Assessment of myeloperoxidase activity by the conversion of hydroethidine to 2-chloroethidium.

Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells.

CYLD, a deubiquitinating enzyme (DUB), is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses, via regulating multiple key signaling cascades such as nuclear factor kappa B (NF-κB) pathway. CYLD has been shown to inhibit vascular lesion formation presumably through suppressing NF-κB activity in vascular cells. However, herein we report a novel role of CYLD in mediating pro-inflammatory responses in vascular smooth muscle cells (VSMCs) via a mechanism independent of NF-κB activity. Adenoviral knockdown of Cyld inhibited basal and the tumor necrosis factor alpha (TNFα)-induced mRNA expression of pro-inflammatory cytokines including monocyte chemotactic protein-1 (Mcp-1), intercellular adhesion molecule (Icam-1) and interleukin-6 (Il-6) in rat adult aortic SMCs (RASMCs). The CYLD deficiency led to increases in the basal NF-κB transcriptional activity in RASMCs; however, did not affect the TNFα-induced NF-κB activity. Intriguingly, the TNFα-induced IκB phosphorylation was enhanced in the CYLD deficient RASMCs. While knocking down of Cyld decreased slightly the basal expression levels of IκBα and IκBß proteins, it did not alter the kinetics of TNFα-induced IκB protein degradation in RASMCs. These results indicate that CYLD suppresses the basal NF-κB activity and TNFα-induced IκB kinase activation without affecting TNFα-induced NF-κB activity in VSMCs. In addition, knocking down of Cyld suppressed TNFα-induced activation of mitogen activated protein kinases (MAPKs) including extracellular signal-activated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 in RASMCs. TNFα-induced RASMC migration and monocyte adhesion to RASMCs were inhibited by the Cyld knockdown. Finally, immunochemical staining revealed a dramatic augment of CYLD expression in the injured coronary artery with neointimal hyperplasia. Taken together, our results uncover an unexpected role of CYLD in promoting inflammatory responses in VSMCs via a mechanism involving MAPK activation but independent of NF-κB activity, contributing to the pathogenesis of vascular disease.

Role of 3beta-hydroxysteroid-delta 24 reductase in mediating antiinflammatory effects of high-density lipoproteins in endothelial cells.

OBJECTIVE: The purpose of this study was to investigate the ability of high-density lipoproteins (HDLs) to upregulate genes with the potential to protect against inflammation in endothelial cells. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for 16 hours before being activated with tumor necrosis factor-alpha (TNF-alpha) for 5 hours. rHDLs decreased vascular cell adhesion molecule-1 (VCAM-1) promoter activity by 75% (P<0.05), via the nuclear factor-kappa B (NF-kappaB) binding site. rHDLs suppressed the canonical NF-kappaB pathway and decreased many NF-kappaB target genes. Suppression of NF-kappaB and VCAM-1 expression by rHDLs or native HDLs was dependent on an increase in 3beta-hydroxysteroid-Delta 24 reductase (DHCR24) levels (P<0.05). The effect of HDLs on DHCR24 is dependent on SR-BI but not ABCAI or ABCGI. Silencing DHCR24 expression increased NF-kappaB (1.2-fold, P<0.05), VCAM-1 (30-fold, P<0.05), and NF-kappaB p50 (4-fold, P<0.05) and p65 subunits (150-fold, P<0.05). TNF-alpha activation of siDHCR24-treated cells increased expression of VCAM-1 (550-fold, P<0.001) and NF-kappaB (9-fold, P<0.001) that could no longer be suppressed by rHDLs. CONCLUSIONS: Results suggest that antiinflammatory effects of rHDLs are mediated partly through an upregulation of DHCR24. These findings raise the possibility of considering DHCR24 as a target for therapeutic modulation.

Dipeptidyl-peptidase-4 inhibitor, alogliptin, attenuates arterial inflammation and neointimal formation after injury in low-density lipoprotein (LDL) receptor-deficient mice.

BACKGROUND: The results of recent studies suggest that dipeptidyl-peptidase-4 inhibitors have antiatherogenic effects. However, whether or not dipeptidyl-peptidase-4 inhibitors could suppress arterial inflammation and intimal hyperplasia after injury remains undetermined. The present study aims to clarify the anti-inflammatory effects of the dipeptidyl-peptidase-4 inhibitor, alogliptin (AGP), on the arteries of atherogenic low-density lipoprotein receptor-deficient (LKO) mice. METHODS AND RESULTS: We compared intimal hyperplasia in LKO mice 2 weeks after femoral artery injury using an external vascular cuff model. All mice received oral injection of AGP (20 mg/kg per day) or normal saline (control) once daily for 14 days. Fasting blood sugar levels, serum cholesterol levels, or blood pressure did not significantly differ between the 2 groups. Plasma levels of active glucagon-like peptide-1 were higher in the AGP than in the control LKO mice (22.2±1.9 versus 15.6±0.9 pg/mL; P<0.05). Compared with saline, AGP significantly reduced intimal hyperplasia (1087±127 versus 1896±140 µm(2); P<0.001) as well as the intima/media ratio (0.08±0.01 versus 0.16±0.02; P<0.001). Immunostaining showed that AGP reduced proliferating cells (proliferating cell nuclear antigen-positive nuclei; P<0.001), percent smooth-muscle cell area (α-SMA-positive cells; P<0.001), inflammatory cells infiltration (lymphocyte antigen 6 complex-positive cells; P<0.05), tumor necrosis factor-α expression (P<0.05), and percent phospho-NF-κB-positive cell compared with saline. Levels of tumor necrosis factor -α (0.5-fold P<0.05), monocyte chemoattractant protein 1 (0.3-fold P<0.01), and interleukin-1ß (0.2-fold P<0.05) mRNA were lower in the injured arteries of the AGP than in the control group. CONCLUSIONS: AGP appeared to suppress neointimal formation by inhibiting inflammation, independently of its effects on glucose or cholesterol metabolism in atherogenic LKO mice.

Secretory group II phospholipase A2 in human atherosclerotic plaques.

Atherosclerotic plaques exhibit a series of features that are similar to those of chronic inflammation. Based on the fact that during inflammation several cell types synthesize and secrete a group II phospholipase A2 (PLA2), an immunohistochemical study was undertaken to explore whether this enzyme can be identified in human atherosclerotic lesions. Tissue specimens obtained from 13 patients who had undergone arteriectomy and three specimens with advanced atherosclerotic plaques obtained at autopsy were analyzed and compared to arteries free of atherosclerosis. The results showed that in all areas with atherosclerotic lesions, a staining with monoclonal antibodies raised against group II PLA2 was evident. In normal arteries without thickened intima, this immunostaining was completely negative. With the use of specific monoclonal antibodies against macrophages (anti-KP-1) and smooth muscle cells (anti-alpha-actin), PLA2-positive cells were identified as foam cells mainly derived from macrophages. In addition to these cells, other regions of the thickened intima gave a partially positive reaction with anti-PLA2 antibodies, but could not be stained with either anti-KP-1 or anti-alpha-actin. Some of these regions were localized on edges of calcification and cell necrosis. Other PLA2-positive regions seem to be associated with extracellular matrix structures. In summary, the findings of this study may be regarded as further evidence to support the link between atherosclerosis and chronic inflammatory processes. In view of the fact that the in vitro modification of lipoproteins by PLA2-treatment induces lipid deposition in macrophages, the results of this study suggest that group II PLA2 may actively be involved in the formation of foam cells in vivo.

[The biochemical and clinical aspects of the participation of granulocytes and their proteinases in a vascular wall lesion]. / Biokhimicheskie i klinicheskie aspekty uchastiia granulotsitov i ikh proteinaz v porazhenii stenki sosudov.

Some components of plasma and granulocyte proteolytic system, granulocyte activation degree were evaluated in 15 patients with nonspecific aortic arteritis (NAA) and 12 hereditary hypercholesterolemia (HHC) patients. Catepsin G activity in blood plasma of NAA and HHC patients was higher than in donors 8-9 and 6-fold, respectively (p < 0.001). Granulocyte collagenase activity, spontaneous NBT test values in them were higher than in donors 6 and 4-fold (p < 0.02 and p < 0.01, respectively), 2-fold (p < 0.005 and p < 0.001, respectively). The data obtained confirm the suggestion on the presence of an inflammatory component not only in pathogenesis of NAA, but also in HHC causation.

Cigarette smoke-induced proinflammatory alterations in the endothelial phenotype: role of NAD(P)H oxidase activation.

