Localization of platelet-derived growth factor receptor subunit expression in chronic venous leg ulcers

Cellular responses to platelet-derived growth factor, which affects all phases of the wound healing process, are dependent on the interaction of the growth factor with its cell surface receptors. Recently, we have shown that the platelet-derived growth factor-receptor was not expressed in uninjured human skin. In acute human wounds healing by secondary intention, both platelet-derived growth factor-receptor subunits were coordinately expressed, whereas no expression was found after reepithelialization at day 47. Even though impaired wound healing may be due to uncoordinated expression or the failure to express platelet-derived growth factor-receptor subunits, little is known regarding their expression in chronic ulcers. We studied the localization of platelet-derived growth factor-receptor expression in chronic venous leg ulcers of 15 patients with a median age of 73 years. Cryostat sections of biopsy specimens were immunostained with the use of antibodies against the alpha- and the beta-platelet-derived growth factor subunits. RNA was extracted from biopsy specimens and subjected to Northern blot analysis with the use of oligolabeled complementary DNA for the platelet-derived growth factor-receptor. Platelet-derived growth factor-receptor alpha- and beta-subunit expression was found in fibroblast-like cells within the wound bed and in cells beneath the epidermis of the wound edge. Platelet-derived growth factor-receptor beta-subunit expression was detected in endothelial cells of the vessels, in the granulation tissue, and the wound edge, whereas platelet-derived growth factor-receptor alpha-subunit was not expressed in endothelial cells of the uninjured skin. This finding suggests that the platelet-derived growth factor alpha-subunit may be involved in vessel formation during tissue repair. Both platelet-derived growth factor-receptor subunits were expressed at the messenger RNA level indicating that the synthesis is at least partly regulated at a pretranslational level. As the cellular responsiveness to growth factors depends on their specific receptors, our finding that both platelet-derived growth factor-receptor subunits are expressed in chronic venous ulcers substantiates the concept of therapeutic trials with recombinant platelet-derived growth factor.     

Prognostic role of factor XIII gene variants in nonhealing venous leg ulcers

OBJECTIVE: Many factors impair healing of chronic venous ulcer (CVU), and many theories have been proposed to explain their pathogenesis. Coagulation factor XIII (FXIII) influences tissue regeneration and angiogenesis with effects on wound healing. Because FXIII properties depend upon its genetic variants, we investigated whether intragene polymorphisms may have modulating effects on the CVU area. METHODS: The study included 121 patients with nonhealing CVUs (CEAP clinical class C6) that included 67% with primary chronic venous disease (CVD), 26% with post-thrombotic ulcers, and 7% with mixed ulcer origin. Polymerase chain reaction was used to genotype them for Val34Leu, Pro564Leu, and Tyr204Phe variants in the FXIII-A subunit gene and for His95Arg variant in the FXIII-B subunit gene. The same variants were analyzed in 102 controls, healthy subjects who were case-matched by age and gender. RESULTS: Genotype distribution for all polymorphisms investigated was not significantly different between cases and controls. Conversely, our CVU cases had a mean ulcer area inversely related with the presence of both Leu34 and Leu564 alleles (ValVal, 12.3 +/- 22.4 cm2 vs LeuLeu, 3.9 +/- 2.6 cm2, P = .002; ProPro, 10.2 +/- 21.2 cm2 vs LeuLeu, 2.9 +/- 1.4 cm2, P = .002). In combined analysis, those cases who were wild-type for both variants (ValVal34/ProPro564) had a further increase in mean ulcer size compared with cases carrying both variants (Leu34/Leu564) (13.3 +/- 27.1 cm2 vs 5.2 +/- 5.6 cm2; P = .034). CONCLUSIONS: No correlation exists between FXIII genotypes and the prevalence of chronic venous ulcers, thus demonstrating that FXIII polymorphisms have no role in ulcer development. In contrast, FXIII-gene variants, in particular the non-wild-type alleles Leu34 and Leu564, were associated with a smaller venous ulcer surface and might have favorable effects on reparative processes.     

Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers

The purpose of this study was to determine the biochemical composition of fluid taken from chronic wounds, to compare these values with that of serum, and therefore to assess whether the wound fluid is representative of the extracellular environment of the wound. Paired wound fluid and blood samples were collected from eight patients with chronic leg ulcers in a nonhealing and healing phase. Wound fluid and serum samples were screened for a profile of general biochemical analyses, including electrolytes, lactate, glucose, and protein analyses. Electrolyte levels were essentially identical in wound fluid and serum samples. Lactate and lactate dehydrogenase levels were significantly greater and glucose and bicarbonate levels were significantly lower in wound fluid when compared with the paired serum samples. Albumin and total protein levels in wound fluid were on average half those of serum levels. In this small sample of eight patients, wound fluid collected from chronic leg ulcers is an exudate with the biochemical composition expected in extracellular fluid. In addition, bicarbonate and glucose levels increase and C-reactive protein levels decrease in wound fluid, but remain unchanged in serum, during healing. These results suggest changes in the state of hypoxia and the inflammatory process in the healing wound.     

Management of mixed arterial and venous leg ulcers

BACKGROUND: The aim was to assess healing in patients with mixed arterial and venous leg ulcers after protocol-driven treatment in a specialist leg ulcer clinic. METHODS: The study included consecutive patients referred with leg ulceration and venous reflux over 6 years. Legs without arterial disease (ankle : brachial pressure index (ABPI) above 0.85) were treated with multilayer compression bandaging and patients with severe disease (ABPI 0.5 or less) were considered for immediate revascularization. Those with moderate arterial compromise (ABPI above 0.5 up to 0.85) were initially managed with supervised modified compression and considered for revascularization if their ulcer did not heal. Healing rates were determined using life-table analysis. RESULTS: Of 2011 ulcerated legs, 1416 (70.4 per cent) had venous reflux. Of these 1416, 193 (13.6 per cent) had moderate and 31 (2.2 per cent) had severe arterial disease. Healing rates by 36 weeks were 87, 68 and 53 per cent for legs with insignificant, moderate and severe arterial disease respectively (P < 0.001). Seventeen legs with moderate and 15 with severe arterial disease were revascularized. Of these, ulcers healed in four legs with moderate and seven with severe disease within 36 weeks of revascularization (P = 0.270). Combined 30-day mortality for revascularization was 6.5 per cent. CONCLUSION: A protocol including supervised modified compression and selective revascularization achieved good healing rates for mixed arterial and venous leg ulceration.     

Randomized, double-blind, placebo-controlled, dose- ranging study of granulocyte-macrophage colony stimulating factor in patients with chronic venous leg ulcers

Chronic venous leg ulcers are a common ailment with no ideal treatment. Recent reports have shown granulocyte- macrophage colony stimulating factor to be of use in the healing of these chronic wounds. Therefore, we conducted a double-blind, randomized, placebo-controlled study which enrolled 60 patients with chronic venous leg ulcers, whom we treated with placebo or with 200 or 400 microg of granulocyte-macrophage colony stimulating factor by perilesional injections of the drug in four weekly treatment episodes. Observations were conducted at each treatment visit, at weeks 5, 9, 13, and six months after the inclusion in the protocol. The number of healed wounds in the placebo and the treated arms were significantly different (p = 0.05), with 4 of 21 (19%) in the first group having healed at week 13, as compared to 12 of 21 (57%) and 11 of 18 (61%), in the 200 microg and the 400 microg groups, respectively. There were only minor side-effects attributable to the treatment, and the reobservation at 6 months showed that none of the treated ulcers recurred during that period. We conclude that granulocyte-macrophage colony stimulating factor injected perilesionally may be a useful drug for the treatment of chronic venous leg ulcers.     

