Regulation of substance P mRNA expression in human dermal microvascular endothelial cells.

Vascular endothelial cells have been identified as a source of substance P (SP) which may act in an autocrine/paracrine fashion to bring about nitric oxide (NO)-dependent vasodilatation and mitogen-induced cell division or immunologic and inflammatory responses. Whilst SP is localised in and released from endothelial cells, an endothelial mRNA expression of SP has not previously been shown. In the present study, mRNA expression of SP in human dermal microvascular endothelial cells is demonstrated using in situ hybridisation techniques with enhancement procedures. Incubation of microvascular endothelial cells with nerve growth factor (NGF) under conditions of increased shear stress increases the mRNA expression and release of SP Endothelin (ET) release is also enhanced. These changes are pertinent to circulatory, events that may occur in Raynaud's phenomenon in systemic sclerosis.

Vascular involvement in systemic sclerosis (SSc).

The vascular endothelium came into view almost three decades ago following the introduction of the vascular hypothesis in scleroderma pathogenesis by Dr LeRoy. Since that time, the endothelial cells, and other vascular cells, became the focus of investigations aimed at eludicating the etiology, pathogenesis and treatment of scleroderma. This review will summarize Dr LeRoy's commitment to the disease, the relevant progress made since the introduction of the vascular hypothesis, and what we have learned since then about the vascular disease in scleroderma.

Initial evidence of endothelial cell apoptosis as a mechanism of systemic capillary leak syndrome.

BACKGROUND: Systemic capillary leak syndrome (SCLS) is a rare disorder of unknown etiology that is characterized by acute recurrent attacks of hypovolemic shock commonly following an inflammatory stimulus such as a viral illness. Prophylactic therapy is generally ineffective, and the outcome is frequently fatal. METHODS: In order to investigate the cellular mechanisms leading to SCLS, we examined the effects of sera from two patients with active SCLS on microvascular endothelial cell apoptosis in vitro. Apoptosis was determined by morphologic criteria, DNA fragmentation, annexin V stain, and by a quantitative photometric assay. The apoptotic pathway was investigated by Western blot of endothelial cells lysate after exposure to SCLS sera. RESULTS: The sera from patients with active SCLS mediated profound apoptosis of microvascular endothelial cells shortly after exposure. The exposed microvascular endothelial cells underwent immediate apoptosis as evidenced by morphologic changes, plasma membrane phosphatidylserine exposure, and by DNA fragmentation. Increased Bax/Bcl-2 ratio in endothelial cells exposed to SCLS sera was observed and suggested an oxidation injury as the possible mechanism for endothelial apoptosis. This potential mechanism was further explored by measuring intracellular reactive oxygen species (ROS) following SCLS serum exposure. Sera from both patients caused marked increases in ROS, initially detectable at 1 h and persisted for at least 12 h, with control serum from healthy subjects showing no effect on basal endothelial cell ROS concentrations. CONCLUSION: Components from the sera of patients with active systemic capillary leak syndrome in contrast to healthy subject sera mediate early and extensive endothelial apoptosis in vitro that is associated with oxidation injury. These data represent compelling initial evidence for oxidation-induced apoptosis as a likely mechanism for endothelial injury leading to SCLS.

Reproducible gene expression measurement among multiple laboratories obtained in a blinded study using standardized RT (StaRT)-PCR.

BACKGROUND: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. METHODS AND RESULTS: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. CONCLUSION: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc.

Autoimmunity and vascular involvement in systemic sclerosis (SSc).

Endothelial injury, obliterative microvascular lesions, and increased vascular wall thickness are present in all involved organs in scleroderma. The vascular pathology is associated with altered vascular function with increased vasospasm, reduced vasodilatory capacity and increased adhesiveness of the blood vessels to platelets and lymphocytes. The extent of injury and dysfunction is reflected by changes in the circulating levels of vascular markers. The initial triggers for the vascular pathology are not known. Possible viral triggers are visited here, including cytomegalovirus in view of increased levels of anti-CMV antibodies in scleroderma, and the remarkable similarities between CMV vasculopathies and scleroderma vascular disease. Endothelial apoptosis in scleroderma may be related to viral infection, immune reactions to viral or environmental factors, reperfusion injury or to anti-endothelial antibodies. The impact of the vascular pathology on the evolution of tissue fibrosis is not known; still, cytokines (TGFbeta, IL4), vascular factors (endothelin), and growth factors (PDGF) are possibly crucial signals that link the vascular disease to tissue fibrosis. Knowledge of the regulation of these and other factors will provide the opportunity to develop more rational therapeutic approaches to the disease.

