An in-vitro model of pressure induced fibroblast aging

Stanley, Andrew

Abstract

Chronic venous insufficiency (CVI) affects about 5-10% of the population >65 years old. In its worse form ulcerations develop. These ulcers are chronic, indolent and recur 70% of the time. The treatment of these ulcers is largely outpatient. It involves a variety of compression dressings and paste compression wraps (Unna's boot). Most ulcers require months of therapy before healing. The cost and morbidity suffered as a direct result of CVI is significant. The exact mechanism of the pathophysiologic process is elusive. Several theories have attempted to explain the process but none have resulted in significantly improving the therapy of the disease. All theories cite venous hypertension as the inciting factor which leads to tissue changes and the resultant ulceration. Recent studies have found fibroblasts isolated from the legs of patients suffering from CVI to be prematurely aged in comparison to fibroblast isolated from uninvolved dermal tissue of the same patients. These diseased fibroblasts had a much higher level of senescent cells. To better study the relationship between venous hypertension, CVI and premature aging, a specially constructed pressure incubator has been constructed. With this incubator, it is possible to culture cells at 200mmHg above atmospheric pressure. Preliminary evidence confirms the pressure/aging relationship in that populations of neonatal fibroblasts grown at 80mmHg and 120mmHg above atmospheric pressure had high rates of senescence after only 10-14 days of growth. With this incubator the pressure aging phenomenon can be better studied. After adjustments of pressure and length of time in culture, appropriate dose response data can be generated. With the use of B- galactosidase staining and quantification of fibronectin production senescence rates can be objectively studied. Next, the ability of pressurized fibroblasts to enter mitosis and increase DNA synthesis will be studied using flow cytometry. Finally, the potential to reverse this aging process will be studied by adding external growth factors and reversing conditions.