RESUMEN
OBJECTIVE: Venous ulcer fibroblasts (w-fb) have attenuated growth compared to normal fibroblasts (n-fb). The MAPKp38 pathway mediates stress-responses in various diseases. We hypothesize that p38 pathway is altered in w-fb. METHODS: W-fb were isolated from venous ulcers and n-fb from the ipsilateral thigh. Fibroblasts were analyzed for phosphorylated p38 using immunoblot. The relation between p38 and w-fb proliferation was assessed with SB203580 (p38 inhibitor). Fibroblasts were treated with bFGF, TNF-a, and IL-1 and p38 expression analyzed. RESULTS: Phosphorylated p38 expression was increased in w-fb (AU%=233.5+/-59.7, P=0.039) compared to n-fb (AU%=99.9+/-14.6). W-fb treated with SB203580 demonstrated increased growth compared to untreated w-fb. W-fb treated with bFGF demonstrated decreased p38. TNF-alpha and IL-1beta significantly increase p38 expression. CONCLUSIONS: MAPK p38 is up-regulated in w-fb. Regulation of w-fb proliferation is influenced by p38. Altering the p38 pathway in vivo with growth factors or cytokine inhibition may improve fibroblast proliferation and venous ulcer healing.
Asunto(s)
Proliferación Celular , Fibroblastos/enzimología , Úlcera Varicosa/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adulto , Anciano , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Imidazoles/farmacología , Interleucina-1/metabolismo , Masculino , Persona de Mediana Edad , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Úlcera Varicosa/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Fibroblasts cultured from venous ulcers demonstrate phenotypic characteristics of cellular senescence including slow growth, altered morphology, upregulation of fibronectin, and increased senescence-associated beta-galactosidase activity. In senescent cells, arrest of cell replication is related to overexpression of p21 and underexpression of phosphorylated tumor-suppressor protein retinoblastoma (ppRb). The regulatory mechanisms for cell proliferation in venous ulcer fibroblasts are unknown. In this study, venous ulcer fibroblasts are examined for cell cycle protein expression and modulation by basic fibroblast growth factor (bFGF). Fibroblasts were isolated from the venous ulcer of the distal lower extremity (fb-D) of patients with chronic venous insufficiency. A control biopsy was obtained from the proximal ipsilateral thigh (fb-P). Paired cultures were plated at 100,000 cells/plate and the cells synchronized. After 24 hr, one culture set was treated with bFGF (20 ng/mL) and the other was kept in culture medium only (untreated). All cultures, treated and untreated, were lysed following 24 hr of incubation, and the lysate was used to perform immunoblot analysis for p21, ppRb, and cyclin D1. Immunoblot samples were standardized to protein content. In all patients analyzed (n = 4), at basal levels (untreated) fb-D demonstrated significant overexpression of p21 versus fb-P (p = 0.016). Treatment with bFGF resulted in significant downregulation of p21 levels for fb-D (p = 0.008) and fb-P (p = 0.037) compared to untreated fibroblasts. ppRb was underexpressed in fb-D versus fb-P (p = 0.069). Treatment with bFGF increased ppRb significantly in fb-D (p = 0.030) and in fb-P (p = 0.027) compared to untreated fibroblasts. No differences were observed in cyclin D1 with respect to basal levels in fb-P versus fb-D or in treated versus untreated groups. Venous ulcer fibroblasts show phenotypic similarity to senescent cells, with overexpression of p21 as well as down regulation of phosphorylated pRb. The aberrations seen in the cell cycle proteins in fb-D are similar to those seen in senescent cells; however, bFGF can modulate important cell cycle regulatory proteins, promoting a proliferative environment in fb-D that is not possible in a senescent cell. The role of bFGF may be useful in the clinical treatment of venous ulcer pathology.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Úlcera Varicosa/patología , Insuficiencia Venosa/patología , Adulto , Proliferación Celular , Células Cultivadas , Ciclina D , Ciclinas/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana Edad , FosforilaciónRESUMEN
Cyclic AMP (cAMP) appears extracellularly in a variety of tissues including brain, liver, and kidney; whether it appears in adipose tissue and responds to physiological perturbation is unknown. The purpose of this study was to examine adipose tissue extracellular cAMP appearance and metabolism in situ and in vitro in physiologically challenged animals. Littermate swine were either sedentary or exercise trained on a treadmill for 3 months and subjected to acute exercise on experiment day. In situ, microdialysis probes in subcutaneous back fat were perfused before, during, and after animals performed 20 mins of acute exercise, and dialysate was analyzed for cAMP and adenosine. In vitro, isolated adipocytes were hormonally stimulated to provoke cAMP synthesis and efflux, and plasma membrane phosphodiesterase and 5'-nucleotidase activities were measured. Extracellular cAMP and adenosine levels in adipose tissue of sedentary swine averaged 5.2 +/- 1.7 and 863 +/- 278 nM, respectively. Exercise training tended to increase extracellular cAMP (11.3 +/- 1.7 nM) and reduce extracellular adenosine (438 +/- 303 nM), although neither change was statistically significant. Acute exercise caused a significant 3-fold and 16-fold increase in extracellular cAMP and adenosine, respectively, compared to rest. These changes occurred despite a 2- to 3-fold increase in adipose tissue blood flow during acute exercise. In vitro, cAMP efflux from exercise-trained swine was 42% greater than that from adipocytes of sedentary swine, yet adipocyte plasma membranes from exercise-trained and sedentary swine did not differ in maximal phosphodiesterase and 5'-nucleotidase activities. We conclude that cAMP appears extracellularly in swine adipose tissue and that the levels of extracellular cAMP and adenosine in intact swine adipose tissue are influenced by both acute and chronic exercise. The subsequent impact of the changes in these biochemicals on local cellular metabolism and growth remains to be determined.