Mechanism of serum-mediated endothelial injury in scleroderma: identification of a granular enzyme in scleroderma skin and sera.

Circulating endothelial cell growth-inhibitory factor with a molecular weight of 40-60 kDa was described in scleroderma (SSc) sera and shown to have a proteolytic action. In view of the recent demonstration of cellular immune activation in SSc, and because of the description of novel serine proteases in the granules of activated cytolytic T cells (granzymes), we hypothesized that granzymes represent the endothelial inhibitory principal in SSc sera. Granular enzymes were isolated from IL-2-activated nonadherent normal lymphocytes, and a 60-kDa granzyme was isolated using benzamidine-affinity column and molecular sieve column. A polyclonal antiserum was generated by immunizing rabbits with the isolated granzyme. Anti-granzyme antibody abolished SSc serum-mediated EC growth inhibition. Furthermore, a circulating protein similar to isolated granzyme was identified as a 60-kDa band on Western blots of benzamidine column-purified SSc sera. Immunofluorescence studies of SSc skin biopsies using anti-granzyme antibody demonstrated the presence of granzyme reactivity, while healthy control tissues were negative. Moreover, granzyme A gene expression was identified in SSc skin biopsies by a PCR method. The data suggest cytolytic mechanism involvement in the pathogenesis of scleroderma.

Effect of cytokines on the production of endothelin by endothelial cells.

OBJECTIVE: Circulating levels of endothelin (ET), a potent vasoconstrictor peptide and a mitogen for smooth muscle cells and fibroblasts, are reported to be increased in a variety of human diseases characterized by vascular pathology. In view of the probable immune bases for vascular injury in connective tissue disorders, we examined the effect of the cytokines IL-1 alpha, IL-4, IL-6, TNF-alpha and lymphotoxin on the production of ET-1 by cultured vascular endothelial cells. RESULTS: ET levels in endothelial cell conditioned media were measured by radioimmunoassay. IL-4 and lymphotoxin had no effect on ET release by endothelial cells, while IL-6, TNF-alpha and IL-1 alpha stimulated ET mRNA expression and ET release in a dose dependent fashion. IL-6 was the most potent stimulator and IL-1 was the least effective. The addition of neutralizing antibodies to the cytokines inhibited the observed increase in ET release. CONCLUSIONS: These results suggest that cytokines may play a significant role in the control of vascular tone. Furthermore, cytokines may indirectly contribute to the development of proliferative vascular lesions by stimulating smooth muscle and interstitial cell proliferation through their effects on endothelin release by the vascular endothelium.

Raynaud's phenomenon and the vascular disease in scleroderma.

Raynaud's phenomenon (RP) is the most common feature of vascular disease in scleroderma. The diagnostic methods and mechanisms involved in the development of RP continue to be defined, and deficiency in the endothelial-dependent vasodilatory mechanism is suggested. Neuronal involvement in the pathogenesis of vasospasm is illustrated by a defective neuromediated vasodilatory mechanism in both spontaneous RP and vibration white-finger syndrome. The exact neuronal-related mechanism(s) is not known, but the development of scleroderma-like dermal changes following spinal cord injury suggests an important role for neuronal participation in the development of scleroderma. Mechanisms involved in endothelial injury are still not known, but the complement system may be involved, as suggested by the finding of decreased endothelial expression of the complement protective molecular system in scleroderma. Occurrence of vasculitis in the digital arteries in patients with digital ischemic lesions suggests that immune-mediated vascular injury occurs in the disease. The role of antiendothelial antibodies is questioned in view of the lack of endothelial specificity for the antibodies, but the association of anticentromere antibodies with vascular occlusion was confirmed. Therapeutic interventions stressing an individualized approach are recommended. Spinal cord stimulation and continued iloprost infusion are some of the newer proposed therapies for patients with difficult vascular complications.

Raynaud's phenomenon and vascular disease in scleroderma.

Raynaud's phenomenon is the most common sign of vascular involvement in scleroderma. Careful clinical evaluation using a simple definition of Raynaud's phenomenon is the most reliable and reproducible method in the diagnosis. The assessment of microvascular function by laboratory methods is still not specific or sensitive enough for individual patient evaluation. The study of mechanisms involved in the pathogenesis of primary and secondary Raynaud's phenomenon offers the best window for investigation of the early pathogenetic stages in scleroderma. The structural vascular disease in scleroderma is well documented. Still, the impact of endothelial involvement on organ functions is just beginning to be identified and appreciated. Dysregulation of vascular tone control and deficiency of the vasodilatory neuropeptides in scleroderma is proposed as a mechanism in the development of Raynaud's phenomenon. Decreased fibrinolysis and enhanced platelet aggregation is documented and undoubtedly contributes to microvascular thrombosis. The nature of endothelial injury is still elusive, yet markers of endothelial activation and injury continue to be described. Therapy directed toward the vascular disease continues to focus on the alleviation of vascular spasm. Calcitonin gene-related peptide is the newest agent in our therapeutic armamentarium.

Mast cell changes in scleroderma. Presence of MCT cells in the skin and evidence of mast cell activation.

OBJECTIVE: To analyze the concentration and distribution of the MCT (tryptase-positive, chymase-negative) and MCTC (tryptase-positive, chymase-positive) types of mast cell in cutaneous lesions of scleroderma. METHODS: Biopsy specimens were obtained from skin lesions in 24 patients with scleroderma, and subjected to double immunohistochemical analysis using mouse monoclonal anti-tryptase and anti-chymase antibodies. RESULTS: Dermal mast cell concentrations were below the normal range in 12 of the specimens, most of which were obtained between 1 and 4 years after disease onset. All other specimens contained normal concentrations of mast cells. MCT cells were present in 12 specimens and comprised between 8% and 100% of the total mast cells. Extracellular tissue deposits of tryptase-positive and/or chymase-positive granular material were observed in 8 specimens, suggesting possible mast cell degranulation. CONCLUSION: These findings are in contrast to those in normal skin, where MCTC cells are essentially the only type of mast cell present in the dermis. The results suggest that mast cells are involved in the pathogenesis of cutaneous lesions in scleroderma.

The role of vascular endothelium in fibroblast activation and tissue fibrosis, particularly in scleroderma (systemic sclerosis) and pachydermoperiostosis (primary hypertrophic osteoarthropathy).

The vascular endothelium is an organ that is distinguished by its versatility and ability to modulate its surroundings. Endothelial cells (EC) interact with a variety of cell types including fibroblasts (FB), smooth muscle cells, circulating mononuclear cells, platelets and other cell types. This paper will focus on the interaction between endothelial cells and fibroblasts in the process of fibrosis. Vascular changes are described in most human fibrotic models, i.e. radiation, bleomycin, wound healing and particularly in scleroderma. FB migration to the perivascular spaces and proliferation is seen in the early settings of tissue fibrosis. The role of EC in this process is not precisely known; however, the EC contribution to fibrosis is likely to be multifactorial and may involve a spectrum of delicate mechanisms and an array of chemical signals. The induction of FB chemotaxis and FB proliferation by the EC's release of chemotactic factors and mitogens is one possible pathway of FB activation. Another major mechanism involves EC mobilization, guidance and regulation of mononuclear cell infiltration in the perivascular spaces and the subsequent impact of that process on FB activation. Histologic studies of human fibrotic disorders have emphasized early FB proliferation. The role of EC in the induction of FB proliferation should be evaluated carefully in order to understand human fibrosis.

Treatment of systemic sclerosis with extracorporeal photochemotherapy. Results of a multicenter trial.

BACKGROUND AND DESIGN: In a pilot study of extracorporeal photochemotherapy, two patients with systemic sclerosis who received this therapy experienced significant clinical improvement. These results prompted the development of a multicenter trial to examine the benefit of extracorporeal photochemotherapy in the treatment of systemic sclerosis. Seventy-nine patients with systemic sclerosis of recent onset (mean symptom duration, 1.83 years) and progressive skin involvement during the preceding 6 months entered a randomized, parallel-group, single-blinded clinical trial comparing extracorporeal photochemotherapy treatments given on 2 consecutive days monthly with treatment with D-penicillamine at a maximum dose of 750 mg/d. Blinded clinical examiners evaluated skin severity score (thickness), percent surface area involvement, oral aperture, and hand closure. Serial skin biopsies and pulmonary function studies were also performed. RESULTS: Following 6 months of treatment, significant improvement in skin severity score occurred in 21 (68%) of 31 patients receiving photochemotherapy and in eight (32%) of 25 receiving D-penicillamine treatment, while significant worsening occurred in three (10%) of 31 receiving photochemotherapy and in eight (32%) of 25 receiving penicillamine treatment, thus indicating a significantly higher response rate for individuals who received photochemotherapy (P = .02). At both the 6- and 10-month evaluation points, the mean skin severity score, mean percent skin involvement, and mean oral aperture measurements were significantly improved from baseline among those who received photochemotherapy. Mean right and left hand closure measurements had also improved significantly by 10 months of therapy. By comparison, among the patients treated with D-penicillamine, none of the parameters of cutaneous disease had improved significantly after 6 months of therapy, although for those individuals in whom treatment was continued, the mean skin severity score and mean percent skin involvement had improved by 10 months. Skin biopsy studies revealed a correlation between clinical improvement and decreased thickness of the dermal layer. Adverse effects of extracorporeal photochemotherapy were minimal and did not require discontinuation of treatment in any of the patients receiving this therapy; six patients permanently discontinued the use of D-penicillamine treatment due to adverse effects. CONCLUSIONS: For patients with systemic sclerosis of recent onset, extracorporeal photochemotherapy is a well-tolerated treatment that may partially reverse the process that results in cutaneous sclerosis.

Enhanced expression of high-affinity interleukin-2 receptors in scleroderma: possible role for IL-6.

Scleroderma (systemic sclerosis) is characterized by tissue fibrosis, a distinctive vascular and microvascular disorder, and a perivascular mononuclear cell infiltration of involved organs. The pathogenesis of scleroderma is not known; however, there is evidence for a cell-mediated immune mechanism in the disease. Enhanced IL-2 production has been documented both in vivo and in vitro. In this study, the effect of IL-2 on lymphocyte proliferation in vitro was examined. An enhanced proliferative response to IL-2 was seen in scleroderma lymphocytes over that in matched control lymphocytes. Since high-affinity IL-2 receptors (HIL-2-R) mediate the growth-promoting activity of IL-2, we examined HIL-2-R expression on lymphocytes from 13 scleroderma and 11 matched control subjects by a radioiodinated IL-2 binding assay. Significantly higher numbers of HIL-2-R were noted in scleroderma cells (3054 +/- 618 in scleroderma vs 1721 +/- 181 in control cells, mean +/- SD; P less than 0.001). The addition of IL-6 to control cell cultures 24 hr prior to binding determination led to changes in IL-2 binding that were identical to scleroderma cell binding characteristics, while the addition of neutralizing IL-6 antibody to scleroderma cells led to a reduction in HIL-2-R expression. Other cytokines (IL-1, IL-3, IL-4, IL-5, TNF, LT, IFN-gamma, and TGF-beta) had no effect on IL-2 binding, suggesting that IL-6 may mediate the enhanced expression of HIL-2-R. This conclusion was further supported by the finding that scleroderma lymphocytes released in vitro 10- to 20-fold higher concentrations of IL-6 than control cells. The data demonstrate an amplification of IL-2 binding in scleroderma and suggest IL-6 as the mediator of this phenomenon.

Endothelin, an endothelial-dependent vasoconstrictor in scleroderma. Enhanced production and profibrotic action.

The vascular endothelium is an important functional unit in the regulation of the vascular and perivascular environment. Various chemical and physical stimuli mediate an endothelial-dependent vasoconstriction through the release of endothelial soluble factors, such as the recently recognized endothelium-derived vasoconstrictor peptide called endothelin. The presence of circulating endothelin and the effect of cold exposure on plasma endothelin levels were investigated in patients with scleroderma and in healthy control subjects. Radioimmunoassay demonstrated a mean +/- SD plasma level of 10.7 +/- 7.3 pg/ml in the patients (n = 19) and 3.7 +/- 2 in the control subjects (n = 16) (P less than 0.005). These levels were also assessed in 5 control subjects and 5 scleroderma patients before and after 30 minutes of total body cooling (to 15 degrees C). The endothelin level did not change significantly in either group; however, 2 scleroderma patients showed a significant increase after cooling. The effects of endothelin on fibroblast proliferation and collagen synthesis were evaluated in order to assess the impact of released endothelin on the interstitium. A significant mitogenic effect and a collagen synthesis-enhancing effect, which were dose-dependent, were seen. The strong, characteristically prolonged, vasoconstrictor activity coupled with the profibrotic effect demonstrated here make it likely that disturbances in the control of endothelin production can contribute to the pathogenesis of scleroderma.

Soluble immunologic products in scleroderma sera.

To investigate the role of immune mechanisms in scleroderma (systemic sclerosis, SSc), we measured the levels of selected cytokines and soluble immune markers in patient sera. Forty-two patients and 14 matched healthy controls are the subject of this report. In the SSc group, tumor necrosis factor (TNF) was found in 8/42 (29 +/- 539 pg/ml, mean level +/- SD) and lymphotoxin in 36/42 (1:409-1:200, serum dilution). Interleukin beta (IL-1 beta) was observed in 23/42 (44 +/- 29, U/ml). IL-2 was identified in 36/42 patients with a mean level of 286 +/- 406 U/ml, soluble interleukin-2 receptor in 42/42 (1055 +/- 393, U/ml), soluble CD4 antigen in 27/42 (1:10-1:320, serum dilution), and CD8 in 42/42 (470 +/- 134, U/ml). TNF, lymphotoxin, IL-1 beta, Il-2, and CD4 were not detected in the control group. IL-2 receptor levels in control subjects were 520 +/- 171 U/ml, significantly lower than those of scleroderma (P less than 0.001), and CD8 levels (582 +/- 140) were significantly higher than in scleroderma (P less than 0.05). The data suggest an ongoing activation of immune cells, particularly the CD4+ subset in SSc and indicate a potential role for the released mediator TNF, IL-1 beta, and lymphotoxin in the disease process.

The role of vascular endothelium in the pathogenesis of connective tissue disease: endothelial injury, activation, participation and response.

Vascular involvement in the pathogenesis of connective tissue diseases is well documented but poorly understood. Endothelial cell activation and injury play a central role in the disease process. The interaction of endothelium with circulating inflammatory and immune cells may trigger a series of events that include the expression of adhesion molecules and MHC antigens in association with the synthesis and release of various active peptides that have the potential for inflaming the surrounding tissues. The understanding of the nature of endothelial functional changes in the disease process may provide opportunities for intervention and prevention.

Vascular disease in scleroderma. Endothelial T lymphocyte-fibroblast interactions.

The accumulated body of evidence suggests a role for a cell-mediated immune mechanism in the pathogenesis of scleroderma vascular disease. The most likely target for immune injury is either the endothelial cell itself or components of its basal lamina, which include type IV collagen and laminin. Whatever the specific target, the net effect is persistently altered endothelial cell dysfunction. However, the molecular basis for the development of endothelial cell injury is not known. Direct investigations of perivascular infiltrating cells have not been possible yet; published studies have focused on the in vitro effects of peripheral blood mononuclear cells and selected cytokines on endothelial cell behavior and function. Understanding the multiple cellular effects of various cytokines on endothelial cells may further the knowledge of the vascular disease. Systematic study of interactions between endothelial cells and cells of the immune system may provide the molecular basis for vascular injury and open yet unidentified avenues for therapy. Furthermore, monitoring parameters of endothelial cell injury may help to define the disease in an earlier and more meaningful fashion. Circulating levels of EC products such as von Willebrand factor, plasminogen activator, and prostacyclin/thromboxane metabolites may permit a precise definition of disease activity and assist the clinician in monitoring responses to therapy.