Estrogen increases expression of vascular alpha 2C adrenoceptor through the cAMP/Epac/JNK/AP-1 pathway and potentiates cold-induced vasoconstriction.

Cutaneous cold-induced vasoconstriction is a normal physiological reaction mediated by alpha 2C-adrenergic receptors (α2C-ARs) expressed in vascular smooth muscle cells (VSMCs). When this reaction is exaggerated, Raynaud's phenomenon (RP) ensues. RP is more prevalent in females compared to age-matched men. We previously established that 17-ß estradiol (estrogen) upregulates α2C-ARs in human VSMCs via a cAMP/Epac/Rap pathway. We also showed that cAMP acts through JNK to increase α2C-AR expression. However, whether estrogen employs JNK to regulate α2C-AR is not investigated. Knowing that the α2C-AR promoter harbors an activator protein-1 (AP-1) binding site that can be potentially activated by JNK, we hypothesized that estrogen regulates α2C-AR expression through an Epac/JNK/AP-1 pathway. Our results show that estrogen (10-10 M) activated JNK in human VSMCs extracted from cutaneous arterioles. Pretreatment with ESI09 (10 µM; an Epac inhibitor), abolished estrogen-induced JNK activation. In addition, pre-treatment with SP600125 (3 µM; a JNK specific inhibitor) abolished estrogen-induced expression of α2C-AR. Importantly, estrogen-induced activation of α2C-AR promoter was attenuated with SP600125. Moreover, transient transfection of VSMCs with an Epac dominant negative mutant (Epac-DN) abolished estrogen-induced activation of α2C-AR promoter. However, co-transfection of constitutively active JNK mutant overrode the inhibitory effect of Epac-DN on α2C-AR promoter. Moreover, estrogen caused a concentration-dependent increase in the activity of AP-1-driven reporter construct. Mutation of AP-1 site in the α2C-AR promoter abolished its activation by estrogen. This in vitro estrogen-increased α2C-AR expression was mirrored by an increase in the ex vivo functional responsiveness of arterioles. Indeed, estrogen potentiated α2C-AR-mediated cold-induced vasoconstriction, which was abolished by SP600125. Collectively, these results indicate that estrogen upregulates α2C-AR expression via an EPAC-mediated JNK/AP-1- dependent mechanism. These results provide an insight into the mechanism by which exaggerated cold-induced vasoconstriction occurs in estrogen-replete females and identify Epac and JNK as potential targets for the treatment of RP.

Effects of phosphodiesterase type 5 inhibitors on Raynaud's phenomenon.

Raynaud's phenomenon (RP) is commonly observed in fingers and toes of patients with connective tissue diseases (CTDs). However, existing vasodilators have very limited efficacy. In this study, phosphodiesterase type 5 inhibitors (PDE-5Is) were administered to evaluate efficacy on RP. Three patients with mixed connective tissue disease and three patients with systemic sclerosis having RP were enrolled. Oral sildenafil, vardenafil, or tadalafil was administered. The fingertip temperature was measured by thermography before and 120 min after administration. To evaluate longer effects, vardenafil was administered daily for 12 weeks; the fingertip temperature was measured by thermography before and 12 weeks after administration. As compared with the pre-administration of sildenafil, vardenafil, and tadalafil, the mean fingertip temperature increased by 2.17, 3.47, and 3.59 °C, respectively, in 120 min. In the 12-week trial with vardenafil in 3 patients, the mean fingertip temperature increased by 3.04, 7.96, and 3.32 °C from baseline in each patient. PDE-5Is significantly increased fingertip temperature within 120 min, and the effect of vardenafil lasted for 12 weeks under daily use. PDE-5Is were safe and would be an effective treatment for RP with CTDs.

Diagnostic criteria of systemic sclerosis.

Systemic sclerosis (SSc) is a multisystem disease characterized by vascular abnormalities, immune system activation manifested by SSc-specific autoantibodies and disturbances in fibroblast function. The clinical manifestations are highly heterogeneous and commonly include skin thickening, Raynaud's phenomenon, digital ulcers, gastroesophageal reflux disease, interstitial lung disease and cardiac diastolic dysfunction. The diagnosis of SSc in a patient with typical end-organ disease is relatively straight-forward, but is unsatisfactory because it implies that the diagnosis is delayed until irreversible tissue damage is present. Diagnostic criteria are generally designed to facilitate the clinical process and to allow early institution of therapy to relieve symptoms and possibly prevent irreversible damage. Several attempts at defining diagnostic criteria for SSc have been made in the past. Raynaud's phenomenon, SSc-specific autoantibodies and nailfold capillary abnormalities are among the most promising items likely to be retained in a final set of diagnostic criteria. The EULAR Scleroderma Trial and Research group (EUSTAR) is currently in the process of prospectively validating a set of diagnostic criteria for the very early diagnosis of SSc and results are expected in 2015.

Antisynthetase syndrome presenting as rheumatoid-like polyarthritis.

BACKGROUND: The antisynthetase syndrome is a systemic inflammatory disease associated with anti-tRNA synthetase antibodies and consisting of the clinical features of inflammatory myopathy arthritis, interstitial lung disease (ILD), fever, Raynaud syndrome, and rash. It rarely presents with symmetric arthritis as the initial manifestation of the disease. OBJECTIVE: The aim of the study was to describe the clinical, laboratory, and radiographic characteristics of patients with antisynthetase syndrome who presented with symptoms of inflammatory arthritis, mimicking rheumatoid arthritis (RA) at the time of initial evaluation. METHODS: Six cases derived from a single university-based rheumatology clinic in Wisconsin are presented. The major clinical, laboratory, radiographic, and histopathologic data are described. RESULTS: All 6 patients demonstrated symmetric synovitis involving the hands. Five patients met the American College of Rheumatology classification criteria for RA. Three patients had nail-fold capillary abnormalities, and 4 patients were observed to have Raynaud phenomenon. Three patients demonstrated a cytoplasmic pattern when testing for antinuclear antibodies by immunofluorescent assay, and all had t-RNA synthetase antibodies. Two patients had positive rheumatoid factors, but none had strongly positive cyclic citrullinated peptide antibodies. None of the patients demonstrated radiographic erosions. All patients had evidence of ILD by imaging or pulmonary function testing. Prognosis was generally favorable, although disease severity and treatment varied considerably. CONCLUSION: In patients who present with features mimicking but atypical for RA, such as early ILD, nail-fold capillary abnormalities, Raynaud phenomenon, cytoplasmic antinuclear antibody pattern, negative cyclic citrullinated peptide antibody status, and nonerosive arthritis, the antisynthetase syndrome should be considered.

Phosphodiesterase-5 inhibitors for the treatment of Raynaud's: a novel indication.

BACKGROUND: Raynaud's phenomenon is a common clinical condition characterized by significant morbidity, especially in patients who have an underlying connective tissue disorder. These patients suffer from a worse clinical phenotype that at times can lead to digital vessel occlusion. Current therapies are not optimal, and better therapeutic options are needed. OBJECTIVE: To describe the up-to-date data available on the use of phosphodiesterase 5 inhibitors in the treatment of Raynaud's phenomenon. METHODS: We performed a literature search of PubMed from 1990 - 2008, as well as a search of the abstracts presented at the American College of Rheumatology scientific meeting and the European League Against Rheumatism scientific meeting for the years 2001 - 2007. The search terms used were 'Raynaud's', 'phosphodiesterase 5 inhibitors', ' tadalafil', 'sildenafil' and 'vardenafil'. RESULTS/CONCLUSION: Based on current data from small clinical trials, open-label pilot studies and case series and reports, phosphodiesterase 5 inhibitors may help some patients with very serious Raynaud's. A large, well-conducted multicenter, double-blind study is needed to determine the benefit and risk of these agents in Raynaud's phenomenon.

Regulation of vascular reactivity in scleroderma: new insights into Raynaud's phenomenon.

Because of the role of the RhoA/Rho kinase (ROCK) pathway in regulating numerous pathologic processes including vasoconstriction, vascular remodeling, and fibrosis, ROCK inhibitors may be especially beneficial in treating Raynaud's phenomenon and scleroderma.

Cooling-induced contraction and protein tyrosine kinase activity of isolated arterioles in secondary Raynaud's phenomenon.

