Increase In Number Of Fibroblasts Research Articles

Morphological features of histogenic differon cells in connective tissue of guinea pigs’ lungs after sensitization with ovalbumin

An urgent issue of modern morphology is establishing a number of patterns of morphological changes and reactivity of connective tissue components of lungs in case of experimental sensitization with allergens. The aim is to estimate morphological features of histogenic differon cells in connective tissue of guinea pigs’ lungs after sensitization with ovalbumin. Materials and methods. Using morphometric and histological method, we have estimated the lung connective tissue of 48 male guinea pigs with experimental ovalbumin-induced allergic inflammation, simulated by subcutaneous sensitization and aeroallergization with ovalbumin. The number of fibrocytes, fibroblasts per 5000 μm2 and their ratio – fibroblast/fibrocyte coefficient were determined. Results. We have established the regularity of morphological changes dynamics in the cellular elements of pulmonary connective tissue. Experimental sensitization and inhalated allergization with ovalbumin leads to a statistically significant increase in the average number of fibroblasts and fibrocytes throughout the observation period in all experimental groups. It has been proved that the dynamics of cells has a multidirectional character, demonstrated by indicators of the fibroblast/fibrocyte coefficient, which shows the disproportion in the fibroblast/fibrocyte ratio and proves the tendency to the development of fibrosis in guinea pigs’ pulmonary connective tissue in case of experimental sensitization with ovalbumin. Conclusions. A gradual increase in the number of fibrocytes, against the background of a decrease in the number of fibroblasts is observed from the 23rd day to the completion of experimental sensitization with ovalbumin in the lungs of guinea pigs, compared with control group. A decrease of fibroblast/fibrocyte coefficient from 1.37 ± 0.03 in the early period to 0.82 ± 0.03 in the late period of the allergic inflammation demonstrates multidirectional nature of the dynamics in the number of connective tissue cells and indicates a tendency towards the development of fibrosis in pulmonary connective tissue.

The healing effect of bone marrow-derived stem cells and aquatic activity in Achilles tendon injury

The common treatment recommended for Achilles tendon rupture as the most common tendon injury during exercise is surgical intervention, while it eventually causes various clinical problems. This study assessed the healing effect of bone marrow-derived stem cells (BMSCs) and aquatic activities in Achilles tendon injury. Forty rats were randomly divided into 5 equal groups. Group 1 underwent aquatic activity, 72 h after a crush lesion formed on Achilles tendon, group 2 received 1×106 intra-articular BMSCs post-tendon injury, group 3 had aquatic activity together with BMSCs transplantation after tendon damage, group 4 just experienced tendon injury without any treatment intervention and group 5 was considered as the control group while did not undergo any tendon injury and did not receive any treatment measure. After 8 weeks, the animals were sacrificed and the tendons were transferred in 10% formalin for histological evaluation. There was a significant increase in fibroblast number in group 3 in comparison to other groups. However, there was a significant increase in collagen deposition in groups 2, 3 and 5 in comparison to group 1 and 4. A significant decrease was noted for cellularity in group 2 when compared to groups 1 and 4. Regarding tendon diameter in group 3; a significant healing was observed when compared to groups 2, 4 and 5. It was shown that aquatic activity together with cell transplantation was an effective therapeutic measure enhancing the healing in tendon injuries. These findings can open a window in sport medicine in treatment of tendon injuries.

Deep Learning With Sampling in Colon Cancer Histology.

This study applied a deep-learning cell identification algorithm to diagnostic images from the colon cancer repository at The Cancer Genome Atlas (TCGA). Within-image sampling improved performance without loss of accuracy. The features thus derived were associated with various clinical variables including metastasis, residual tumor, venous invasion, and lymphatic invasion. The deep-learning algorithm was trained using images from a locally available data set, then applied to the TCGA images by tiling them, and identifying cells in each patch defined by the tiling. In this application the average number of patches containing tissue in an image was ~900. Processing a random sample of patches greatly reduced computation costs. The cell identification algorithm was applied directly to each sampled patch, resulting in a list of cells. Each cell was labeled with its location and classification (“epithelial,” “inflammatory,” “fibroblast,” or “other”). The number of cells of a given type in the patch was calculated, resulting in a patch profile containing four features. A morphological profile that applied to the entire image was obtained by averaging profiles over all patches. Two sampling policies were examined. The first policy was random sampling which samples patches with uniform weighting. The second policy was systematic random sampling which takes spatial dependencies into account. Compared with the processing of complete whole slide images there was a seven-fold improvement in performance when systematic random spatial sampling was used to select 100 tiles from the whole-slide image for processing, with very little loss of accuracy (~4% on average). We found links between the predicted features and clinical variables in the TCGA colon cancer data set. Several significant associations were found: increased fibroblast numbers were associated with the presence of metastasis, venous invasion, lymphatic invasion and residual tumor while decreased numbers of inflammatory cells were associated with mucinous carcinomas. Regarding the four different types of cell, deep learning has generated morphological features that are indicators of cell density. The features are related to cellularity, the numbers, degree, or quality of cells present in a tumor. Cellularity has been reported to be related to patient survival and other diagnostic and prognostic indicators, indicating that the features calculated here may be of general usefulness.

Abstract 985: Stromal PDGFR-α activation stalls mammary ductal development and increases tumorigenic potential of mammary epithelia

Abstract One of the major confounding factors in the treatment of breast cancer is the cellular heterogeneity of the breast. An important cellular component of the normal breast as well as tumor stroma is the fibroblast. Fibroblasts are cells of mesenchymal origin that express platelet derived growth factor receptor alpha (PDGFR-α), a pharmaceutically targetable receptor tyrosine kinase. While activation of PDGFR-α was shown to be a poor prognostic factor in glioblastoma and gastro-intestinal stromal tumors, the role of stromal PDGFR-α signaling in mammary development and carcinomas is yet to be explored. To elucidate the role of stromal PDGFR-α signaling in this context, we utilized mice harboring the activating Pdgfra-D842V mutation knocked into the endogenous Pdgfra locus under a Lox-STOP-Lox cassette. To obtain selective activation of PDGFR-α in the fibroblastic stroma, these mice were crossed with fibroblast specific Fsp-cre mice. Whole mount carmine alum staining revealed that the D842V mutants have severely impaired mammary ductal development. Further analyses at the histological level revealed that the D842V glands have increased extra-cellular matrix (ECM) deposition (Masson's trichrome) likely due to the observable increase in fibroblasts (vimentin positivity). Immuno-fluorescent dual staining for keratin 8 and Ki67 demonstrated that stromal PDGFR-α activation increases the proliferation of neighboring mammary ductal cells, potentially making them more tumorigenic. To gain insight into the molecular mechanisms underlying this stromal-epithelial cross-talk, primary mammary epithelial cells and fibroblasts were isolated for downstream assays. Western blot analyses showed that mutant mammary fibroblasts have increased levels of activated PLC-γ, AKT and JNK which are known oncogenic pathways in the stroma. Gene expression arrays followed by global pathway analyses showed that pro-tumorigenic processes such as proliferation and metastasis are enriched in the epithelium from the mutant mice, while pro-tumor pathways such as KRAS and JNK were found to be enriched in the mutant fibroblasts. Interestingly, Pdgfra-D842V mutant mice have significantly increased stiffness as measured by atomic force microscopy of naïve tissue. This result implies that the observed increase in fibroblast number and resulting ECM deposition is sufficient to alter the entire mammary tissue. Since breast density correlates with higher breast cancer risk and tumor invasiveness, this phenotype reveals a novel function for stromal PDGFR-α signaling in breast cancer susceptibility. Studies are underway to evaluate whether these mice are predisposed to mammary tumor formation. Given our present data, we conclude that activation of stromal PDGFR-α signaling not only abrogates mammary development, but also makes the mammary ductal epithelium more tumorigenic. Citation Format: Anisha Mathur, Gina M. Sizemore, Subhasree Balakrishnan, Vasudha C. Shukla, Maria Cuitino, Anthony J. Trimboli, Samir Ghadiali, Gustavo W. Leone, Michael C. Ostrowski. Stromal PDGFR-α activation stalls mammary ductal development and increases tumorigenic potential of mammary epithelia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 985. doi:10.1158/1538-7445.AM2015-985

Use of Spongostan™ for Prevention of Cranial Subdural Adhesions Following Craniotomy in an Experimental Rabbit Model.

Spongostan™ is a sterile, water-insoluble, porcine gelatin absorbable sponge, which is widely used as a hemostatic material. The aim of this study is to test the anti-fibrotic capacity of Spongostan™, using a craniotomy model in an experimental rabbit model. Eighteen rabbits were divided into two groups: Each group consisted of 9 rabbits, duratomy plus Spongostan™ (group 1), and duratomy without Spongostan™ (group 2). Right parietal bone was removed via trephine and low speed drill and dura was opened. On the group 1 rabbits, an appropriate piece of Spongostan™ was meticulously placed under dural layer. On group 2 rabbits, same procedures were repeated without Spongostan™. Histological sections were taken from each group and evaluated for degree of fibrosis and collagen fibers. There was marked increase in number of fibroblasts and collagen fibers in group 2 rabbits, however most of the rabbits in Spongostan™ group demonstrate scarce histopathological findings for fibrosis. We conclude that an appropriately placed subdural Spongostan™ over cerebral tissue may prevent postoperative surgical adhesions after neurosurgical operations.

17 Cardiomyocytes Influence Fibroblast Proliferation and α-Smooth Muscle Actin Expression via the Secretion of Paracrine Mediators

IntroductionCardiac fibroblasts are known to modulate cardiomyocyte structure and function through soluble mediators. While less studied, the ability of cardiomyocytes to influence fibroblast properties, such as proliferation and activation, is...

Abstract 174: The Origin Of Cardiac Fibroblasts During Normal Development And After Injury

Fibroblasts are the most abundant cells in the heart. They play a critical role in maintaining normal myocardial function as well as mediating fibrosis that occurs upon pathological injury. However, the source of cardiac fibroblasts during development and after the injury in the adult heart remains poorly characterized. The lack of cardiac fibroblast-specific markers has limited our ability to isolate or track them during normal development and in response to cardiac injury. Here, we define cardiac fibroblasts as mesenchymal cells with a surface-marker profile of CD45-/CD31-/Ter119-/CD11b-/Thy1.1+ /Thy1.2+. We used lineage tracing, transplantation studies, and parabiosis surgeries to identify the cellular origins of cardiac fibroblasts and their contribution to fibrosis during pathological injury induced by pressure overload. We show that fibrosis is associated with proliferation of fibroblasts within 7 days after pressure overload injury with no evidence of bone marrow or hematopoietic stem cells contributing to the cardiac fibroblast population. We then used the transgenic mouse models Tie2cre;mTmG, Tbx18cre;mTmG, and Pax3cre;mTmG to lineage trace the origin of fibroblasts from the epicardium, endothelial and the neural crest, respectively. By using fluorescent activated cell sorting (FACS) and immunostaining, we demonstrate that the majority of cardiac fibroblasts are derived from the epicardium, a minority arises from endothelial cells, and a small fraction from the neural crest. Although we observed an increase in the total number of fibroblasts after pressure overload injury, this response was not restricted to any single developmental subset. In summary, our findings show that the majority of the cardiac fibroblasts, during normal development as well as in response to pathological injury, are derived from the epicardium via epithelial-to-mesenchymal transformation. Endothelial to mesenchymal transition and neural crest sources also contribute to fibroblast generation and the progression of pressure overload-induced cardiac fibrosis.

Inibição da expressão de ciclooxigenase 2 em feridas cutâneas de camundongos NOD submetidos à terapia a laser de baixa intensidade

