Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1.

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p < 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.

Significance of cell cycle for wound stratification in clinical trials: analysis of a pressure ulcer clinical trial utilizing cyclin D/cdk4.

During the past 5 years, progress in the treatment of pressure ulcers appears to have reached a plateau. Several factors in the design of recent clinical studies may have contributed to this situation. These factors include the criteria chosen for patient selection, small sample size, and lack of a concisely defined final clinical outcome. Hunt noted that wound stratification according to a patient's physiological characteristics may be a key to quantifying differences among clinical treatments. To address this issue, we examined the expression of cyclin D/cdk4 in pressure ulcer fibroblasts taken from tissues during a recent clinical trial. The treatment groups included patients treated with the following regimens: placebo, granulocyte macrophage-colony stimulating factor alone, basic fibroblast growth factor alone, or granulocyte macrophage-colony stimulating factor and basic fibroblast growth factor given in sequence. Immunocytochemical colocalization of cyclin D and cdk4 showed that, before the initial treatment began, patients were distributed disproportionately among treatment subgroups in regards to the initial expression of these markers. For example, we found that compared with other subgroups, ulcer fibroblasts in the basic fibroblast growth factor treatment group showed a much lower expression of cyclin D/cdk4 at day 0. However, this group exhibited higher levels of expression of this complex after 35 days of treatment. This study shows that measurement of cyclin D/cdk4 expression permits more accurate stratification of patients within treatment subgroups, measurement of a cell's ability to detect the presence of functional cytokines, identification of area(s) of failure within the G1 of the cell cycle, and a basis for critical evaluation of various treatments.