Targeting PI3Kδ function for amelioration of murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.

Blockade of p38 Mitogen-Activated Protein Kinase Inhibits Murine Sclerodermatous Chronic Graft-versus-Host Disease.

Bone marrow transplantation (BMT) of B10.D2 mice into sublethally irradiated BALB/c mice across minor histocompatibility loci is a well-established animal model for human sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) and systemic sclerosis (SSc). The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammation and cytokine production. Furthermore, the activation of p38 MAPK plays an important role in collagen production in SSc. We investigated the effects of p38 MAPK inhibitor, VX-702, on Scl-cGVHD mice. VX-702 was orally administered to Scl-cGVHD mice from day 7 to 35 after BMT. We compared skin fibrosis of Scl-cGVHD mice between the VX-702-treated group and control group. Allogeneic BMT increased the phosphorylation of p38 MAPK in the skin. The administration of VX-702 attenuated the skin fibrosis of Scl-cGVHD compared to the control mice. Immunohistochemical staining showed that VX-702 suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the dermis of Scl-cGVHD mice compared to the control mice. VX-702 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines, such as IL-6 and IL-13, in the skin of Scl-cGVHD mice. In addition, VX-702 directly inhibited collagen production from fibroblasts in vitro. VX-702 was shown to be a promising candidate for use in treating patients with Scl-cGVHD and SSc.

Nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis.

OBJECTIVES: Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis. METHODS: Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid). RESULTS: In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT. CONCLUSIONS: The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.

Matrix metalloproteinases in venous tissue remodeling and varicose vein formation.

Matrix metalloproteinases (MMPs) play a major role in extracellular matrix (ECM) turnover under both physiological and pathological conditions. Studies on venous tissues from experimental animals and humans identified several MMP subtypes, and showed significant changes in the expression and activity of specific MMPs during vein wall remodeling. Also, significant research has focused on the role of MMPs in chronic venous disease (CVD) and varicose vein formation in the lower extremities and their progression to thrombophlebitis and venous leg ulcer. Several hypotheses have been forwarded regarding the pathophysiological mechanisms underlying the relation between MMPs and the formation, progression and complications of varicose veins. The effects of MMPs on ECM degradation could result in significant venous tissue remodeling and degenerative and structural changes in the vein wall, leading to venous dilation and valve dysfunction. MMPs may also induce early changes in the endothelium and venous smooth muscle function in the absence of significant ECM degradation or structural changes in the vein wall. In addition, evidence suggests increased activity of MMPs in the advanced stages of chronic venous insufficiency (CVI) associated with skin changes and leg ulceration as well as in the wound fluid environment. Several pharmacological therapies and surgical strategies are being utilized in the management of varicose veins, with variable success and recurrence rates. Inhibition of MMPs may represent a novel therapeutic intervention to limit the progression of varicose veins to CVI and leg ulceration.

Role of cytokines and proteases in murine scleroderma.

Scleroderma is a fibrotic condition characterized by immunological abnormalities, vascular injury and increased accumulation of extracellular matrix proteins in the skin. Although the etiology of scleroderma has not been fully elucidated, a growing body of evidence suggests that the overproduction of extracellular matrix proteins by activated fibroblasts results from an imbalance between synthesis and degradation of connective tissues. A number of mediators, cytokines, chemokines and growth factors secreted by inflammatory cells and mesenchymal cells (fibroblasts and myofibroblasts) play an important role in the fibrotic process of scleroderma. In this article, we describe recent advances concerning immunological aspects in the pathogenesis of bleomycin-induced murine scleroderma, laying stress on the involvement of interleukin-13 (IL-13) and plasminogen activator inhibitor-1 (PAI-1).

Clinical significance of serum matrix metalloproteinase-13 levels in patients with localized scleroderma.

