transforming-growth-factor-beta and Venous-Insufficiency

Topics: Adult; Aged; Aged, 80 and over; Cell Line; Chronic Disease; Endoglin; Female; Glycosaminoglycans; Humans; Male; Middle Aged; Protein Isoforms; Transforming Growth Factor beta; Varicose Ulcer; Venous Insufficiency; Wound Healing

Hepatocyte growth factor (HGF) is a multifunctional cytokine that is involved in recovery process after organ injuries.. We studied HGF and the membrane bound receptor, c-met locally in patients who suffered from chronic leg ulcers (> or =1 year) caused by venous insufficiency.. Skin biopsies from the edge of the ulcers were taken from patients (n=13) and studied by immunohistochemical staining for detection of HGF and c-met. Skin biopsies from healthy volunteers (n=10) were used as the control material. Ulcer secretion from chronic ulcers (n=11) was examined for the presence of HGF by ELISA and the concentration of HGF was compared with acute ulcers in healthy controls (n=10) and in patients operated for a non-invasive breast cancer (n=12).. We observed that c-met expression in the ulcer area increased significantly in chronic ulcers compared to controls (p=0.005). Concentration of ulcer-HGF in the patients with chronic ulcer was significantly higher than acute ulcers (p<0.01). The biological activity of HGF in ulcer secret was assessed in-vitro in transferred, mouse skin epithelial cell monolayer. Enhanced migration and morphologic changes were seen after adding ulcer secret from acute ulcers (> 1 ng/mL) that was inhibited by anti-HGF antibodies. No biological activity was observed by adding ulcer secret from chronic ulcers irrespective HGF concentration.. We conclude that in chronic skin ulcers decreased biological activity of endogenous HGF and overexpression of c-met is seen which might explain fibrosis and delayed recovery. Administration of exogenous active HGF might contribute to accelerated healing in these patients.

Topics: Adult; Animals; Biopsy; Blotting, Western; Breast Neoplasms; Case-Control Studies; Cell Line; Cell Movement; Cytokines; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Leg Ulcer; Male; Mice; Proto-Oncogene Proteins c-met; Skin; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ulcer; Venous Insufficiency; Wound Healing

Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI.. One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC).. Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC.. MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.

Topics: Blotting, Western; Female; Gene Expression; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Venous Insufficiency

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.. 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.. 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

Topics: Animals; Cell Line; Collagen; Dermis; Disease Progression; Extracellular Matrix Proteins; Fibrosis; Gene Expression; Humans; Immunohistochemistry; Mink; Muscle, Smooth, Vascular; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transforming Growth Factor beta; Venous Insufficiency

Ultrastructural assessments of the dermal microcirculation in patients with chronic venous insufficiency have been limited to qualitative morphologic descriptions of venous ulcer edges or venous stasis dermatitis. The purpose of this investigation was to quantify differences in endothelial cell structure and local cell type with emphasis on leukocytes and their relationship to arterioles, capillaries, and postcapillary venules (PCVs).. Two 4.0 mm punch biopsies were obtained from areas of dermal stasis skin changes in the gaiter region of the leg, as well as from noninvolved areas of skin in the ipsilateral thigh, from 35 patients: CEAP class 4 (11 patients), class 5 (9 patients), class 6 (10 patients), and five normal skin biopsies from patients without chronic venous insufficiency. Electron microscopy was performed on sections at 6700x and 23,800x magnification. At 6700x endothelial cell thickness was determined, and the number of fibroblasts, leukocytes, and mast cells were recorded relative to their proximity to arterioles, capillaries, and PCVs. Similarly, at 23,800x endothelial cell vesicle density, interendothelial junctional widths, and basal lamina thickness (cuff width) were measured. Preliminary evaluation for the presence of transforming growth factor-beta 1 (TGF-beta 1) was performed on three patients using reverse transcriptase-polymerase chain reaction (RT-PCR).. Quantitative measurements demonstrated increased mast cell content for class 4 and 5 patients around arterioles and PCVs and increased macrophage numbers for class 6 patients around PCVs (p < 0.05). Fibroblasts were the most common cells observed; however, no differences were demonstrated between groups. No differences were observed in interendothelial junctional widths or vesicle densities in arterioles, capillaries, or PCVs. Basal lamina thickness was increased only at the capillary level (p < 0.05). The results of RT-PCR for TGF-beta 1 messenger RNA were positive in the three patients studied.. Our data suggest that (1) mast cells play a role in the pathogenesis of chronic venous insufficiency; (2) the effects of mast cells, macrophages, or both may be mediated in part by TGF-beta 1; and (3) capillary cuff formation is not associated with widened interendothelial gap junctions, but may be a result of enhanced vesicular transport rate or conformational changes in the interendothelial glycocalyx.

Topics: Aged; Arterioles; Biopsy, Needle; Cell Count; Chronic Disease; Cytokines; Endothelium, Vascular; Fibroblasts; Humans; Leg; Leukocytes; Macrophages; Male; Mast Cells; Microcirculation; Middle Aged; Polymerase Chain Reaction; Skin; Transforming Growth Factor beta; Venous Insufficiency; Venules

Treatment of leg ulcers should consider two aspects, i.e. the exact underlying condition (main cause and contributing factors) and local conditions. Compression therapy remains the corner-stone of the therapeutic concept. A compression of 35 mmHg at the distal calf improves insufficient venous function. A systolic ankle pressure of < or = 80% of blood pressure (ankle-arm-index < or = 0.8) requires reduction of compression therapy. At an ankle pressure below 80 mmHg compression should not be used. If superficial reflux is the major cause of chronic venous insufficiency, vein stripping should be considered. Contributing diseases like heart insufficiency, anemia or diabetes may require general medical care. Local contributing factors like reduced mobility of the ankle joint and lymphostasis may require physical therapy, and calcification of the wound bed should be excised. Local treatment considers ulcer bed and border. The ulcer bed needs debridement and moist wound care. Infection is treated with systemic antibiotics, according to the antibiogram. Tetanus immunization is required for all leg ulcer patients. Some centers report good results with endoscopic subfascial decision of perforator veins, paratibial fasciotomy and excision of fibrous tissue. Local application of recombined growth factors is currently under clinical evaluation. Adjuvant pharmacotherapy plays a minor role in the treatment of venous leg ulcers. An efficient treatment of the underlying cause combined with optimal wound care are the key to therapeutic success.

Topics: Anti-Bacterial Agents; Bandages; Debridement; Humans; Leg Ulcer; Platelet-Derived Growth Factor; Practice Guidelines as Topic; Pressure; Tetanus Toxoid; Transforming Growth Factor beta; Venous Insufficiency; Wound Infection