CONTEXTO: A terapia a laser de baixa intensidade (LLLT) tem sido relatada como importante moduladora da cicatrização de feridas cutâneas aumentando a proliferação fibroblástica associada ao aumento da expressão da citocina fator transformador de crescimento- β2 (TGF-βB2). OBJETIVO: No presente estudo foram avaliados os efeitos da LLLT sobre a expressão da enzima ciclooxigenase 2 (COX2) no sítio do reparo tecidual utilizando o modelo experimental com camundongos diabéticos não obesos (NOD) para estudar a cicatrização de feridas cutâneas. MÉTODOS: Foram utilizados 30 camundongos NOD, destes 14 ficaram diabéticos e foram divididos em dois grupos: o grupo I (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e o grupo II (n=7) foi submetido a um procedimento cirúrgico de feridas cutâneas e tratados com LLLT. O grupo II foi submetido à LLLT nos seguintes parâmetros: 15 mW de potência, dose de 3,8 J/cm² e tempo de aplicação de 20 segundos. Após sete dias do ato cirúrgico e após aplicação do laser, os animais foram eutanasiados com sobredose de anestesia e amostras das feridas foram colhidas para posterior análise histopatológica, histomorfométrica e imuno-histoquímica. RESULTADOS: A LLLT promoveu a inibição da expressão da COX2 em feridas cutâneas de camundongos diabéticos. CONCLUSÃO: Em conjunto, os resultados sugeriram que a LLLT é capaz de modular negativamente a expressão da enzima COX2 contribuindo para o controle da resposta inflamatória em feridas cutâneas de camundongos NOD.

Fibroblasts and Phagocytic Cells in Phenytoin-induced Connective Tissue Proliferation

Objective: To evaluate the relationship of phenytoin-induced gingival enlargement and inflammation to find out if there is any significant correlation between hyperplastic index and periodontal parameters, the number of fibroblasts and phagocytic cells. Background: The introduction of phenytoin as an anti-epileptic drug in 1938 marked the beginning of a new era in the treatment of grandmal epileptic patients decreasing significantly not only the epileptic attacks but also improving the quality of life. However, there is concern in dentistry regarding gingival overgrowth as a side-effect. A histological study of this tissue can shed some light on the changes taking place. Materials and methods: Twenty-four epileptic patients on phenytoin therapy were divided into two groups as follows: • Group I or test group of individuals who had been suffering from gingival enlargement, • Group II or control patients taking phenytoin without any gingival enlargement. Plaque, gingival and hyperplastic indices of anterior teeth were determined in all the subjects. Biopsy specimens of all patients were taken and subjected to histopathological examination to determine the number of fibroblasts, macrophages, lymphocytes, and the level of inflammation, capillary proliferation and collagenation. Results: There is a significant increase in number of fibroblasts, macrophages, lymphocytes,collagenation and capillary proliferation in the enlarged gingiva of test group as compared to the control group. Conclusion: The degree of inflammation increases with the degree of enlargement. There is an increase in angiogenesis and the number of phagocytic cells thus indicating that phenytoin exerts growth promoting effects on the connective tissues.

Effects of PerClot® on the healing of full-thickness skin wounds in rats

PerClot® is a hemostatic material made of polysaccharide from modified starch and has been shown to assist in topical hemostasis. The principal goal in treating surgical and non-surgical wounds is the need for rapid closure of the lesion. This study investigated whether topical application of PerClot® could improve impaired wound healing in Sprague–Dawley (SD) rats. Full-thickness skin wounds were created on the back of the rats. Immediately, PerClot® was introduced into the wound bed, while wounds receiving starch or nothing served as controls. Wound closure was monitored using well-recognized wound-healing parameters: histological examination for inflammatory cells and fibroblast infiltration, newly formed capillaries, and collagen deposition. Meanwhile, transforming growth factor (TGF-β1) was measured by immunochemistry. Wound closure was significantly accelerated by local application of PerClot®. Furthermore, PerClot®-treated wounds showed significantly increased fibroblast numbers at 5 days post-wounding, and newly formed capillaries at 7 days post-wounding, and collagen regeneration at 7 and 14 days post-wounding. The number of infiltrating fibroblasts expressing TGF-β1 was significantly higher than that in the controls at 7 and 14 days post-wounding. PerClot® can improve the wound healing and this effect might involve an increase in the activity of fibroblasts and increased release of TGF-β1.

Epithelial-mesenchymal transition in primary human bronchial epithelial cells is Smad-dependent and enhanced by fibronectin and TNF-α

BackgroundDefective epithelial repair, excess fibroblasts and myofibroblasts, collagen overproduction and fibrosis occur in a number of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Pathological conversion of epithelial cells into fibroblasts (epithelial-mesenchymal transition, EMT) has been proposed as a mechanism for the increased fibroblast numbers and has been demonstrated to occur in lung alveolar epithelial cells. Whether other airway cell types also have the capability to undergo EMT has been less explored so far. A better understanding of the full extent of EMT in airways, and the underlying mechanisms, can provide important insights into airway disease pathology and enable the development of new therapies. The main aim of this study was to test whether primary human bronchial epithelial cells are able to undergo EMT in vitro and to investigate the effect of various profibrotic factors in the process.ResultsOur data demonstrate that primary human bronchial epithelial cells (HBECs) are able to undergo EMT in response to transforming growth factor-beta 1 (TGF-β1), as revealed by typical morphological alterations and EMT marker progression at the RNA level by real-time quantitative polymerase chain reaction and, at the protein level, by western blot. By using pharmacological inhibitors we show that this is a Smad-dependent mechanism and is independent of extracellular signal-related kinase pathway activation. Additional cytokines and growth factors such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL1β) and connective tissue growth factor (CTGF) were also tested, alone or in combination with TGF-β1. TNF-α markedly enhances the effect of TGF-β1 on EMT, whereas IL1β shows only a very weak effect and CTGF has no significant effect. We have also found that cell-matrix contact, in particular to fibronectin, an ECM component upregulated in fibrotic lesions, potentiates EMT in both human alveolar epithelial cells and HBECs. Furthermore, we also show that the collagen discoidin domain receptor 1 (DDR1), generally expressed in epithelial cells, is downregulated during the EMT of bronchial epithelium whereas DDR2 is unaffected. Our results also suggest that bone morphogenetic protein-4 is likely to have a context dependent effect during the EMT of HBECs, being able to induce the expression of EMT markers and, at the same time, to inhibit TGF-β induced epithelial transdifferentiation.ConclusionsThe results presented in this study provide additional insights into EMT, a potentially very important mechanism in fibrogenesis. We show that, in addition to alveolar epithelial type II cells, primary HBECs are also able to undergo EMT in vitro upon TGF-β1 stimulation via a primarily Smad 2/3 dependent mechanism. The effect of TGF-β1 is potentiated on fibronectin matrix and in the presence of TNF-α, representing a millieu reminiscent of fibrotic lesions. Our results can contribute to a better understanding of lung fibrosis and to the development of new therapeutic approaches.

Laser induced collagen remodeling: A comparative study in vivo on mouse model

Many lasers have claimed the clinical efficacy on skin rejuvenation. In this study, the mechanisms of laser induced collagen remodeling were explored systematically on a Kunming (KM) mouse model in vivo by comparing the different non-ablative laser effects using four different laser treatment modalities. The dorsal skin of KM mice was exposed by depilation before the laser treatments. Four laser treatment modalities were used: the 595-nm pulsed dye laser (PDL) (10 ms), 1,320-nm neodymium-yttrium-aluminum garnet (Nd:YAG) laser (0.35 ms), 1,064-nm Nd:YAG laser with Q-switched (5 ns), and long-pulsed (0.3 ms) mode. Each modality exposed one side of the mouse dorsal skin leaving the other side as the contralateral control. Then skin histology, fibroblast number, and the genesis of collagen type I and III were studied by comparing the treatment site and control site at 1 hour, 1 day, 1 week, 3 weeks, 4 weeks, and 8 weeks after laser treatment. Hydroxyproline content of the skin tissue was measured 4 weeks and 8 weeks after laser exposure. All laser treatments led to marked improvements in dermal layer thickness and collagen fiber density, and the increase in fibroblast number and hydroxyproline content compared with their own controls. Collagen synthesis and remodeling induced by the Q-switched 1,064-nm laser was most effective 4 weeks after treatment, while there was no significant difference among the other three modalities. Among the new collagen genesis after the different laser treatments, collagen type III increased sharply after the Q-switched 1,064-nm laser treatment whereas more collagen type I was elicited by the other laser treatment modalities. The efficacy of photo-mechanical effects in promoting more effectively the synthesis of collagen type III, whereas the photo-thermal effect favored more the formation of collagen type I.