OBJECTIVES: To investigate the clinical significance of serum matrix metalloproteinase-13 (MMP-13) levels in patients with localized scleroderma (LSc). METHODS: Serum MMP-13 levels were determined by using a peptide substrate cleavage assay in 10 patients with generalized morphea, 10 with linear scleroderma, 10 with morphea, and 10 normal controls. RESULTS: The serum MMP-13 levels in patients with LSc were lower than those in normal controls, but there was no significant difference (64.9 +/- 19.9 versus 73.2 +/- 11.5, p = 0.058). Serum MMP-13 levels in patients with generalized morphea were significantly lower than those in normal controls (54.0 +/- 18.7 versus 73.2 +/- 11.5 ng/ml; p < 0.01). Serum levels of MMP-13 were comparable among normal controls, the patients with linear scleroderma, and those with morphea. The prevalence of muscle involvement was significantly greater in the LSc patients with decreased MMP-13 levels compared with those with normal MMP-13 levels (50% versus 8%, p < 0.05). Serum MMP-13 levels were significantly inversely correlated with the number of linear lesions (r = 0.366, p < 0.05) and the number of involved body areas (r = 0.552, p < 0.005) in patients with LSc, while there was no significant correlation between serum MMP-13 levels and the number of plaque lesions. Furthermore, there was significant inverse correlation between serum MMP-13 levels and the number of involved body areas in patients with generalized morphea (r = 0.631, p < 0.05). CONCLUSION: The serum MMP-13 levels may reflect the disease severity in patients with LSc, especially generalized morphea, the severest form of this disorder.

Novel autoantibody to Cu/Zn superoxide dismutase in patients with localized scleroderma.

Abnormal production of reactive oxygen species (ROS) induces tissue damage and superoxide dismutase (SOD) that converts superoxide radicals to hydrogen peroxide functions as defense against ROS. Cu/Zn SOD administration has been shown to be effective for various fibrotic conditions by inhibiting the fibrogenic effects of ROS. We hypothesized that autoimmune background in localized scleroderma induced anti-Cu/Zn SOD autoantibodies that inhibited SOD activity and thereby contributed to fibrosis by increasing ROS. ELISA using human purified Cu/Zn SOD revealed that IgG or IgM anti-Cu/Zn SOD Ab was detected in the serum of 89% of localized scleroderma patients, especially 100% of patients with generalized morphea, the severest form of localized scleroderma, but was positive only in the serum of less than 15% of patients with other autoimmune disorders, including systemic sclerosis, systemic lupus erythematosus, dermatomyositis, and autoimmune bullous disorders. The immunoblotting analysis confirmed the presence of IgG anti-Cu/Zn SOD Ab in sera from localized scleroderma patients. Remarkably, anti-Cu/Zn SOD autoantibody could inhibit Cu/Zn SOD enzymatic activity. Collectively, these results indicate that anti-Cu/Zn SOD Ab is a novel, major autoantibody in localized scleroderma, and also suggest that the autoantibody may play a role in the development of fibrosis by directly inhibiting SOD activity.

Activation of tissue inhibitor of metalloproteinases-3 (TIMP-3) mRNA expression in scleroderma skin fibroblasts.

Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases-3 (TIMP-3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP-3 mRNA were elevated up to 2.5-fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP-1 mRNA expression was elevated up to 1.8-fold in two and TIMP-2 mRNA expression up to 1.8-fold in two systemic sclerosis strains. Using in situ hybridization, TIMP-3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP-1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP-3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6- and 8.1-fold, respectively) by transforming growth factor-beta, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor-alpha. Specific activation of TIMP-3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP-3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP-3 expression by transforming growth factor-beta.

Lipodermatosclerosis is characterized by elevated expression and activation of matrix metalloproteinases: implications for venous ulcer formation.

Lipodermatosclerosis refers to skin induration of the lower extremities and is associated with patients preceding venous ulcerations. To better understand the pathogenesis of ulcer formation we investigated the expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in lipodermatosclerosis. By preparing biopsies from healthy skin and liposclerotic lesions, MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were analyzed by using reverse transcriptase-polymerase chain reaction, western blot, zymography, hydrolysis of [3H]labeled collagens, and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of MMP-1, MMP-2, and TIMP-1 were significantly increased in lipodermatosclerosis, whereas the total amount of MMP-9 and TIMP-2 mRNA and protein was not altered. Western blot of liposclerotic lesions revealed an inactive proMMP-1-TIMP-1 complex, whereas MMP-2 was prominent as an active 66 kDa band. Increased proteolytic activity of MMP-2 could be proven in lesional in comparison with healthy skin by zymography and [3H] collagen degradation. Increased diffuse staining was found for MMP-1 in the epidermis and dermis in comparison with controls. In lipodermatosclerosis, MMP-2 was predominantly localized in the basal and suprabasal layers of the epidermis, in perivascular regions, and in the reticular part of the dermis. Furthermore, MMP-2 was imbalanced by locally reduced expression of TIMP-2 in the basement membrane zone of lesional skin. Our findings indicate lipodermatosclerosis to be characterized by elevated matrix turnover.

