Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-beta1 gene expression and protein production

Abstract

Purpose: Pathologic dermal degeneration in patients with chronic venous insufficiency (CVI) is characterized by aberrant tissue remodeling that results in stasis dermatitis, tissue fibrosis, and ulcer formation. The cytochemical processes that regulate these events are unclear. Because transforming growth factor-beta(1) (TGF-beta(1)) is a known fibrogenic cytokine, we hypothesized that the increased production of TGF-beta(1) would be associated with CVI disease progression.

Methods: Seventy-eight punch biopsy specimens of the lower calf (LC) and the lower thigh (LT) of 52 patients were snap frozen in liquid nitrogen and stratified into four groups according to the Society for Vascular Surgery/International Society for Cardiovascular Surgery CEAP classification (C, clinical; E, etiologic; A, anatomic distribution; and P, pathophysiology). One set of LC biopsy specimens were analyzed for TGF-beta(1) gene expression with quantitative reverse transcriptase-polymerase chain reaction: healthy skin, n = 6; class 4, n = 6; class 5, n = 5; and class 6, n = 7. A second set of biopsy specimens from the LC and LT were analyzed for the amount of bioactive TGF-beta(1) with a certified cell line 64 mink lung epithelial bioassay: healthy skin, n = 8; class 4, n = 23; class 5, n = 13; and class 6, n = 10. The location of TGF-beta(1) was determined at the light and electron microscopy level with immunocytochemistry and immunogold (IMG) labeling. Multiple comparisons were analyzed with a one-way analysis of variance and the Student-Newman-Keuls post hoc tests. The LC and LT comparisons were analyzed with a two-tailed unpaired t test.

Results: The TGF-beta(1) gene transcripts for control subjects and patients in classes 4, 5, and 6 were 7.02 +/- 7.33, 43.33 +/- 9.0, 16.13 +/- 7.67, and 7.22 +/- 0.56 x 10(-14) mol/microg total RNA, respectively. The transcripts were significantly elevated in class 4 patients only (P </=.05). The amount of active TGF-beta(1) in picograms/gram of tissue from LC and LT biopsy specimens as compared with healthy skin biopsy specimens were as follows: healthy skin, <1. 0 pc/g; class 4: LC, 5061 +/- 1827 pc/g; LT, 317.3 +/- 277 pc/g; class 5: LC, 8327 +/- 3690 pc/g; LT, 193 +/- 164 pc/g; and class 6: LC, 5392 +/- 1800 pc/g; LT, 117 +/- 61 pc/g. Differences between healthy skin and the skin of the patients in classes 4 and 6 were significant (P </=.05 and P </=.01, respectively). Differences between the LC and LT biopsy specimens within each CVI group were also significant: class 4, P </=.003; class 5, P </=.008; and class 6, P </=.02. Immunocytochemistry results of healthy skin showed TGF-beta(1) staining of epidermal basal cells only. CVI dermal biopsy results demonstrated positive staining in epidermal basal cells, fibroblasts, and leukocytes. Many leukocytes had positive staining of intracellular granules, which appeared morphologically similar to mast cells. IMG labeling results demonstrated gold particles in the leukocytes and collagen fibrils of the extracellular matrix.

Conclusion: Our study indicated that activated leukocytes traverse perivascular cuffs and release active TGF-beta(1). Positive TGF-beta(1) staining results of dermal fibroblasts were observed and suggest that fibroblasts are the targets of activated interstitial leukocytes. Increased protein production, despite normal levels of gene transcripts in patients in classes 5 and 6, suggests that alternate mechanisms other than gene transcription regulate protein production. A potential mechanism for quick access and release is storage of TGF-beta(1) in the extracellular matrix. IMG labeling to collagen fibrils support this possibility. Furthermore, TGF-beta(1) was exclusively elevated in areas of clinically active disease, indicating a regionalized response to injury. These data suggest that alterations in tissue remodeling occur in patients with CVI and that dermal tissue fibrosis in CVI is regulated by TGF-beta(1).