Although the cardiovascular morbidity and mortality induced by cigarette smoking exceed those attributable to lung cancer, the molecular basis of smoking-induced vascular injury remains unclear. To test the link between cigarette smoke, oxidative stress, and vascular inflammation, rats were exposed to the smoke of five cigarettes per day (for 1 wk). Also, isolated arteries were exposed to cigarette smoke extract (CSE; 0 to 40 microg/ml, for 6 h) in organoid culture. We found that smoking impaired acetylcholine-induced relaxations of carotid arteries, which could be improved by the NAD(P)H oxidase inhibitor apocynin. Lucigenin chemiluminescence measurements showed that both smoking and in vitro CSE exposure significantly increased vascular O(2)(*-) production. Dihydroethidine staining showed that increased O(2)(*-) generation was present both in endothelial and smooth muscle cells. CSE also increased vascular H(2)O(2) production (dichlorofluorescein fluorescence). Vascular mRNA expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and that of inducible nitric oxide synthase was significantly increased by both smoking and CSE exposure, which could be prevented by inhibition of NAD(P)H oxidase (diphenyleneiodonium and apocynin) or scavenging of H(2)O(2). In cultured endothelial cells, CSE elicited NF-kappaB activation and increased monocyte adhesiveness, which were prevented by apocynin and catalase. Thus we propose that water-soluble components of cigarette smoke (which are likely to be present in the bloodstream in vivo in smokers) activate the vascular NAD(P)H oxidase. NAD(P)H oxidase-derived H(2)O(2) activates NF-kappaB, leading to proinflammatory alterations in vascular phenotype, which likely promotes development of atherosclerosis, especially if other risk factors are also present.

[Immunohistochemical and histoenzymological characteristics of the arterial intimal cells in nonspecific aorto-arteritis]. / Immunogistokhimicheskaia i gistoénzimologicheskaia kharakteristika kletok intimy arterii pri nespetsificheskom aorto-arteriite.

Enzymatic activity of cells, antigenic cellular markers and extracellular matrix of the hyperplastic intima of the aorta and carotid arteries was investigated in non-specific aorto-arteritis by immunomorphological and histochemical techniques. The cells of subendothelial layer of thickened arterial intima contained smooth muscle cell myosin, gave positive reactions to myosin ATP-ase and revealed high activity of thiamine pyrophosphatase. Fibronectin and type IV and V collagen were located in close proximity to these cells. The data obtained make it possible to consider these cells as modified smooth muscle cells. Type III collagen was the prevalent type of extracellular matrix of the thickened intima. A great number of blood vessels of the capillary and precapillary types have been found to penetrate into the intima from the adventitia. A possible role of pericytes surrounding newly formed capillaries as the precursors of subendothelial cell population in the hyperplastic intima is discussed.

Association of alkaline phosphatase with an autoantigen recognised by circulating anti-neutrophil antibodies in systemic vasculitis.

A solid phase radioimmunoassay has been developed to detect circulating autoantibodies to neutrophil cytoplasmic antigens in systemic vasculitis. After fractionation of these antigens by size, with gel filtration high performance liquid chromatography, sera from patients with clinically different forms of systemic vasculitis, Wegener's granulomatosis (WG) and microscopic polyarteritis (MP), showed contrasting specificities of binding. WG sera bound to 100, 6.2, and 1.8 kD components, whereas MP sera bound only to the 100 kD component, allowing immunological distinction between the syndromes. The 100 kD component recognised by both WG and MP sera also showed alkaline phosphatase activity. Further evidence for this association was obtained by direct binding experiments between systemic vasculitis sera and calf-intestinal or human neutrophil alkaline phosphatase and by the cross-reactivity of W8, a monoclonal antibody raised to a neutrophil cytoplasmic autoantigen, with various preparations of the enzyme.

Role of inflammation and metalloproteinases in plaque disruption and thrombosis.

