Vincent Falanga

The "trap" hypothesis of venous ulceration

The pathogenesis of venous ulceration is unknown. We propose that macromolecules leaking into the dermis as a result of venous hypertension bind to or trap growth factors and matrix material, which then become unavailable for tissue repair and for the maintenance of tissue integrity.

Inhibition of cell proliferation by chronic wound fluid

It has been proposed that occlusive wound dressings may enhance chronic wound repair by the stimulatory action of the fluid accumulating beneath the dressings. In this report, we investigated the in vitro proliferative effects of chronic wound fluid obtained from under a polyurethane membrane applied for 24 hours to venous ulcers in the ambulatory setting. By measuring cell counts and DNA synthesis, we found that chronic wound fluid inhibited the proliferation of human dermal fibroblasts (p = 0.008) and failed to stimulate the proliferation of microvascular endothelial cells (p = 0.03) and keratinocytes (p = 0.03). The inhibitory activity of chronic wound fluid on fibroblast proliferation was blocked after the fluid was heated to 100° C, but not 56° C, and was mainly restricted to a fraction of chronic wound fluid enriched in components less than 30 kd in molecular weight (p = 0.028). At concentrations ranging from 1% to 4% and in the presence of serum, chronic wound fluid decreased the viability of fibroblasts, as shown by a decreased ability of the cells to exclude trypan blue (p = 0.02), and the viability of endothelial cells, as shown by an increased release of tritiated adenine (p = 0.03). We conclude that the wound fluid obtained from beneath occlusive dressings applied to chronic wounds inhibits cell proliferation.

Human wound fluid from acute wounds stimulates fibroblast and endothelial cell growth

One proposed mechanism for the beneficial effect of occlusive dressings on healing is the maintenance of contact between the wound bed and accumulated wound fluid, which is thought to contain growth stimulatory substances. We have examined the effect of human wound fluid on the in vitro growth of human dermal fibroblasts and umbilical vein endothelial cells. Acute wound fluid was collected from six patients undergoing split-thickness skin grafting. The acute wound fluid was sterilely collected daily from underneath a vapor-permeable membrane applied to the donor site and changed every 24 hours for 3 days postoperatively. After seeding in optimal growth media (control) on day 0, cultures of human dermal fibroblasts and umbilical vein endothelial cells were supplemented with or without acute wound fluid on the next day (day 1) and on day 3. As determined by cell counts, 2% acute wound fluid stimulated the growth of human dermal fibroblasts (p less than 0.05) and umbilical vein endothelial cells (p less than 0.01) when these cells were cultured in 2% fetal bovine serum and endothelial growth medium, respectively. Wound fluid from postoperative days 1 or 3 caused the same level of stimulation. The addition of an anti-platelet-derived growth factor antibody to wound fluid resulted in a 45% mean reduction in its stimulatory effect on fibroblast growth (p less than 0.02), suggesting that platelet-derived growth factor contributes to the observed effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue engineering and the development of Apligraf, a human skin equivalent.

In recent years, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This review details the pioneering work of numerous investigators that led to the following precursors of tissue-engineered skin replacement: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts; autologous/allogeneic composites, acellular collagen matrices, and cellular matrices. It also discusses the rationale for the development of the newer products and describes the technical advances leading to the development of Apligraf, a tissue-engineered human skin product.

Linear Scleroderma: Clinical Spectrum, Prognosis, and Laboratory Abnormalities

The clinical features and natural history of linear scleroderma in 53 patients and the laboratory tests helpful in the management of this disease are described. No patient had Raynauds phenomenon or signs of systemic connective tissue disease in a mean follow-up of 10 years. Blood eosinophilia (greater than 300 cells/mm3) was present in half the patients, usually those with clinically active disease rather than inactive disease (p less than 0.02). An elevated serum IgG level correlated with the presence of joint contractures (p less than 0.02). Antinuclear antibodies, commoner in patients with extensive and prolonged disease, were present in 31% and 46% of patients whose sera were tested on mouse kidney and HEp-2 cells, respectively. Antibodies to single-stranded DNA, present in 50% of patients, were associated with extensive disease, joint contractures (p less than 0.001), and active disease of greater than 2 years duration (p less than 0.001). Discordance in immune reactivity indicates that at least three serum autoantibodies exist in these patients: antibodies to single-stranded DNA and antinuclear antibodies with homogeneous and nucleolar immunofluorescence patterns.

Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-β 11

Failure to reepithelialize is the major clinical problem in venous ulcers. It is not clear whether the problem resides with keratinocytes or with inadequate and improper formation of extracellular matrix. In this study, we characterized the biosynthetic activity and response to transforming growth factor-beta 1 (TGF-beta) of dermal fibroblast cultures isolated from biopsies of venous ulcers and from normal thigh skin (controls) of seven patients. We found that baseline 3H-proline incorporation was similar in fibroblasts from venous ulcers and control skin (p= 0.1716). No difference was detected by ELISA between ulcer and control fibroblasts in the synthesis of total TGF-beta (p = 0.2309), and the mRNA levels for alpha 1(I) procollagen and TGF-beta were comparable in both groups. However, TGF-beta (0.1-5 ng/ml) enhanced collagen protein synthesis by more than 60% and in a dose-dependent manner (r = 0.997) in control fibroblast cultures, while failing to stimulate collagen production by venous ulcer fibroblasts (p = 0.0001). This unresponsiveness to TGF-beta was associated with up to a fourfold decrease in TGF-beta Type II receptors. We conclude that fibroblasts from the edge of non-healing venous ulcers are unresponsive to the action of TGF-beta, and that this blunted response may cause faulty deposition of the extracellular matrix needed for reepithelialization and wound healing.

