Alterations in Cellular Proliferation Motility and Regulation

Fibroblasts are an important cell in wound healing in acute and chronic wounds and, in microscopic analysis, have been determined to be a major cell type in dermal biopsies from venous ulcer and lipodermatosclerotic skin.25 Interest in alterations in fibroblast growth and growth factor response from patients with venous ulcers was evaluated by biopsies taken from the ulcer margin and compared to normal ipsilateral thigh fibroblasts of the same patients. The authors found a significant reduction in proliferation and the fibro-blasts were morphologically larger and polygonal with less uniform nuclear features. However, response to growth factors (basic fibroblast growth factor, epidermal growth factor) was maintained in venous ulcer fibroblasts, albeit not to the same magnitude of control fibroblasts. These results indicated a functional abnormality with dermal fibroblasts in venous ulcers and suggested that cellular senescence may contribute to the pathophysiology of venous ulcer forma-tion.29 Other investigators also have determined that venous ulcer fibroblasts have diminished proliferative rate and an attenuated response to growth factors including platelet derived growth factor (PDGF). In addition, the fibroblasts from patients with ulcers older than three years grew significantly slower than those with ulcers less than three years.30 Certain characteristics of cellular senescence were elucidated in subsequent experiments. Venous ulcer fibroblasts contained more cells that stained positive for senescent associated^ galactosidase (specific marker for senescent state) and had increased expression of protein and mRNA product for cellular fibronectin. The authors speculated that increased accumulation of senescent cells in venous ulcers may lead to the observed impaired healing.31 Of interest, taking ulcer fibroblasts and subjecting them to progressive passage had a significant effect on the expression of senescent associated-P galactosidase compared to normal fibroblast or fibroblasts cultured from patients with varicose veins only. Not only did the ulcer fibroblasts have an increased mean number of senescent associated-P galactosidase expressing cells (63.8 ± 8.9% vs. 11.2 ± 3.1%), but after six passages nearly all the ulcer fibroblasts were senescent (>95%). These data indicated that venous ulcer fibroblasts were significantly advanced in cellular age and closer to replicative exhaustion, suggesting that the accumulation of senescent cells in venous ulcer wounds may lead to recalcitrant healing.32 In experiments evaluating the effect of bFGF on fibronectin and MMP-2 expression, fibroblasts from venous ulcer and CVI patients and in normal controls were found to increase the expression of these proteins. The implications were that bFGF mediated its effects by increasing both extracelluar matrix protein and matrix proteinase, indicating that the up-regulation of fibronectin and MMP-2 may be a normal, transient, and inducible response of these cells to bFGF. Furthermore, that ulcer fibroblasts at baseline have higher levels of fibronectin may not signify that they possess more of a senescent-like phenotype, but rather that they have been subjected to more mitogenic stimuli as a result of their slow growth or location in the ulcer environment.33

Functional studies evaluating fibroblast motility by time lapse digital photoimaging were performed in both venous ulcer fibroblasts and fibroblasts cultured from the medial malleolar skin of patients with varicose vein. The findings demonstrated a significant reduction in venous ulcer fibroblast motility compared to ipsilateral normal thigh fibroblasts and in fibroblasts from patients without any CVI, and interestingly fibroblasts from varicose vein patients also had significant lower motility. The decreased fibroblast motility was associated with the expression of a-sma a marker for myofibroblast differentiation. These data supported that altered motility in CVI fibroblasts and myofibroblast differentiation are important functional characteristics and provide further explanation in altered wound healing.34

The response to PDGF by venous ulcer fibroblast previously has been demonstrated to be attenuated.30 Although these authors were unable to demonstrate any differences in PDGF receptors, a recent report demonstrated that venous ulcer fibroblasts had no growth response to PDGF AB, and the basal levels of PDGF a and PDGF P receptors were decreased.35 A possible explanation for these differences is that in the latter, fibroblasts were cultured from biopsies taken from the ulcer margin,35 and in the former, biopsies were from the central portion of granulation tissue and from lipodermatosclerotic skin.30

The regulatory mechanisms for fibroblast-reduced growth and attenuated response to growth factors remain unknown. In a recent report the mitogen-activated protein kinase pathway (MAPK) ERK1 and -2 were studied in venous ulcer fibroblasts treated with PDGF AB. The ulcer fibroblasts were found to activate MAPK and inhibition of the upstream kinase MEK1 significantly reduced fibroblast proliferation, which was reversible with the addition of PDGF. In addition venous ulcer wound fluid inhibited MAPK directly. These data suggest the importance of the MAPK ERK pathway in regulating venous ulcer fibroblasts proliferation.36

Key cell-cycle regulatory proteins for proliferation and apoptosis specifically involved with epithelialization have been investigated. In biopsies of venous ulcers, diabetic ulcers, and control subjects no major differences in kerati-nocyte immunohistochemical staining was observed for cell-cycle regulatory proteins or apoptosis-related proteins.37 In a follow-up study these investigators compared the edge of venous ulcer to that of the central granulation tissue for growth factors and cytokines in keratinocytes and endothe-lial cells by immunohistochemistry and phenotype characterization. Significant findings were that on the ulcer margin, keratinocytes and endothelial cells retained their secretory potential for growth factors and cytokines, whereas the ulcer bed was significant for very few fibroblasts and mainly scavenging cells (macrophages) presence.38 From these data the authors speculated that the wound bed organization was altered by chronic infections, and impaired nutrition inhibited keratinocyte migration. It is well known that fibronectin is an important protein of the extracellular matrix and involved in keratinocyte reepithelialization. A study evaluating biopsies from venous ulcer wound margin, acute wounds, and normal skin determined that the transcription product for fibronectin was increased significantly in venous ulcer. However, immunostaining for alpha5beta1 integrin, the cell surface receptor for fibronectin, was undetectable in venous ulcer biopsies. The authors concluded that although fibronectin mRNA was expressed, the lack of integrin receptor may prevent keratinocyte migration and wound closure.39

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