Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures (original) (raw)
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The American Journal of Pathology, 2008
Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serumfree, 0.1% ITS؉ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two-to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease in-hibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions , such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here , may have therapeutic utility in the prevention of keloid scarring.
Journal of Investigative Dermatology, 1996
Using a 3-dimensional fibrin gel model system simulating fibroplasia of wound repair, we investigated the interaction between keloid fibroblasts and fibrin matrix and compared it with that of normal fibroblasts. Normal skin fibroblasts caused fibrin gel degradation under serum-free conditions, whereas keloid fibroblasts did not cause microscopically detectable gel degradation. Fibrin gel degradation occurred through plasmin-mediated fibrinolysis, which was initiated by fibroblasts exhibited high uPA but low plasminogen activator inhibitor-1 (PAI-1) activities, and transforming growth factor-beta 1 prevented fibrinolysis of normal fibroblasts by upregulating PAI-1 while downregulating uPA activities. In contrast, keloid fibroblasts exhibited an intrinsically high level of PAI-1 and a low level of uPA. This change in the ratio of activator and inhibitor activities was attributed to altered fibrin degradation by keloid fibroblasts. The PAI-1 increase was also demonstrated at the RNA level by Northern analysis. In terms of the pivotal role of the plasmin/plasminogen activator system in matrix remodeling, the elevated PAI-1 level exhibited by keloid fibroblasts may have significant consequences not only in altered fibrin degradation, but also in subsequent repair steps that lead to keloids and fibrosis.
Experimental Cell Research, 1999
Synthesis of the major negative physiologic regulator of plasmin activation [plasminogen activator inhibitor type-1 (PAI-1)] is elevated during progressive cellular senescence, in premature aging disorders (e.g., Werner's syndrome), and in conditions associated with tissue fibrosis and excessive fibrin accumulation (e.g., sclerosis, keloid formation). Dermal fibroblasts derived from Werner's patients as well as from keloid lesions markedly overexpress PAI-1 mRNA transcripts compared to normal skin fibroblasts. Such cell type-related differences in steady-state PAI-1 mRNA content, and variances in the relative abundance of the 3.0-compared to the 2.2-kb PAI-1 mRNA species, served to discriminate normal from Werner's and keloid fibroblasts. This disparity in PAI-1 mRNA levels paralleled transcriptional activities of the PAI-1 gene; de novo synthesis of PAI-1 protein among the three cell types, moreover, closely approximated the respective differences in total PAI-1 mRNA content. Despite the markedly elevated levels of PAI-1 mRNA and protein evident in newly confluent keloid fibroblasts, these cells effectively suppressed PAI-1 synthesis (as did normal dermal fibroblasts) upon culture in serum-free medium. Werner's syndrome skin fibroblasts, in contrast, continued to maintain high-level PAI-1 expression even after 3 days of growth arrest. Adhesion-mediated attenuation of serum-stimulated PAI-1 expression, a characteristic of normal cells involving sequences which mapped to the distal 5 flanking region of the PAI-1 gene, was retained in keloid but not Werner's fibroblasts. Collectively, these data suggest that (1) specific controls on PAI-1 gene expression are fundamentally different between these two clinically significant high PAI-1-synthesizing cell types and (2) the localized keloid may define the emergence of a distinct profibrotic dermal fibroblastoid phenotype in genetically predisposed individuals.
AJP: Cell Physiology, 2004
Keloids are characterized as an “overexuberant” healing response in which disequilibrium between production and catabolism of extracellular matrix (ECM) occurs. Previous studies from our laboratory and others demonstrate an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) expression in keloid tissues and cultured fibroblasts compared with normal bordering skin. These findings support the concept that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis. Vascular endothelial growth factor (VEGF) has been implicated as a critical factor in regulating angiogenesis and inflammation under both physiological and pathological conditions. This study was designed to assess whether VEGF plays a role in keloid fibrosis. We report that VEGF was expressed at higher levels in keloid tissues and their derived fibroblasts compared with their ...
Keloids ar .e histologically characterized by an abundance of the extracellular matrix of connective tissue. In the present study, we examined the connective tissue composition of keloids, and analyzed the details of collagen metabolism utilizing fibroblast cultures established from keloid tissue. Quantitative connective tissue analyses indicated that collagen was the predominant extracellular matrix component in keloids.