Characterization of Fibroblasts withSon of Sevenless-1Mutation

Although non-syndromic hereditary gingival fibromatosis (HGF) is genetically heterogeneous, etiologic mutations have been identified only in the Son of Sevenless-1 gene (SOS1). To test evidence of increased cell proliferation, we studied histological, morphological, and proliferation characteristics in monolayer and three-dimensional cultures of fibroblasts with the SOS1 g.126,142-126,143insC mutation. Histological assessment of HGF gingiva indicated increased numbers of fibroblasts (30%) and increased collagen (10%). Cell proliferation studies demonstrated increased growth rates and 5-bromo-2-deoxyuridine incorporation for HGF fibroblasts. Flow cytometry showed greater proportions of HGF fibroblasts in the G2/M phase. Attachment of HGF fibroblasts to different extracellular matrix surfaces demonstrated increased formation of protrusions with lamellipodia. HGF fibroblasts in three-dimensional culture showed greater cell proliferation, higher cell density, and alteration of surrounding collagen matrix. These findings revealed that increased fibroblast numbers and collagen matrix changes are associated with mutation of the SOS1 gene in vitro and in vivo.

Morphogenesis of dermal–epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C

In skin, cohesion between the dermis and the epidermis is ensured by the dermal-epidermal junction which is also required for control of epidermal growth and differentiation. Here we showed that addition of vitamin C optimized the formation of the dermal-epidermal junction in an in vitro human reconstructed skin model leading to a structure closer to that of normal human skin. Compared with controls, vitamin C treatment led to a better organization of basal keratinocytes, an increase in fibroblast number and a faster formation of the dermal-epidermal junction. Vitamin C also accelerated deposition of several basement membrane proteins, like type IV and VII collagens, nidogen, laminin 10/11, procollagens I and III, tenascin C and fibrillin-1 at the dermal-epidermal junction. The mechanism of action of vitamin C was investigated by quantitative polymerase chain reaction in fibroblasts and keratinocytes respectively. Vitamin C effects passed in part through an increase in col I alpha1, col III alpha1 and fibrillin-1 mRNA levels. Effects on the other markers appeared to happen at the translational and/or post-translational level, as illustrated for tenascin C, col IV alpha2 and col VII alpha1 mRNA levels which were reduced by vitamin C in both cell types.

Increased and Prolonged Pulmonary Fibrosis in Surfactant Protein C-Deficient Mice Following Intratracheal Bleomycin

Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.

Pleural fluid transforming growth factor-beta1 correlates with pleural fibrosis in experimental empyema.

Transforming growth factor-beta1 (TGF-beta1) is a growth factor that is implicated in fibrosis of many organs. The purpose of this study was to determine the sequential levels of TGF-beta1 in the pleural fluid of rabbits that had undergone empyema induction, as fibrosis of the pleural space develops. Thirty-seven rabbits underwent empyema induction. Rabbits were sacrificed on Days 1, 2, 3, 4, 5, 6, and 8. Pleural fluid and viscera pleura specimens were collected at autopsy. TGF-beta1 levels were measured in pleural fluid using a commercially available ELISA kit, and pathologic specimens were scored for evidence of fibrosis (pleural thickness and number of fibroblasts). The median levels of pleural fluid TGF-beta1 increased from 8,100 pg/ml (Days 1 and 2) to 39,600 pg/ml (Day 8). Pleural fluid TGF-beta1 levels closely correlated with microscopic pleural thickness (r = 0.7, p < 0.001) and number of fibroblasts present in the visceral pleura (r = 0.68, p < 0.001). The first increase in pleural fluid levels of TGF-beta1 (Day 3) occurred before the increase in pleural thickness (Day 4) and before the increase in number of fibroblasts (Day 4). In conclusion, pleural fluid levels of TGF-beta1 rise in experimental empyema as pleural fibrosis develops. The rise in empyemic pleural fluid TGF-beta1 levels correlates with markers of pleural space fibrosis.

Induction of telomerase activity in fibroblasts from bleomycin-injured lungs.