Euthyroid sick syndrome and inhibitory effect of sera on the activity of thyroid 5'-deiodinase in systemic sclerosis.

OBJECTIVE: Our aim was to demonstrate the occurrence of euthyreoid sick syndrome in patients with systemic sclerosis (SSc). Furthermore, the presence of anti-thyroid antibodies and their relationship to thyroid 5'-deiodinase activity was investigated. METHODS: The activity of thyroid 5'-deiodinase was measured by 5' outer ring-deiodination using the sera of patients with SSc (n = 21), undifferentiated connective tissue disease (n = 12), and secondary (n = 19) and primary (n = 11) Raynaud's syndrome (RP). Patients with acute cardiovascular events at the time of the study (n = 16) were investigated as controls. RESULTS: Low FT3 (FT3 < 2.5 pg/ml) was frequently demonstrated in all the patient groups (9/21, 3/12, 10/19 and 8/11, respectively). The high frequency of a FT3/FT4 ratio < 0.2 representing euthyreoid sick syndrome was also often found in SSc (14 cases) and primary (12 cases) and secondary (6 cases) RP patients. Anti-thyroid peroxidase antibody was detected in 17 patients with SSc and in 7, 8 and 3 cases with undifferentiated connective tissue disease, secondary and primary Raynaud's phenomenon, respectively, and in none of the controls. The inhibiting effect of sera on the activity of thyroid 5'-deiodinase was higher in patients with anti-thyroid peroxidase antibodies compared to antibody negative cases (P < 0.01). An inverse correlation was shown between the levels of anti-thyroid peroxidase antibodies and the decreased activity of thyroid 5'-deiodinase (r = -0.6111, P < 0.02) in patients with low FT3. CONCLUSION: The low FT3 or FT3/FT4 ratio observed supports the hypothesis that euthyroid sick syndrome is often present in SSc. Anti-thyroid antibody is also frequently detected in SSc and the positive sera inhibit the activity of thyroid 5'-deiodinase, which can contribute to the low FT3 or FT3/FT4 ratio. Anti-thyroid peroxidase antibodies may play an additive role in the development of low FT3 levels via the inhibiting effect of thyroid 5'-deiodinase. The low FT3 levels may directly influence the already impaired microcirculation in SSc by increasing the systemic vascular resistance.

Matrix metalloproteinases and venous leg ulceration.

Metalloproteinase-mediated proteolysis plays an important role during the phase of venous ulcer formation and wound repair. Venous ulcers manifest as a breakdown of the collagenous stromal tissue and are highly associated to chronic venous insufficiency. A major change in our understanding of the pathogenesis of venous ulcers occurred with the demonstration of extracellular matrix-degrading activity of matrix metalloproteinases to generate a dermal-epidermal skin defect. These proteases were intensely investigated in preceding stages and during wound repair of venous ulcerations. Different studies have revealed their significance in the process of proteolytic remodeling and recognized their potential importance in finding therapeutic rationales to manage late complications of chronic venous ulcers.

Case report. The nonspecific rheumatoid subcutaneous nodule: its presence in fibrositis and scleroderma.

"Rheumatoid" subcutaneous nodules that were at one time considered diagnostic of rheumatoid arthritis or rheumatic fever have also been found in individuals withoug known disease, in patients with systemic lupus erythematosus, and in other conditions. In this report, subcutaneous nodules were described in one patient with fibrositis and in another scleroderma. Multiple pathogenic mechanisms are probably responsible for development of these nodules.

[Serum lysosomal hydrolases in various forms of scleroderma]. / Hydrolases lysosomiques sériques au cours des différentes formes de sclérodermie.

The activities of several lysosomal hydrolases (beta-galactosidase, beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase and acid phosphatase) were determined in serum and blood lymphocytes from patients affected with scleroderma. Statistical comparisons between means of patient and control groups (Student's t test) showed a significant difference in serum beta-galactosidase and acid phosphatase between patients and controls (serum beta-galactosidase: 36.5 +/- 22 nmol/h/ml in the scleroderma group versus 24 +/- 13 nmol/h/ml in the control group; serum acid phosphatase 853 +/- 345 nmol/h/ml in the scleroderma group versus 634 +/- 295 nmol/h/ml in the control group). These differences were significant (p less than 0.01). Both enzyme activities were also significantly increased in the group with systemic scleroderma, but the difference was less in the group with localized scleroderma (this is discussed in terms of statistics and pathophysiology). The other enzyme activities determined were not significantly modified. The validity of these results is discussed, together with their diagnostic value and with the pathophysiological hypotheses put forward to explain the high levels found.

