Chronic wound fluid suppresses proliferation of dermal fibroblasts through a Ras-mediated signaling pathway.

Wound fluid collected from chronic venous leg ulcers (chronic wound fluid (CWF)) has been shown to inhibit the growth of dermal fibroblasts by interfering with cell-cycle progression from G1 into S phase. Specifically, CWF was shown to downregulate the levels of hyperphosphorylated retinoblastoma tumor-suppressor gene (Rb) and cyclin D1, known to be critical for entering the S phase of the cell cycle. To further elucidate the effects of CWF, a Ras-mediated signaling pathway involving the mitogen-activated protein kinase kinase (MEK), known to modulate the expression of these cell-cycle-regulatory proteins, was examined. Transient transfection of dermal fibroblasts with constitutively active Ras abrogated the growth suppressive effects of CWF on hyperphosphorylated Rb (ppRb) and cyclin D1. In contrast, an MEK inhibitor PD 98059 mimicked the effects of CWF on these cell-cycle-regulatory proteins. Concurrent treatment with PD 98059 and CWF produced additive effects. Taken together, these results suggest that CWF inhibits the growth of dermal fibroblasts at least in part by decreasing the level of active Ras, resulting in decreased levels of ppRb and cyclin D1. Therefore, a Ras-dependent signaling pathway may mediate the growth inhibitory effect of CWF, and reconstitution of Ras activity may overcome this growth inhibitory effect.

Atopy, serum IgE, and interleukin-13 in steroid-responsive nephrotic syndrome.

Earlier studies have demonstrated a strong association of steroid-responsive nephrotic syndrome (SRNS), atopy, and elevated serum IgE levels. Interleukin (IL-13) gene expression is significantly increased in children with SRNS in relapse. As interferon (IFN)-gamma, IL-13, and IL-4 have regulatory effects on IgE synthesis, we examined the relationship between intracellular cytokine production and serum IgE levels in children with SRNS, in order to further define the reported association with atopy. The median serum IgE levels in nephrotic patients in relapse with (492 U/ml) or without atopy (561 U/ml) were significantly higher than those in remission (221 U/ml, P<0.002 or 90 U/ml, P<0.001, respectively) and non-atopic controls (177 U/ml) (P<0.001). The percentage of CD3+ IL-13-producing cells was significantly higher in nephrotic children in relapse, and correlated with the serum IgE levels during the active phase of the disease (r=0.90, P<0.001). These data suggest that the elevated serum IgE levels during relapses of SRNS were the result of upregulation of IL-13. This probably reflects some common immune activation following various stimuli, rather than a direct association with atopy.

bFGF-induced alterations in cellular markers of senescence in growth-rescued fibroblasts from chronic venous ulcer and venous reflux patients.

Fibroblasts cultured from the distal lower extremity of chronic venous insufficiency (CVI) patients exhibit characteristics of cellular senescence. Basic fibroblast growth factor (bFGF) has been shown to improve growth rates in these fibroblasts. In bFGF-treated fibroblasts, levels of fibronectin and matrix metaloproteinase-2 (MMP-2), known to be up-regulated in senescence, were examined to determine whether bFGF induces changes in these markers of senescence with rescue of cellular proliferation. Fibroblasts were isolated from the distal leg of patients with CVI with and without ulcers (fb-D). In all patients, a control was obtained from the proximal ipsilateral thigh (fb-P). Cells were plated at 3000 cells/plate and treated with bFGF (20 ng/mL) on days 1, 5, 8, and 11. Total cell number was obtained on days 5 and 12 using the Coulter particle counter, and concurrently cells were plated at 10,000 cells/plate and treated with bFGF on the same schedule; cell lysate was harvested on day 12 for immunoblot analysis for MMP-2 and fibronectin. In all patients (n = 7), fb-P grew faster than fb-D (p = 0.039). fb-D showed a mean 3.3-fold increase in growth in response to bFGF, and immunoblot analysis demonstrated an up-regulation of fibronectin and MMP-2 in response to bFGF. This represents the possibility that by stimulating growth, bFGF may drive cells toward senescence. This suggests clinical implication for the use of bFGF and other growth factors in general.