Expression of apoptosis- and cell cycle-related proteins in epidermis of venous leg and diabetic foot ulcers

BACKGROUND: Epithelialization of cutaneous ulcers is a long-lasting process. To study the pathomechanism of impaired epithelialization, we evaluated the role of cell cycle- and apoptosis-related proteins in the regenerating epidermis. We characterized immunohistochemically the expression of cell cycle regulators p63, CD29, PCNA, p53, pro- and antiapoptotic proteins bcl2, bax, caspase 3 and DNA breaks, as well as keratin 10, 16 and 17. METHODS: Studies were carried out in 12 patients with diabetic foot, and 10 patients with varicose ulcers of the calf. Skin biopsy specimens were obtained from the border area of ulcers and the topographically corresponding sites of normal skin of patients undergoing orthopedic surgery. Biopsy specimens were stained by use of specific primary antibodies, a kit based on biotin-avidin-peroxidase complex technique, and DAB substrate. Results were expressed as a mean staining intensity. RESULTS: At the edge of both types of ulcers, keratinocytes were p63+, CD29+, PCNA+ and p53-. The mean intensity of p63 and CD29 staining was slightly higher than in controls. The intensity of bcl2 staining was higher at the edge of diabetic ulcers compared with venous ulcers, whereas the intensity of bax staining was similar. The expression of caspase 3 was lower at the edge of venous ulcers and higher in diabetic ulcers and the intensity of TUNEL staining was lower at the edge of both types of ulcers compared with controls. Keratinocytes at the edge and distally to both types of ulcers expressed cytokeratin 16 and 17. There was no expression of cytokeratin 10 at the edge of ulcers. Together, there was a slight tendency for higher expression of cell cycle-related proteins in venous and of apoptosis-related proteins in diabetic ulcers epidermis; however, the differences were minor. CONCLUSIONS: The impaired epithelialization of chronic leg ulcers is not caused by an inadequate epidermal stem cell proliferation, differentiation, or apoptosis. It may rather reflect the distorted organization of wound bed, caused by infection and impaired nutrition supply, altering keratinocyte migration. To accelerate healing of an ulcer, modeling of the granulation tissue by regulatory cytokines but not stimulation of keratinocyte growth seems to be indicated.     

Economic analysis of VenUS I, a randomized trial of two bandages for treating venous leg ulcers

BACKGROUND: The study investigated the cost-effectiveness of four-layer and short-stretch compression bandages for treating venous leg ulcers. METHODS: Cost-effectiveness and cost-utility analyses were performed using patient-level data collected alongside the VenUS I leg ulcer study. The perspective for the economic analysis was that of the UK National Health Service (NHS) and Personal Social Service. The time horizon for the analysis was 1 year after recruitment. Health benefit was measured as differences in ulcer-free days and quality-adjusted life years (QALYs). RESULTS: The mean healing time for ulcers treated with four-layer bandages was 10.9 (95 per cent confidence interval (c.i.) -6.8 to 29.1) days less than that for ulcers treated with short-stretch bandages. Mean average difference in QALYs between compression systems was -0.02 (95 per cent c.i. -0.08 to 0.04). The four-layer bandage cost a mean of pound 227.32 (95 per cent c.i. pound 16.53 to pound 448 .30) less per patient per year than the short-stretch bandage. CONCLUSION: On average, four-layer bandaging was associated with greater health benefits and lower costs than short-stretch bandaging.     

Epidermal growth factor receptor gene analysis in renal cell carcinoma

The epidermal growth factor receptor binds the mitogens epidermal growth factor and transforming growth factor-alpha. Increased expression of the epidermal growth factor receptor has been noted in many types of tumors and is associated with gene amplification in several including epidermoid carcinoma, lung carcinoma, breast carcinoma and glioblastoma. We have recently observed increased expression of the epidermal growth factor receptor messenger RNA in neoplastic tissue relative to normal kidney tissue from patients with renal cell carcinoma. To determine if epidermal growth factor receptor gene amplification was present in renal cell carcinoma, DNA was extracted from renal cell carcinoma cell lines and from normal kidney and renal cell carcinoma tissues derived from radical nephrectomy specimens from thirty patients. DNA was analyzed by Southern blot hybridization. There was no epidermal growth factor receptor gene amplification detected in the renal cell carcinoma samples studied, indicating the increased epidermal growth factor gene expression observed in renal cell carcinoma does not occur through gene amplification. Unlike other tumors with enhanced epidermal growth factor receptor gene expression, amplification of this gene does not appear to be a common feature of renal cell carcinoma.