Venous ulcers affect up to a million Americans.The investigators have previously reported that a striking feature of venous ulceration is the presence of dermal and subcutaneous fibrosis (termed lipodermatosclerosis or LDS) in and around the non-healing wound. LDS represents a fundamental aspect of venous disease. Evidence linking LDS to the pathogenesis of venous disease include the findings that ulcers almost always are preceded by LDS, develop and recur within LDS, their failure to heal correlates with the severity of LDS, and grafts often fail when placed over a fibrotic ulcer bed. Although the specific mechanisms of excessive collagen deposition in LDS are unknown, the investigators believe it may in part be due to elevated levels of transforming growth factor-beta-1 (TGF-beta), a potent and general mediator of fibrosis. Investigators present preliminary evidence that the fibrotic dermis in venous ulcers is characterized by increased amounts of TGF-beta. The cause of this enhanced deposition of TGF-beta is unclear, but they have shown a dramatic upregulation of both TGF-beta synthesis and collagen mRNA level in dermal fibroblasts exposed to levels of hypoxia well within the range consistently found in LDS. Their hypothesis is that LDS fibroblasts synthesize excessive amounts of collagen and TGF-beta, at least in part through the stimulatory action of low oxygen tension.
Their first aim i s to determine the distribution and expression of collagen and TGF-beta in LDS sites, ulcer sites, acute wound sites and normal skin using in situ hybridization and immunohistochemistry.
The second aim i s to measure by Northern analysis and run-off experiments TGF-beta and collagen synthesis by dermal fibroblasts cultured from normal and LDS tissue.
The third aim i s to determine the effect of hypoxia/re-oxygenation and lactate on TGF- beta and collagen synthesis (types I and II) in normal and LDS cultured dermal fibroblasts by Northern blot and run-off studies. The level of collagenases will also be quantitated. Finally, the fourth aim is to identify cis-acting elements responsive to hypoxia using transient transfection with TGF-beta promoter-CAT chimeric plasmids.
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