Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-β1 gene expression and protein.

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Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-β1 gene expression and protein production Peter J. Pappas, MD, Raul You, MD, PhD, Pranela Rameshwar, PhD, Rhaguram Gorti, MD, David O. DeFouw, PhD, Courtney K. Phillips, MD, Frank T. Padberg, MD, Michael B. Silva, MD, Gregory T. Simonian, MD, Robert W. Hobson, MD, Walter N. Durán, PhD Journal of Vascular Surgery Volume 30, Issue 6, Pages 1129-1145 (December 1999) DOI: 10.1016/S0741-5214(99)70054-6 Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Quantitative reverse transcriptase–polymerase chain reaction for transforming growth factor–β1 (TGF-β 1) . The target, endogenous TGF-β1 messenger RNA, is represented by complementary DNA bands at 161 base pairs (bp) . A 131-bp complementary DNA, developed by removing 18% bp from endogenous TGF-β1 bp sequence, served as internal standard for TGF-β1. Figure shows experimental approach in which target TGF-β1 was loaded at constant amount and compared with decreasing amount of internal standard. Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Molecular ratio of target to internal standard (mimic) as function of initial amount of internal standard. Regression equation allows determination of initial amount of target transforming growth factor–β1 messenger RNA at point where log of molecular ratio of target to internal standard is zero. Note excellent correlation coefficient. Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Bar graph shows differences in transforming growth factor–β1 (TGF-β 1) messemger RNA levels in skin biopsy specimens of healthy control subjects and patients with chronic venous insufficiency (CVI) according to disease classification. Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Bar graph shows differences in active transforming growth factor–β1 (TGF-β 1) protein concentration between and within chronic venous insufficiency groups as compared with control subjects. CON, Control; C4, class 4; LC, lower calf; LT, lower thigh. Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Immunohistochemistry. A, Positive transforming growth factor–β1 (TGF-β1) reaction is shown by dark brown staining in pig spleen. Leukocytes and trabeculae stain positively for TGF-β1. Pig spleen is used as a positive control because it is tissue rich in TGF-β1 (576×). B1, Negative control. Vertical arrow indicates basal epidermal layer. Horizontal arrow points to a perivascular cuff (400×). B2, Positive control demonstrating mild TGF-β1 staining of epidermal basal layer. Note absence of perivascular cuffs and leukocytes and lower density of fibroblasts (400×). C, Lower thigh biopsy specimen from patient with class 4 chronic venous insufficiency. This section illustrates presence of perivascular cuffs and pronounced positive staining for TGF-β1 in basal layer of epidermis (368×). D, Lower calf biopsy specimen shows TGF-β1 staining in basal layer of epidermis, fibroblasts, and perivascular leukocytes. Perivascular cuff appears to be free of TGF-β1 staining. However, neighboring leukocytes are stained. Box is magnified in F (295×). E1, Spindle-shaped cells stain positively for TGF-β1 in patient in class 4. These cells appear to be fibroblasts (arrows) and are surrounded by collagen (575×). E2, Spindle-shaped cells seen in E1 stain positively for vimentin. Red staining shows presence of vimentin in these cells and confirms they are fibroblasts (arrows indicate positive staining in fibroblasts). Blue stain is nuclear Mayer’s hematoxylin counterstain (400×). Magnification of box in D shows TGF-β1 staining of leukocytes near perivascular cuff (575×). G, TGF-β1 staining of leukocytes traversing perivascular cuff (arrows; 750×). Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 A, Clusters of immunogold-labeled particles attached to intracapillary leukocyte (34,800×). B, Gold-labeled particle attached to collagen fibril in extracellular matrix (arrows ; 69,600×). Journal of Vascular Surgery 1999 30, 1129-1145DOI: (10.1016/S0741-5214(99)70054-6) Copyright © 1999 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions