagenase, may facilitate a more normal arrangement of collagen. Once the wound has been repopulated and the chemotactic gradient that was established by inflammatory cells is decreased, fibroblast migration will cease. It is at this point that myofibroblasts appear and play a key role in the production of hypertrophic scars, given that their prolonged presence and over-representation are hallmarks of this pathology. One of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars and scar contractures may be a lack (or late induction) of myofibroblast apoptotic cell death. The combined contribution of fibroblasts and myofibroblasts to abnormal extracellular matrix protein production results in an excessive and rigid scar. The isometric application of contractile forces by myofibroblasts probably contributes to the formation of the whorls, nodules, and scar contractures characteristic of hypertrophic scars. Because the prolonged presence of myofibroblasts, producing an imbalance in extracellular matrix proteins and proteases, probably exacerbates hypertrophic scars and wound contraction, accelerating the rate of apoptotic cell death to reduce the cell number to that seen in normal scar may be a useful strategy for providing effective and efficient treatment of scar contracture.

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-beta1 mRNA and protein than normal skin and cells.

Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment. The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs. 97 +/- 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Mannose-6-phosphate/IGF-II receptors mediate the effects of IGF-1-induced latent transforming growth factor beta 1 on expression of type I collagen and collagenase in dermal fibroblasts.

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor beta 1 (LTGF-beta 1) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-beta 1 has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-beta 1 neutralizing antibody or mannose-6-phosphate (M6P) and levels of mRNAs for TGF-beta 1, collagenase and the pro alpha 1(I) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-beta 1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro alpha 1(I) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-beta 1 neutralizing antibody at a concentration of 0.5-2.0 micrograms/ml. As the TGF-beta 1 induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold increase (201.9 +/- 38 vs 81.8 +/- 13, p < 0.05) in mRNA for the pro alpha 1(I) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-beta 1 and the pro alpha 1(I) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-beta 1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-beta 1 in conditioned medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-beta 1 and that the matrix-modulating autocrine effects of LTGF-beta 1 on dermal fibroblasts are facilitated by M6P/IGF-II receptors on these cells.

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.

Delayed appearance of decorin in healing burn scars.

AIMS: We have previously shown that hypertrophic scar tissue from burn patients contains abnormally high amounts of the proteoglycans versican and biglycan and reduced amounts of decorin, in comparison with normal dermis or mature scar. The lack of decorin may account for the poor organization of collagen fibrils in the nodular areas of these scars. Decorin has also been reported to neutralize the fibrogenic growth factor TGF-beta1. This study was conducted to monitor the time-course of expression of decorin in healing burn wounds by in-situ hybridization to determine whether its absence from hypertrophic scars could result from reduced synthesis. METHODS AND RESULTS: Scar tissue from 19 patients and normal dermis from six patients, was fixed in paraformaldehyde, embedded in paraffin and sectioned. Digoxigenin-labelled cRNA probes were prepared from a plasmid containing a 622-bp insert of human decorin cDNA and used for in-situ hybridization. Total numbers of connective tissue cells and cells positive for decorin mRNA were counted in 10 random fields in the upper (papillary), middle and lower (reticular) one-thirds of the dermis. In all regions the number and percentages of cells with decorin mRNA were low during the first 12 months after injury (eight samples), much higher between 12 and 36 months (seven samples) and low and similar to those in normal skin after 36 months (five samples). The differences between intermediate and early or late stage samples were statistically significant (one-way ANOVA). Immunohistochemistry showed little staining for decorin in early stage samples and much stronger staining in mid-stage. Late stage tissue showed intense staining for decorin, almost comparable to that in normal dermis. CONCLUSION: Expression of decorin in burn wounds is suppressed for about 12 months and then increases at a time when resolution of hypertrophic scarring is generally considered to occur.

A role for decorin in the structural organization of periodontal ligament.

Decorin is a small leucine-rich proteoglycan that interacts with several matrix molecules, including various types of collagen and growth factors, and suppresses the growth of neoplastic cells by an epidermal growth factor (EGF) receptor-mediated pathway. Decorin is abundantly expressed in the periodontal connective tissues during development and tissue maintenance. In periodontal disease, which is one of the most common diseases in the human kind, the level of decorin is decreased in the periodontal connective tissue. Abnormal expression of decorin may also associate with certain inherited disorders that involve increased susceptibility to severe periodontal disease in the early childhood. Therefore, we investigated the periodontal tissues of mice with targeted disruption of the decorin gene. Gross and microscopic analyses showed that decorin-deficient mice appeared to have normal tooth development and eruption, and there were no signs of periodontal disease. However, electron microscopic analysis revealed abnormal morphology and organization of the collagen fibrils in the periodontal ligament. The number of periodontal ligament fibroblasts in the decorin-deficient mice was also increased about two-fold as compared with the wild-type mice. In cell culture, ectopic overexpression of decorin in NIH 3T3 fibroblasts or decorin added exogenously to periodontal fibroblasts suppressed cell growth. However, blocking the EGF receptor tyrosine kinase activity did not prevent the decorin-elicited growth suppression in periodontal fibroblasts. Additionally, decorin did not induce a marked increase in the relative expression of p21 mRNA in periodontal fibroblasts. Therefore, decorin appeared to regulate growth of normal periodontal fibroblasts by a mechanism distinct from that reported for neoplastic cells. The findings demonstrate that decorin plays a role in the organization of collagen fibrils and regulates cell proliferation in the periodontal ligament.

The effect of interferon-alpha2b on an in vitro model Dupuytren's contracture.