OBJECTIVE: To investigate the response of skin arterioles from control subjects and patients with scleroderma and Raynaud's phenomenon (RP/SSc) to cooling and modulators of protein tyrosine kinase (PTK) activity. METHODS: We used the microvessel perfusion technique to characterize the response of isolated dermal arterioles (100-200 microm, outside diameter) from normal (n = 17) and RP/SSc (n = 17) subjects to cooling from 37 degrees to 31 degrees C. Fluorescent immunohistochemistry was used to measure tyrosine phosphorylation. RESULTS: Arterioles from control subjects exhibited dilation in response to cooling from 37 to 31 degrees C whereas those from RP/SSc subjects contracted (+4.3 +/- 1.7 vs -16.7 +/- 3.1%, P < 0.05, n = 6). In the presence of the protein tyrosine phosphatase inhibitor sodium orthovanadate (SOV, 10 microM), the response of arterioles from control subjects did not change; however, arterioles from RP/SSC subjects exhibited a significantly greater contraction (-72.6 +/- 19.7%; P < 0.05, n = 6). Tyrosine phosphorylation of arterioles at 37 degrees C from control and RP/SSc subjects was similar. In response to cooling to 31 degrees C, however, arterioles from RP/SSc subjects exhibited a significantly greater increase in tyrosine phosphorylation compared with those from control subjects (43 +/- 7.0% vs 10 +/- 3.8%; P < 0.01). SOV increased tyrosine phosphorylation in arterioles from both groups (73 +/- 11.6% vs 42 +/- 5.6%; P < 0.05, n = 5). Arterioles from RP/SSC subjects precontracted with norepinephrine exhibited greatly attenuated relaxation to acetylcholine compared with those from control subjects. CONCLUSION: The results of this study support the view that the hallmark of Raynaud's phenomenon associated with scleroderma, cooling-induced vasospasm, appears to be mediated by an increase in PTK activity possibly exacerbated by impaired endothelium-dependent vasodilation.

Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)-9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP-9 and MMP-2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP-9 activity, but not MMP-2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP-9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP-9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti-phospholipid antibodies. Changes in activity levels of MMP-9, but not of MMP-2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP-9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP-9 plays a role in the pathogenesis of SLE.

Serum TIMP-1, TIMP-2, and MMP-1 in patients with systemic sclerosis, primary Raynaud's phenomenon, and in normal controls.

BACKGROUND: Excess tissue matrix accumulates in systemic sclerosis (SSc), accounting for both visceral and dermal fibrosis. It is suggested that decreased serum levels of matrix metalloproteinases (MMPs) or increased levels of tissue inhibitors of matrix metalloproteinases (TIMPs) may account for this matrix accumulation. OBJECTIVE: To measure serum levels of tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and collagenase-1 (MMP-1), in patients with diffuse cutaneous systemic sclerosis (dcSSc), limited cutaneous systemic sclerosis (lcSSc), primary Raynaud's phenomenon (RP), and in normal controls. METHODS: Serum samples from patients with dcSSc (n=83), lcSSc (n=87), RP (n=80), and normal controls (n=98) were analysed using enzyme linked immunosorbent assays (ELISAs) for total TIMP-1, TIMP-2, and MMP-1. Results from each assay were analysed by the Kruskal-Wallis test. Dunn's multiple comparison post-test was then applied between groups. RESULTS: TIMP-1 levels were significantly raised in dcSSc and lcSSc groups compared with the RP group and normal controls (p<0.01 to p<0.001). In the dcSSc group, TIMP-1 levels were significantly higher in early disease (<2 years) than in late stage disease (>4 years) (p<0.05). This was not found for the lcSSc group. Serum TIMP-2 and MMP-1 levels in dcSSc and lcSSc did not differ significantly from those in normal controls. Increased levels of TIMPs were not convincingly associated with organ disease. No assay result correlated with autoantibody status (anti-topoisomerase 1 (anti-Scl-70), anticentromere antibody, or anti-RNA polymerase). No significant differences in serum TIMP-1, TIMP-2, or MMP-1 levels were shown in the RP group compared with normal controls. CONCLUSIONS: Raised TIMP-1 levels in the SSc groups support the hypothesis that matrix accumulation occurs in SSc at least in part owing to decreased degradation. Moreover, the variation in TIMP-1 levels between the early and late disease stages of dcSSc seems to reflect the early progressive course of dermal fibrosis seen clinically. The expected reduction in serum MMP-1 levels in the SSc groups was not found. This suggests that tissue matrix accumulation is due to increased inhibitors rather than to decreased MMPs.

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.

Investigation of sialic acids and sialyltransferase activity in blood of patients with systemic scleroderma.

Systemic scleroderma (SSd) is a connective tissue disorder accompanied by generalized fibrosis. A disturbance of the synthesis and production of matrix glycoproteins, such as collagens, fibronectin, and proteoglycans, by connective tissue cells is typical for this disease. We previously demonstrated a decrease in the ganglioside content of cultured skin fibroblasts from patients with SSd. In this work the contents of sialoglycoproteins and sialoglycolipids in blood sera of patients with SSd were estimated. Simultaneously, the level of asialofetuin-sialyltransferase activity in blood plasma of three groups of patients--those with SSd, Raynaud's phenomenon, and with localized scleroderma--was investigated. CMP-5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid was used as a substrate for the enzyme assay. It was shown that the concentration of total sialic acid was increased and the concentration of lipid-bound sialic acid was slightly decreased in the blood sera of patients with SSd. A correlation between the lipid-bound sialic acid level and the severity of disease was observed; there was no correlation between severity of disease and total sialic acid. Sialyltransferase assay showed a decrease in the activity level in all three groups of patients. The greatest decrease (2-fold) of this activity was observed in patients with Raynaud's phenomenon. Our data suggest that in SSd and similar diseases the process of glycoconjugate sialylation is disturbed. These changes may considerably affect the mechanisms of regulation of metabolism and cellular interactions.

