Morphology Of Dermal Fibroblasts Research Articles

Tamoxifen Upregulates Collagenase Gene Expression in Human Dermal Fibroblasts.

Tamoxifen is a known inhibitor of fibroblast transforming growth factor beta biosynthesis and wound scar formation. Tamoxifen is also known to be an estrogen antagonist and protein kinase C (PKC) inhibitor. Cells treated with tamoxifen and other PKC/calmodulin inhibitors depolymerize their membrane focal adhesion complexes and cytoskeletal protein structures. These effects result in substrate detachment, cell shape rounding, and upregulation of collagenase synthesis and extracellular matrix degradation. The purpose of our study was to test the hypothesis that tamoxifen treatment of human foreskin fibroblasts results in alteration of cytoskeletal protein organization, cell detachment and rounding, and increased collagenase synthesis similar to known PKC/calmodulin inhibitors such as H-7. We characterized the effects of PKC/calmodulin inhibitors tamoxifen and H-7 on human dermal fibroblast morphology, cytoskeletal protein organization, and collagenase gene expression in monolayer culture and within collagen gels. We found that fibroblasts responded to tamoxifen by initiation of actin filament depolymerization followed by alteration from spindle to spheroidal shapes. This change in cell shape led to increased collagenase synthesis in cells treated with either tamoxifen or H-7 compared with controls. There was also a 23% increase of hydroxyproline release from tamoxifen-treated fibroblast-populated collagen matrices. Tamoxifen may reduce scarring by inhibiting fibroblast PKC/calmodulin activity, which down-regulates pro-fibrotic transforming growth factor beta signaling and upregulates collagenase production. These effects mimic those of the known PKC/calmodulin inhibitor H-7. Overall, these findings suggest that tamoxifen and its analogues are promising agents for clinical investigation as small molecule regulators of fibrosis and scarring disorders.

Age-related reduction of dermal fibroblast size upregulates multiple matrix metalloproteinases as observed in aged human skin in vivo.

Fragmentation of collagen fibrils, the major structure protein in skin, is a hallmark of dermal ageing. Matrix metalloproteinases (MMPs) are largely responsible for the fragmentation of collagen fibrils. To quantify gene expression of all 23 known mammalian MMPs in sun-protected young and aged human skin in vivo and to investigate the potential mechanism underlying age-related alteration of multiple MMPs. MMP mRNA expression levels and MMP activity in sun-protected young and aged human skin in vivo were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ zymography, respectively. The relative contributions to elevated MMPs in epidermis and dermis were quantified by laser capture microdissection coupled real-time RT-PCR. Dermal fibroblast morphology and collagen fibril fragmentation in human skin in vivo were assessed by second-harmonic generation microscopy and atomic force microscopy, respectively. In vitro cell morphology was assessed by CellTracker® fluorescent dye (Molecular Probes, Eugene, OR, U.S.A.) and phalloidin staining. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (ProteinSimple, Santa Clare, CA, U.S.A.). Multiple MMPs are elevated in aged human skin dermis. Increased MMP activity and collagen fibril fragmentation were observed in aged skin dermis. As dermal fibroblasts are the major MMP-producing cells in the dermis, reduction of dermal fibroblast size, which is observed in aged human skin, contributes to the elevation of age-related multiple MMPs. Reduction of fibroblast size upregulates c-Jun/c-Fos and activates AP-1. Combined actions of the wide variety of MMPs that are constitutively elevated in aged dermis may be involved in the progressive degradation of dermal collagen fibrils. Age-related elevations of multiple MMPs are likely to be a result of the reduction of fibroblast size via activation of AP-1.

Botulinum toxin type A: implications in wound healing, facial cutaneous scarring, and cleft lip repair

Botulinum toxin is a neurotoxin that has been utilized to induce chemo-denervation of muscles. Cutaneous wounds represent a special situation in which the tensile forces applied by these muscles on wound edges might have deleterious effects on the healing process. The aim of this review was to investigate such an effect and to review other mechanisms this toxin might have on the healing process. We also reviewed the role of botulinum toxin in the management of hypertrophic scars and cleft lip repair.