Serum levels of matrix metalloproteinase-13 in patients with eosinophilic fasciitis.

Matrix metalloproteinase-13 (MMP-13), a member of the collagenase family, has been implicated in the pathogenesis of connective tissue diseases characterized by extracellular matrix remodeling. Since serum MMP-13 levels reflect disease severity of systemic sclerosis and localized scleroderma, we evaluated the clinical significance of serum MMP-13 levels in eosinophilic fasciitis (EF). All the EF patients had serum MMP-13 levels lower than the mean - 2SD of healthy controls. Serum MMP-13 levels were also significantly decreased in EF patients compared with diffuse cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, and generalized morphea patients. Although serum MMP-13 levels did not reflect any clinical and serological features of EF, these results indicate that MMP-13 may be involved in the development of this disease.

Matrix metalloproteinases as potential targets in the venous dilation associated with varicose veins.

Varicose veins (VVs) are a common venous disease of the lower extremity characterized by incompetent valves, venous reflux, and dilated and tortuous veins. If untreated, VVs could lead to venous thrombosis, thrombophlebitis and chronic venous leg ulcers. Various genetic, hormonal and environmental factors may lead to structural changes in the vein valves and make them incompetent, leading to venous reflux, increased venous pressure and vein wall dilation. Prolonged increases in venous pressure and vein wall tension are thought to increase the expression/activity of matrix metalloproteinases (MMPs). Members of the MMPs family include collagenases, gelatinases, stromelysins, matrilysins, membrane- type MMPs and others. MMPs are known to degrade various components of the extracellular matrix (ECM). MMPs may also affect the endothelium and vascular smooth muscle, causing changes in the vein relaxation and contraction mechanisms. Endothelial cell injury also triggers leukocyte infiltration, activation and inflammation, which lead to further vein wall damage. The vein wall dilation and valve dysfunction, and the MMP activation and superimposed inflammation and fibrosis would lead to progressive venous dilation and VVs formation. Surgical ablation is an effective treatment for VVs, but may be associated with high recurrence rate, and other less invasive approaches that target the cause of the disease are needed. MMP inhibitors including endogenous tissue inhibitors (TIMPs) and pharmacological inhibitors such as zinc chelators, doxycycline, batimastat and marimastat, have been used as diagnostic and therapeutic tools in cancer, autoimmune and cardiovascular disease. However, MMP inhibitors may have side effects especially on the musculoskeletal system. With the advent of new genetic and pharmacological tools, specific MMP inhibitors with fewer undesirable effects could be useful to retard the progression and prevent the recurrence of VVs.

Role of cathepsin K in the turnover of the dermal extracellular matrix during scar formation.

Cathepsins are a group of cysteine proteinases that are involved in various aspects of extracellular matrix turnover. The collagenolytic activity of cathepsin K plays a pivotal role in bone resorption and lung matrix homeostasis, but so far has not been described in skin. To study the role of cathepsin K in the turnover of the cutaneous extracellular matrix, we studied the expression of cathepsin K in human skin and in cultured primary neonatal skin fibroblasts. Normal skin exhibited only low levels or no expression of cathepsin K. In contrast, dermal fibroblasts in surgical scars showed strong cytoplasmic cathepsin K expression. Cathepsin K expression was most prominent in young scars and declined with time. Cultured neonatal primary fibroblasts showed strong cathepsin K staining in the perinuclear endosomal compartment, consistent with intracellular degradation of internalized collagen in lysosomes. Cathepsin K was also found to be strongly expressed in keloids and dermatofibromas, but not in sclerotic areas of morphea. Our data suggest that cathepsin K may play an important role in the homeostasis of the dermal extracellular matrix and the dynamic equilibrium between matrix synthesis and proteolytic degradation, by counteracting deposition of matrix proteins during scar formation with its matrix-degrading activity.

High-dose UVA1 radiation therapy for localized scleroderma.

BACKGROUND: Fibrotic skin lesions in patients with localized scleroderma can cause muscle atrophy, disfigurement, and flexion contractures. There is no effective therapy for this disease. Skin fibrosis is thought to be caused by decreased collagenase activity. Collagenase activity can be induced in dermal fibroblasts by UVA1 irradiation. OBJECTIVE: Our purpose was to assess whether UVA1 radiation therapy is effective for patients with localized scleroderma. METHODS: Patients with localized scleroderma (n = 17) were exposed 30 times to 130 J/cm2 UVA1 (high-dose UVA1 therapy; n = 10) or 20 J/cm2 UVA1 (low-dose UVA1 therapy; n = 7). Therapeutic effectiveness was assessed by evaluation of (1) clinical features, (2) thickness of sclerotic plaques, and (3) cutaneous elastometry. Sequential biopsy specimens from treated lesions were analyzed for collagenase I messenger RNA (mRNA) expression by semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: In all patients, high-dose UVA1 therapy softened sclerotic plaques, and complete clearance was observed in four of 10 patients. High-dose UVA1 therapy significantly reduced thickness and increased elasticity of plaques. These changes could not be detected in unirradiated control plaques and were still present in 9 of 10 patients 3 months after cessation of therapy. For all factors assessed, high-dose UVA1 was superior to low-dose UVA1 therapy (p = 0.001). High-dose UVA1 therapy increased collagenase I mRNA expression about 20-fold in treated plaques. CONCLUSION: High-dose UVA1 therapy is effective in the treatment of localized scleroderma. Effectiveness is UVA1 dose dependent and is associated with induction of collagenase I expression.

Differential expression of nitric oxide by dermal microvascular endothelial cells from patients with scleroderma.

Vascular abnormalities in scleroderma are fundamental to the pathogenesis of this disease. The objective of this study was to characterize dermal microvascular endothelial cells (DMEC) isolated from scleroderma patients with respect to growth and expression of the constitutive form of endothelial nitric oxide synthase (eNOS). DMEC from patients with both systemic sclerosis (SSc) and localized scleroderma (Loc Scl) contained small intact microvascular structures in contrast to single cell isolations obtained from control skin. Immunoaffinity selection on anti-PECAM-1 beads yielded pure populations of DMEC expressing normal markers. While the morphology and initial growth of SSc DMEC closely paralleled control cells, the growth of SSc DMEC decreased with time in culture (doubling time of 3 days vs. 5 days). Expression of ecNOS mRNA was reduced in both Loc Scl and SSc as shown by semi-quantitative RT-PCR (p < 0.001). Western blots showed variable but generally lower ecNOS protein levels and decreased levels of nitrogen oxides in media were found from both SSc and Loc Scl relative to control cells. The results indicate an intrinsic defect in the mechanism of nitric oxide production in DMEC isolated from scleroderma patients and suggest its possible involvement in the pathophysiology of scleroderma.

Modulation of urokinase-type and tissue-type plasminogen activator occurs at an early stage of progressing stages of chronic venous insufficiency.

Chronic venous insufficiency (CVI) progresses through a series of clinical stages, from healthy skin to poorly healing leg ulcers. The aim of this study was to analyse the distribution pattern and activity level of urokinase-type (uPA) and tissue-type plasminogen activators (tPA) in normal skin and in tissue biopsies of progressing stages of CVI, prior to and including venous ulceration. Biopsies 6 mm thick were taken from 14 healthy volunteers and 37 patients with 5 different stages of CVI: telangiectases; stasis dermatitis; hyperpigmentation; lipodermatosclerosis; and leg ulcer. Changes in the enzymatic activity and spatial localization of uPA and tPA during the progression of CVI were examined using in situ histological zymography. Normal skin and skin with telangiectases showed a punctate PA activity, consisting of both uPA and tPA activity. As CVI progressed, an increase in the distribution of uPA and a decrease in tPA activity was observed. The spatial localization of uPA was widespread within the dermis of biopsies from stasis dermatitis and lipodermatosclerosis and was associated in particular with the dermoepidermal junction. Hyperpigmented skin revealed a pattern of PA expression similar to that of healthy skin. However, leg ulcer specimens exhibited peak levels of uPA with little tPA. Furthermore, a plasminogen-independent protease activity that was not present in any of the earlier stages of CVI appeared. Our results indicate that there are profound changes in PA activity during the progression of CVI and that these changes begin early in CVI, for example, in stasis dermatitis. We hypothesize that the balance or imbalance of the PA activity in the later stages of CVI is an important pathogenic factor for the development of venous leg ulcer.