The effects of interferon-alpha2b (IFN-alpha2b) on Dupuytren's and control palmar fibroblasts were evaluated using the fibroblast-populated collagen lattice model. Three paired strains of Dupuytren's and control fibroblasts were exposed to IFN-alpha2b for 96 hours before incorporation into triplicate collagen lattices. Contraction of the lattices was recorded and Northern blot analysis of cytoskeletal mRNA was performed. Comparisons of Dupuytren's and control fibroblasts revealed significantly increased contractility of the Dupuytren's fibroblasts in 2 of the 3 strains. Treatment with IFN-alpha2b significantly inhibited contraction in both Dupuytren's and control fibroblasts. In Dupuytren's fibroblasts, treatment with IFN-alpha2b significantly down-regulated mRNA expression for cytoplasmic beta-actin and gamma-actin.

The antifibrogenic effects of liposome-encapsulated IFN-alpha2b cream on skin wounds.

Interferons (IFN), including IFN-alpha2b, have been used as antifibrogenic factors to modulate the excessive production of extracellular matrix (ECM) associated with dermal fibroproliferative disorders. This study was conducted to examine the ability of a dermal cream containing liposome-encapsulated IFN-alpha2b (LIPO+IFN) to affect the synthesis of ECM in open and reepithelialized wounds. Full-thickness skin wounds in 32 female Hartley guinea pigs (6 wounds per animal, 3 on each side) were made with an 8-mm biopsy punch. Each wound on the right side received 3,000 U LIPO+IFN, whereas wounds on the left side received cream containing empty liposomes. Histologic examination revealed a significant reduction in scar formation in LIPO+IFN-treated but not in vehicle-treated wounds. Northern analysis showed reductions in type I procollagen mRNA in healed wounds treated with LIPO+IFN (day 4 groups: 1596.9 +/- 207 vs. 3710.2 +/- 493 densitometry units, p < 0.01, n = 8). This was consistent with a reduction in the concentration of collagen in the tissue, assayed as 4-hydroxyproline (day 4 group: 38.5 +/- 3.8 vs. 54.5 +/- 3.9 microg per tissue, p < 0.01, n = 8). Even when applied to reepithelialized wounds, LIPO+IFN caused a marked reduction in type I collagen mRNA (1938.5 +/- 579 vs. 4085.7 +/- 1271 densitometry units, p < 0.01, n = 8). These findings support the concept of the early topical use of this antifibrogenic agent for treatment of dermal fibroproliferative disorders, such as hypertrophic scars.

An immunohistochemical study of the localization of biglycan, decorin and large chondroitin-sulphate proteoglycan in adult rat temporomandibular joint disc.

To analyse regional variations in extracellular matrix components of adult rat temporomandibular joint discs, immunohistochemical techniques were used to examine the localization of two small dermatan-sulphate proteoglycans, biglycan and decorin, and a large chondroitin-sulphate proteoglycan. Staining for biglycan was intense in the posterior band, although it had a rather weak and even distribution throughout the disc. In contrast, staining for decorin was faint in the intermediate zone and the central part of the posterior band, moderate in the anterior and posterior attachments and most intense in the junction between the anterior band and attachment. The upper surface of the disc stained more intensely than the lower. Similarly, there was intense staining for large chondroitin-sulphate proteoglycan in the peripheral band, but both the anterior and the temporal parts of the posterior attachments were faintly stained. These results demonstrate marked regional differences in the expression of biglycan, decorin and large chondroitin-sulphate proteoglycan in the temporomandibular joint discs of adult rats. These variations probably reflect the different biomechanical environments caused by the complicated articulatory functions of the temporomandibular joint.

Transforming growth factor-beta in thermally injured patients with hypertrophic scars: effects of interferon alpha-2b.

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

Effect of interferon-alpha2b on guinea pig wound closure and the expression of cytoskeletal proteins in vivo.

Scar contraction following the healing of deep partial-thickness or full-thickness dermal injury is a leading cause of functional and cosmetic morbidity. The therapeutic use of interferon for the treatment of fibroproliferative disorders associated with scar contraction, including hypertrophic scar, has been suggested because of its antifibrotic properties. Treatment of fibroblasts with interferon has been shown to reduce the rate and extent of contraction using the in vitro fibroblast-populated collagen lattice model. In order to establish the effect of interferon-alpha2b on full-thickness wound contraction in vivo, osmotic pumps loaded with interferon or sterile saline were implanted intraperitoneally in guinea pigs. Seven days following implantation, six full-thickness punch biopsy wounds were created and were monitored by daily assessment of the wound. There was a significant reduction in the rate of wound contraction in the interferon-treated animals after day 3 (p < 0.01). Western blot analysis was used to quantitate selected cytoskeletal proteins in the normal skin and tissue biopsied from the wound at days 7, 14, and 21 postinjury. The amount of vimentin in the contracted wound increased following injury as compared with the amount present in normal skin (p < 0.0001); however, the relative amounts of the myofibroblast-associated cytoskeletal proteins alpha-smooth muscle actin and smooth muscle myosin were less than those found in normal, uninjured skin. By using vimentin to adjust the levels of cytoskeletal proteins for the increase in cellularity in the wounds, both alpha-smooth muscle actin and smooth muscle myosin significantly increased after closure of the wounds on day 14, as compared with the open-wound stage (day 7), before further reductions occurred with remodeling on day 21. Measurement of apoptotic cells using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay revealed an increase in apoptosis in the interferon-alpha2b-treated animals at 21 days following wounding (p < 0.001), which did not colocalize with alpha-smooth muscle actin staining. Taken together, these findings suggest that interferon-alpha2b inhibits wound contraction in vivo, not through an appreciable alteration in myofibroblast number or cytoskeletal protein expression, but possibly through a reduction in fibroblast cellularity by the induction of apoptosis.