Journal of Burn Care & Research, 2011
Fibroblasts, the main cell type of the dermis, are responsible for production and remodeling of extracellular matrix during wound healing. Disruption of either production or degradation of extracellular matrix can lead to abnormal scarring, resulting in hypertrophic scar or keloid scar. Aberrations in proliferation and gene expression have been observed in fibroblasts isolated from abnormal scars, but differences observed may be related to biologic responses to growth conditions and media formulations. This study examined gene expression in primary human fibroblasts from normal skin or abnormal scar in two culture media formulations and three relative cell densities. In general, higher expression of collagen type 1 alpha-1 (COL1A1) and alpha-2 (COL1A2) and matrix metalloproteinase 3 (MMP3) and lower levels of MMP1 were observed in all cell strains cultured in standard medium containing 10% fetal bovine serum compared with cells cultured in medium optimized for proliferation. Normal and scar-derived fibroblasts exhibited differences in gene expression in specific response to media formulations and cell density. COL1A1 and COL1A2 were increased, and MMP1 and MMP3 were decreased, in keloid cells compared with normal fibroblasts under most conditions analyzed. However, expression of plasminogen activator inhibitor 1 in keloid fibroblasts, which was significantly different than in normal fibroblasts, was either increased or decreased in response to the medium formulation and relative cell density. A related gene, plasminogen activator inhibitor 2, was shown for the first time to be significantly increased in keloid fibroblasts compared with normal fibroblasts, in both media formulations and at all three cell densities. The results emphasize the critical role of culture conditions in interpretation of cell behavior and expression data and for comparison of cells representing normal and fibrotic phenotypes. (
Increased Proliferation in Keloid Fibroblasts Woundedin Vitro
Journal of Surgical Research, 1996
A keloid is a pathological overgrowth of scar expanding beyond the boundaries of the initiating skin wound. llitimately, this expansive scar is a result of excess collagen synthesized by fibroblasts within the wound. The processes that lead to this collagen excess remain unknown. An in vitro wound model was developed to test the hypothesis that fibroblasts isolated from keloid tissue and wounded in vitro might proliferate more rapidly than similarly wounded normal dermal fibroblasts. Keloid fibroblasts (KF) and normal human dermal fibroblasts (NDF) were grown to confluence and quiescence in flexible-bottomed culture plates. Wounds were created in a standardized fashion using a specially designed jig. The jig utilized a 25 gauge needle to reproducibly ablate 16-20% of cells from confluent cell sheets. Wounded and nonwounded cells were labeled with 3H-thymidine at 24, 48, 72, and 96 hr postwounding to measure DNA synthesis. Wounded KF and NDF demonstrated increased 3H-thymidine incorporation compared to nonwounded control cultures, and wounded KF demonstrated significantly higher levels of 3H-thymidine incorporation than wounded NDF both 24 and 48 hr after wounding. A similar trend was seen in cell counts. The wounded KF also showed a statistically greater labeling index quantitated by autoradiography than did wounded NDF. The increased commitment to DNA synthesis in response to wounding in vitro in keloid fibroblasts correlates with pathology seen in vivo. Keloid fibroblasts may have a lower inherent threshold for S phase entry than do normal fibroblasts contributing to the increased proliferation of keloid fibroblasts in response to wounding in vitro.
Journal of Investigative Dermatology, 2008
Keloids are benign tumors of the dermis that form during a protracted wound healing process. Susceptibility to keloid formation occurs predominantly in people of African and Asian descent. The key alteration(s) responsible for keloid formation has not been identified and there is no satisfactory treatment for this disorder. The altered regulatory mechanism is limited to dermal wound healing, although several diseases characterized by an exaggerated response to injury are prevalent in individuals of African ancestry. We have observed a complex pattern of phenotypic differences in keloid fibroblasts grown in standard culture medium or induced by hydrocortisone. In this study Affymetrix-based microarray was performed on RNA obtained from fibroblasts cultured from normal scars and keloids grown in the absence and presence of hydrocortisone. We observed differential regulation of approximately 500 genes of the 38,000 represented on the Affymetrix chip. Of particular interest was increased expression of several IGF-binding and IGF-binding related proteins and decreased expression of a subset of Wnt pathway inhibitors and multiple IL-1-inducible genes. Increased expression of CTGF and IGFBP-3 was observed in keloid fibroblasts only in the presence of hydrocortisone. These findings support a role for multiple fibrosis-related pathways in the pathogenesis of keloids.
Biomedicines
Interactions between keratinocytes and fibroblasts in the skin layers are crucial in normal tissue development, wound healing, and scarring. This study has investigated the role of keloid keratinocytes in regulating collagen production by primary fibroblasts in vitro. Keloid cells were obtained from removed patients’ tissue whereas normal skin cells were discarded tissue obtained from elective surgery procedures. Fibroblasts and keratinocytes were isolated, cultured, and a transwell co-culture system were used to investigate the effect of keratinocytes on collagen production using a ‘scar-in-a-jar’ model. Keloid fibroblasts produced significantly more collagen than normal skin fibroblasts in monoculture at the RNA, secreted protein, and stable fibrillar protein level. When keloid keratinocytes were added to normal skin fibroblasts, expression of collagen was significantly upregulated in most samples, but when added to keloid fibroblasts, collagen I production was significantly reduc...