Plasma levels of tumor necrosis factor and endothelial response in patients with chronic arterial obstructive disease or Raynaud's phenomenon.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine that affects endothelial cells' function by changing their antithrombotic potential to a net procoagulant effect. Only a few data have so far been reported for the pathophysiologic role of TNF in vascular diseases in the involvement of microvessels and/or macrovessels and a prothrombotic state. In the present study the authors evaluated plasma TNF (and interleukin-1) levels in 20 patients with chronic arterial obstructive disease (CAOD) with intermittent claudication and 10 CAOD patients with more severe disease (pain at rest/skin ulcers). In addition, they studied 10 patients with Raynaud's phenomenon (RP), suspected to be secondary to a collagen disease. The control group consisted of 20 subjects matched for sex and age with the three groups of patients. TNF levels were assayed by enzyme-linked immunosorbent assay. The antigen levels of von Willebrand factor (vWF), tissue plasminogen activator (t-PA), and its inhibitor (PAI) were also determined as markers of release from the endothelium, while the fragment 1 + 2 of prothrombin (F1 + 2) and thrombin-antithrombin III (TAT) complexes were assessed as indexes of systemic thrombin generation. TNF levels were significantly higher in both groups of CAOD patients than in controls or RP patients, and the same was true for vWF. t-PA was significantly higher only in the CAOD subjects with more severe disease. No differences among groups were seen in PAI antigen/activity or thrombin generation. When data were corrected for age, TNF no longer differentiated CAOD patients from controls and RP subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of Ca(2+)-ATPase in erythrocytes from subjects with essential hypertension or Raynaud's phenomenon.

The increased peripheral vascular resistance in subjects with essential hypertension and in subjects with primary Raynaud's phenomenon is thought to be the consequence of an impaired control of intracellular Ca2+ in vascular smooth muscle cells. Abnormal membrane handling of Ca2+ in human essential hypertension is not limited to vascular smooth muscle cells but has been demonstrated in a number of blood cells, for example, erythrocytes. Ca(2+)-ATPase activity and Ca(2+)-ATPase densities were measured in erythrocyte membranes from healthy volunteers and subjects with essential hypertension or with Raynaud's phenomenon. We found no significant differences between the three groups in the Ca(2+)-ATPase activity and in the number of Ca(2+)-ATPase immunoreactive sites per erythrocyte. Furthermore, we could not demonstrate any influence of sex or age on these two parameters. We conclude that, in essential hypertension and in Raynaud's phenomenon, the Ca2+ extrusion capacity of the erythrocytes is not altered.

[Connective tissue diseases and HEp-2 antinuclear antibodies]. / Connettiviti e anticorpi antinucleari HEp-2.

Some characteristics of antinuclear antibodies that might be of use for diagnostic and/or prognostic purposed were studied using indirect immunofluorescence on HEp-2 cells in 55 cases of various types of connective tissue disease. For each nuclear (homogeneous, speckled, granular, dotted, pulverulent and centromeric) and nucleolar fluorescence pattern (homogeneous, conglutinated and dotted), the following parameters were observed; C3 fixing capacity, degree of antibody affinity and sensitivity to RNAse, DNAse and trypsin. The results were very interesting, especially in relation to the diagnosis of progressive systemic sclerosis and of the related subsets, but were insufficient for reliable, conclusive prognostic evaluation.

[Liver enzyme function and humoral immunity in workers exposed to vinyl chloride]. / Izuchavane enzimnata funktsiia na cherniia drob i khumoralniia imunitet na rabotnitsi, eksponirani na vinilkhlorid.

The work aims at studying the enzyme function of the liver, as well as the humoral immunity of workers engaged in the production of vinyl chloride and polyvinylchloride with proved hepatotropic and Raynaud syndromes. About 191 workers /experimental group/ from the synthesis and polymerization of vinyl chloride and 2 control groups--34 persons from Varna and 54 from G. Traikov were examined. The following biochemical and immunological studies were performed: GOT, GPT, GGTP, alkaline phosphatase, IGG, circulating immune complexes, titer of the complement, cryoglobulins in the serum and identification of the sediments by immunoelectrophoresis. It was established that the immune system was included in the early phases of toxic effect. Disturbances in the enzyme function and humoral immunity were registered both in workers with hepatotropic and Raynaud syndromes.