Numerous pathological, clinical, angiographic and angioscopic studies have demonstrated that acute coronary syndromes (unstable angina, acute myocardial infarction and ischemic sudden death) are most frequently the consequence of plaque disruption (plaque rupture or superficial plaque erosion) and consequent coronary thrombosis. Several serial angiographic studies have demonstrated that nearly 60-70% of acute coronary syndromes evolve from mildly to moderately obstructive atherosclerotic plaques. Coronary plaque disruption appears to be a function of both the composition of the plaque (plaque vulnerability ) as well as extrinsic triggers that may precipitate plaque disruption in a vulnerable plaque. Vulnerability for plaque disruption appears to be largely determined by the size of the lipid-rich atheromatous core, the thickness of the fibrous cap covering the core, and the presence of ongoing inflammation within and underneath the cap. Inflammatory cells may play a critical role in plaque disruption through the elaboration of matrix degrading metalloproteinases or MMPs (collagenases, gelatinases, stromelysins and matrilysin) and by inhibition of function and survival of matrix-synthesizing smooth muscle cells. Inflammatory cells may also play a critical role in triggering thrombosis following plaque disruption through the tissue factor pathway. In addition, stresses resulting from hemodynamic and mechanical forces may precipitate plaque disruption, particularly at points where the fibrous cap is weakest, such as at its shoulders. The degree of thrombosis following plaque disruption is determined by the thrombogenicity of the disrupted plaque, disturbed local rheology and systemic thrombotic-thrombolytic milieu. Surges in sympathetic activity provoked by sudden vigorous exercise, emotional stress -- including anger, or cold weather, may also trigger plaque disruption. These observations have led to the concept of plaque stabilization as a new clinical strategy for the prevention of acute coronary syndromes. Plaque stabilization can be achieved through pharmacologic and lifestyle-modifying interventions that reduce vulnerability to plaque disruption by altering plaque composition and/or inflammatory activity within the plaque.

Nitric oxide inhibition accelerates hypertension and induces perivascular inflammation in rats.

1. Inflammatory changes in peripheral arteries have been reported in animal models of hypertension. Whether they occur in cerebral arteries (CA) with hypertension induced by deprivation of endogenous nitric oxide (NO) remains unknown. 2. In the present study, we compared the arteriolar injury score (AIS) and perivascular inflammation in CA between hypertensive and normotensive rats following NO deprivation with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Five-week-old male spontaneously hypertensive rats (SHR) and Wistar -Kyoto (WKY) rats were fed with L-NAME (1 mg/mL) for 4 weeks. 3. Nitric oxide deprivation resulted in time-dependent elevations in tail-cuff pressure (representing systolic blood pressure (SBP)) in both SHR and WKY rats. The magnitude of increase in SBP was larger in SHR (+81.0 +/- 3.2 vs+25.0 +/- 2.2 mmHg; P < 0.01). Arteriolar hyalinosis and AIS in various segments of the CA were assessed with periodic acid-Schiff staining and inflammatory cells were immunostained with the antibody against macrophage/monocyte marker (ED1). The ED1+ cells appeared in the middle CA of L-NAME-treated SHR as early as 2 weeks after treatment. These cells were not observed in L-NAME-treated WKY rats and untreated SHR. More ED1+ cells were found in L-NAME-treated SHR than L-NAME-treated WKY rats after 4 weeks treatment. 4. The AIS and number of ED1+ cells around the perivascular area of the internal carotid artery were significantly higher in L-NAME-treated compared with untreated rats (AIS: 137 +/- 28 vs 46 +/- 10 for WKY rats, respectively; 169 +/- 18 vs 53 +/- 6 for SHR, respectively (P < 0.01); ED1+ cells: 7.9 +/- 0.6 vs 1.3 +/- 0.9 for WKY rats, respectively; 13.6 +/- 2.7 vs 2.1 +/- 0.9 for SHR, respectively (P < 0.01)), although SBP was higher in untreated SHR than in L-NAME-treated WKY rats (170 +/- 4 vs 137 +/- 4 mmHg, respectively; P < 0.05). 5. These findings suggest that ED1+ cells appeared in the middle CA of L-NAME-SHR as early as 2 weeks after treatment. Chronic inhibition of NO accelerates hypertension and induces perivascular inflammation.

[Potential clinical use of an acid-stable proteinase inhibitor from human urine]. / Vozmozhnosti klinicheskogo primeneniia kislotostabil'nogo ingibitora proteinaz iz mochi cheloveka.

The method for the preparation of a specific proteinase adsorbent (acid-resistant human urinary proteinase inhibitor--UPI--immobilized on sepharose) has been developed. Proteinase adsorption using this adsorbent has proved highly effective in the treatment of acute pancreatitis with plasmasorption. High adsorption capacity of UPI-sepharose from plasma samples of patients with acute nonspecific aortoarteritis has been demonstrated in vitro. Determination of UPI activity in the human urine is an informative test for early tissue damage, as compared to plasma middle molecules assay. The former test may be used as a diagnostic technique in patients with parenchymal kidney injury and increased arterial pressure.