Ultrastructural Analysis of Dermal Fibroblasts in Mucopolysaccharidosis Type I: Effects of Enzyme Replacement Therapy and Hematopoietic Cell Transplantation

In mucopolysaccharidosis type I (MPS I; alpha-L-iduronidase deficiency), glycosaminoglycans (GAGs) accumulate in different cell types, causing characteristic vacuolization. Hematopoietic cell transplantation (HCT) and enzyme replacement therapy (ERT) both aim to restore tissue morphology by delivering alpha-L-iduronidase to the deficient cells. The authors investigated the efficacy of both therapies on dermal fibroblast morphology in 12 patients by electron microscopy of repeated skin biopsies before and during 2 years of ERT as well as before and 6 months after HCT. Cell vacuolization was rated according to a semi-quantitative scoring system. At baseline all patients showed an increased vacuolization score as compared to controls. In addition the vacuolization score was significantly higher in patients with the severe phenotype of the disease (n = 7) compared to patients with attenuated phenotypes (n = 5) (p = .009). After initiation of ERT a significant decrease in cell vacuolization was observed (p = .012). However, the response rate varied among patients, as the vacuolization score remained high during the first year of ERT in 3 patients with the severe phenotype. In all patients who received a successful HCT (n = 3) only minimal disturbances in cell morphology were observed afterward. In conclusion, both ERT and HCT are capable of restoring, at least partially, dermal fibroblast morphology in MPS I.

Dermal Fibroblast Morphology is Affected by Stretching and not by C48/80

Both stretching and C48/80 have been hypothesized to cause disruption of cell-matrix adhesions and thereby affect the dynamics of fluid balance in tissues. We investigated the effect of sinusoidal stretching and/or C48/80 on the morphology of fibroblasts in skin excised from the backs of Wistar-Moller rats in order to assess how these stimuli affect cellular interactions in tissues. Tissue samples were either soaked in Krebs’ buffer with and without C48/80, or sinusoidally stretched (20% strain) in buffer with and without C48/80. Control skin was fixed immediately after excision. All tissues were processed for transmission electron microscopy. Morphometric analyses demonstrated that sinusoidal stretching of the skin results in the retraction or disruption of fibroblast cytoplasmic extensions, rounding up of the cell bodies and subsequently in increased tissue water content. C48/80 had no apparent effect on fibroblast morphology and adherence in tissues.

Bone marrow transplant in a case of mucopolysaccharidosis I Scheie phenotype: skin ultrastructure before and after transplantation

An 11-year-old girl with mucopolysaccharidosis I Scheie phenotype (MPS I-S) received a bone marrow transplant (BMT) from her heterozygous HLA-identical LMC-non-reactive mother. Multidisciplinary studies were carried out and results evaluated 21 months after transplantation. Herein we report the ultrastructural findings pre- and post-BMT in skin. Multidisciplinary studies are commonly used to evaluate the benefits of metabolic correction following BMT in some MPS and other inherited metabolic disorders, and changes in morphology have been described in liver and few other tissues. In this case, we elected skin, since connective tissue is universally involved in MPS and is safely and easily obtainable. Comparison of skin biopsy specimens taken before and after BMT showed a considerable change in dermal fibroblast morphology, with marked reduction in cell size and the number and size of abnormal lysosomes, thus indicating the clearance of storage. Our results demonstrate that dermal cells respond to enzyme replacement therapy in MPS I-S, with the clearance of glycosaminoglycan lysosomal accumulation in connective tissue fibroblasts, which had near-normal morphology 21 months after BMT. Therefore, the practice of skin biopsy after BMT in MPS and other metabolic disorders in which dermal cells are involved should be encouraged.

Osteogenesis imperfecta: a heterogeneous morphologic phenotype in cultured dermal fibroblasts.

Osteogenesis imperfecta (OI) is a phenotype with clinical and biochemical heterogeneity. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n = 6) and OI cell strains (n = 10, representing the four major OI categories, Sillence classification) were compared by measuring the following: (i) days required in culture to reach confluence after plating at uniform density; (ii) cell density at confluence; (iii) width and length of cells (measured on phase contrast micrographs at 300 X magnification). Our results show that: (i) OI fibroblasts take longer (11-27 days, mean 20 days) than control cells (10-19 days, mean 16 days) to reach stationary phase; (ii) all OI phenotypes achieve a lower cell density (0.87 X 10(6) cells/P60, range 0.3-1.6 X 10(6] at stationary phase relative to control cells (2.2 X 10(6) cells/P60, range 1.7-2.6 X 10(6); F4,77 = 56.1, p less than 0.01, indicating that OI cells are larger than normal). Cell shape (expressed as the width : length ratio) was also abnormal in OI cells. (F4,730 = 37.6, p less than 0.01), types I and II OI cells have significantly increased ratios (p less than 0.01) relative to control, type III, and type IV cells. Intra-group phenotypic heterogeneity was also apparent in the OI categories and also within the control population. These findings confirm deviant morphologic phenotypes in OI dermal fibroblasts and further demonstrate interindividual heterogeneity in the expression of genes that determine size and shape of dermal fibroblasts in both OI and normal donors.