Bleomycin-induced lung injury causes increased fibroblast numbers in the lung and pulmonary fibrosis. Studies of fibroblasts isolated from such injured lungs have revealed evidence of increased intrinsic proliferative capacity, but the mechanism is unknown. Telomerase catalyzes the addition of telomeric DNA repeats onto chromosomal ends, which is associated with increased cellular life span or immortality. To examine whether telomerase might play a role in regulating fibroblast proliferative capacity in pulmonary fibrosis, lung fibroblasts were isolated from rats treated with endotracheal injections of phosphate-buffered saline or bleomycin. At selected time points, the rats were killed and lung fibroblasts isolated. The isolated cells and lung tissue were then used in experiments for measurement of telomerase activity. The results show undetectable telomerase activity in fibroblasts isolated from control uninjured lungs, or in the control lung tissue extracts. Similar results were obtained in cells and lung tissue from Days 1, 3, and 28 bleomycin-injured lungs. However, significant telomerase activity was detected in fibroblasts and tissue extracts isolated from Days 7, 14, and 21 bleomycin-treated rat lungs, with maximal activity observed in the Day 14 samples. Analysis of the isolated cells for telomerase messenger RNA or reverse transcriptase expression, combined with alpha-smooth-muscle actin expression by immunohistochemistry, revealed that telomerase expression localized primarily to nonmyofibroblasts. These findings suggest that in addition to elevated growth factor expression, the injured lung fibroblast population may contain cells with increased life span, which could contribute to the observed overall increase in lung fibroblast numbers.

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ¿mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.

The effects of treatment with cyclophosphamide and methylprednisolone on expression of endothelin-1 in unilateral instillation of paraquat-induced pulmonary fibrosis in guinea pigs

Background : The herbicide paraquat can cause severe lung injury and fibrosis in experimental animals. In this study we have investigated the changes in lung endothelin-1(Et-1) levels and immunohistochemical localization in relation to treatment with cyclophosphamide and methylprednisolone in paraquat induced pulmonary fibrosis in guinea pigs. Material and methods : 29 male Hartley guinea pigs were divided into 4 groups. Group I was normal control. Paraquat was instilled into the lung of guinea pig of group II, III and IV unilaterally. Group II was treated with cyclophosphamide and methylprednisolone. Group III was treated with methlprednisolone. Group IV was not treated. The degree of fibrosis was evaluated by H-E stains and Masson's trichrome stains and cell activity was assessed by Et-1 immunohistochemical stains. Statistical evaluation was performed using the Kruskawallis oneway analysis. Results : Paraquat induced an increase in numbers of fibroblasts and total amount of lung collagen in Group IV compared to the normal controls. There was no significant difference in total numbers of fibroblasts between any of paraquat instilled groups, but there was significant increase in total amount of collagen in Group IV compared to group II and III (p

Localization of platelet-derived growth factor and insulin-like growth factor I in the fibrotic lung.

To better understand the mechanisms responsible for the increase in numbers of fibroblasts and increased collagen synthesis in fibrotic intestitial lung diseases, platelet-derived growth factor (PDGF)-A and PDGF-B, PDGF receptor-alpha and -beta, insulin-like growth factor I (IGF-I), and IGF-I receptor were evaluated immunohistochemically. Additionally, the messenger ribonucleic acids (mRNAs) for PDGF-A and PDGF-B, PDGF receptor-alpha and -beta, and IGF-I were investigated by in situ hybridization in alveolar macrophages and lung tissues from patients with interstitial lung disease. In specimens of bronchoalveolar lavage fluid (BALF), PDGF-A, PDGF-B, and IGF-I were local in alveolar macrophages in patients with idiopathic pulmonary fibrosis (IPF), patients with sarcoidosis (Sar), and normal individuals. Although there were no differences between IPF and Sar in terms of the staining intensity or number of positive cells, the number of such cells was smaller in the normal controls. In lung tissues with early-stage IPF, PDGF and IGF-I proteins were localized exclusively in alveolar macrophages, mononuclear phagocytes, fibroblasts, alveolar Type II cells, vascular endothelial cells, and vascular smooth-muscle cells. In lung tissues with late-stage IPF and those from normal controls, only alveolar macrophages contained PDGF and IGF-I proteins. Interestingly, the cellular localizations of PDGF receptor-alpha and -beta, and of IGF-I receptor were the same as those of the PDGF and IGF-I proteins in early-stage IPF, whereas these cells were not positive for any of these substances in late-stage IPF or normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)