Extravasation of macromolecules and possible trapping of transforming growth factor‐β in venous ulceration

The pathogenesis of venous ulceration is thought to involve formation of pericapillary fibrin cuffs as a result of venous hypertension, and a recent hypothesis suggests that extravasated plasma proteins may bind or trap growth factors. We have compared the tissue distribution of fibrin cuffs, plasma proteins, procollagen, and transforming growth factors (TGF‐β1 and TGF‐β2) within venous ulcers and normally healing graft donor sites. In venous ulcers, the papillary dermis and the ulcer bed contained convoluted capillaries with phosphotungstic acid haematoxylin‐positive pericapillary fibrin cuffs. By immunohistochemical staining, the cuffs were positive for actin, and contained massively redundant lamellae of basement membrane material which stained positive for type IV collagen. Extravasated factor XHIa and α2‐macroglobulin were present within the fibrin cuffs. increased numbers of type I procollagen positive fibroblasts, and increased TGF‐β1 immunoreactiv‐ity were present within the fibrin cuffs, but not in the provisional matrix in the ulcer bed around the cuffs. In contrast, in normally healing graft donor sites, tortuous capillaries and fibrin cuffs were absent, factor XHIa and α1‐macroglobulin were restricted to the lumina of vessels, and procollagen and TGF‐/V immunoreactivity were present within the granulation tissue and adjacent dermal matrix at the wound margin. These observations suggest that growth factors critical in wound healing, such as TGF‐β, are present within venous ulcers, but are abnormally distributed. Their distribution within fibrin cuffs and co‐localization with extravasated plasma proteins, particularly α2‐macro‐globulin, which is a recognized scavenger molecule for TGF‐β and other growth factors, provides evidence for a possible ‘trapping’ of growth factors in venous ulcers.

Growth Factors and Chronic Wounds: The Need to Understand the Microenvironment

The care of chronic wounds has become a major health issue in developed countries because of their increasingly elderly populations. There is hope that progress made in understanding and producing growth factors will lead to their successful use to induce faster and better healing of chronic wounds. This report will discuss growth factors in the context of their use in chronic wounds, and will focus on the importance of the wound microenvironment in determining the interactions between growth factors and wounds. We believe that a greater understanding of the chronic wound microenvironment will be of benefit in the optimal use of growth factors. In published studies, we have found that wound fluid taken from acute wounds stimulates fibroblast and endothelial cell proliferation, whereas fluid obtained from chronic non‐healing wounds inhibits the growth of fibroblasts, endothelial cells, and keratinocytes. In this report, we describe the effect of these two types of wound fluid on the synthesis of extracellular matrix components. We hypothesize that the chronic wound microenvironment is generally non‐conducive to cell growth, and that this may prevent a truly successful use of topical growth factors in chronic wounds. Novel approaches in the delivery of growth factors to wounds may be necessary to overcome these obstacles.

Gene Expression in Low Oxygen Tension

Low oxygen tension is a feature of many physiologic and pathologic conditions, including wound healing, fibrosis, and neoplasia. Increasing evidence suggests that low oxygen tension induces the transcription of a number of genes, and that this process depends on the cellular context. The proteins synthesized from these genes enable cells to adapt to the hypoxic environment and/or to fulfill their functional roles. The regulatory regions responsible for the induction of erythropoietin gene transcription and synthesis in response to hypoxia/anemia appear to be cis-acting deoxyribonucleic acid sequences located within the 5 and 3 flanking regions of the erythropoietin gene. Other proteins induced by hypoxia include cytokines (platelet-derived growth factor-beta chain, endothelin-1, transforming growth factor-beta), enzymes (tyrosine hydroxylase, glycolytic enzymes), and stress proteins. The molecular mechanisms of the hypoxia-induced expression of these genes are poorly understood. A heme protein may act as the oxygen tension sensor, or the redox state of certain nuclear transcription factors may function as second messengers.

Frequency, levels, and significance of blood eosinophilia in systemic sclerosis, localized scleroderma, and eosinophilic fasciitis

Blood eosinophilia is a common feature of eosinophilic fasciitis and is variably reported in systemic sclerosis and localized scleroderma. Since these diseases share cutaneous fibrosis as the final outcome and have other clinical and pathologic features that are difficult to differentiate, the presence of blood eosinophilia may be a further source of confusion. In this study, we examined the frequency and level of blood eosinophilia in 715 patients with systemic sclerosis, 72 patients with localized scleroderma, and 22 patients with clinically active eosinophilic fasciitis. When defined as greater than 400 cells/mm3, eosinophilia was present in 7% of patients with systemic sclerosis, 31% of patients with localized scleroderma, and 83% of patients with eosinophilic fasciitis. Greater than 1000 eosinophils/mm3 were present less frequently in systemic sclerosis (1%) and localized scleroderma (8%) than in eosinophilic fasciitis (61%). No difference in the frequency of eosinophilia was present in patients with the limited cutaneous CREST syndrome or the diffuse cutaneous variety of systemic sclerosis, and in these patients the presence of eosinophilia did not correlate with the extent of cutaneous or internal organ involvement or with other laboratory abnormalities. Among patients with localized scleroderma, eosinophilia was more common in those with linear scleroderma and generalized morphea than in those with morphea, and both the frequency and level of eosinophilia were greater in individuals with clinically active disease (p less than 0.02). Eosinophilia was a persistent feature in untreated patients with active eosinophilic fasciitis, even up to 30 months of disease duration.