Ramona Consuelo Maio

Prolonged statin-associated reduction in neutrophil reactive oxygen species and angiotensin II type 1 receptor expression: 1-year follow-up

AIMS Our study investigated reactive oxygen species (ROS) generation and angiotensin II type 1 receptor (AT(1)-R) expression in primed polymorphonuclear leukocytes (PMNs) of dyslipidaemic subjects over prolonged statin treatment. METHODS AND RESULTS Sixteen untreated dyslipidaemic subjects with moderately increased cardiovascular risk (National Cholesterol Education Program, Adult Treatment Panel III) were studied before and during long-term (1 year) simvastatin treatment. Neutrophils from dyslipidaemic subjects generated more ROS in comparison with cells from healthy control subjects. After 1 year of simvastatin treatment, ROS production (delta N-formyl-Met-Leu-Phe-induced generation and area under the curve) was significantly reduced. At baseline, AT1-R mRNA expression was also higher in dyslipidaemic subjects than in healthy controls and it was reduced after clinical treatment with simvastatin. In a subgroup of patients, a reduced angiotensin II-induced ROS generation was also observed upon clinical simvastatin treatment. Moreover, a direct effect of statin on the upregulated AT(1)-R expression was demonstrated in vitro in neutrophils of untreated dyslipidaemic subjects. CONCLUSION A consistent reversion of pro-inflammatory oxidative functional response and reduction of AT(1)-R expression in primed PMNs was observed in patients during long-term statin treatment. The AT1-R reduction over treatment may contribute to the normalization of dysregulated neutrophil activation which occurs in the pre-clinical phase of atherosclerosis.

Simvastatin treatment modifies polymorphonuclear leukocyte function in high-risk individuals: a longitudinal study.

Background Although extensive experimental evidence supports a primary role of polymorphonuclear leukocytes (PMNs) in atherosclerosis, few data exist concerning the functional properties of these cells and their pharmacological modulation in high-risk individuals. Objective The production of the proinflammatory chemokine interleukin-8 (IL-8), migration and chemotaxis, and reactive oxygen species (ROS) generation were investigated in a longitudinal study in PMNs obtained from high-risk individuals during statin treatment. As a secondary endpoint we compared PMN function of high-risk patients with that of controls. Methods and results PMNs were isolated from 21 high-risk individuals before treatment and 3 and 30 days after the beginning of simvastatin treatment, and from healthy controls. During treatment a significant reduction was observed both in resting (P = 0.009) and N-formyl-Met-Leu-Phe (fMLP)-stimulated (P = 0.008) IL-8 production, and in the chemotactic index (P = 0.038), whereas ROS generation did not significantly change. In comparison with cells from controls, PMNs obtained from patients before starting simvastatin treatment showed higher resting and fMLP-stimulated IL-8 release (P = 0.007 and P = 0.002, respectively) and ROS generation (resting, P = 0.009; and fMLP-stimulated, P = 0.046), whereas migration and the chemotactic index did not significantly differ. Conclusions An activation of neutrophils is present in high-risk individuals, shown by the enhanced production of IL-8, and increased ROS generation. The 4-week statin treatment is able to reduce the cell capability to produce IL-8, and to decrease chemotaxis, thus affecting the proinflammatory properties of PMNs.

Scanning electron microscopy examination of endothelium morphology in human carotid plaques

In recent years, experimental and clinical evidence has been provided regarding endothelial dysfunction and its contribution to the inflammatory process leading to atherosclerotic plaque formation. All the techniques employed direct to the study of endothelial dysfunction, however usually require isolation of endothelial cells and therefore tissue manipulation and destruction, with subsequent loss of information regarding morphology and topographical distribution of the lesions. By means of scanning electron microscopy (SEM), we have investigated the characteristics of the endothelial layer in carotid specimens obtained from subjects undergoing carotid endarterectomy. Carotid specimens obtained from 6 patients undergoing carotid endarterectomy for stenosis> or =70% were fixed, prepared and examined by use of SEM in a direct mode. All the plaque specimens showed increased thickness of subendothelial connective tissue with respect to post-mortem tissue obtained from an healthy subject. Endothelial layers were typically detached from the basal lamina surface and infiltrating cells (mainly erythrocytes and, possibly, monocytes) could be identified. Endothelial cells in proximity of the plaques had irregular shape, with prominent nuclei. In several areas, the endothelial layer was completely absent and basal lamina completely uncovered. In the present study, by using SEM analysis, the morphological features of dysfunctional endothelium in human carotid plaques were extensively documented at the ultrastructural level. SEM is a powerful investigational technique which allows tridimensional examination of specimens without disruption of the originary morphology.

Angiotensin II type 1 receptor expression in polymorphonuclear leukocytes from high-risk subjects: changes after treatment with simvastatin.

Statins may directly interfere with the effects of angiotensin (Ang) II, which is a key player in the pathogenesis of atherosclerosis (ATH). Ang II promotes a wide array of detrimental processes including a prominent proinflammatory effect, increasingly regarded as a target for therapeutic intervention. Because the proinflammatory effects of Ang II are exerted mainly through the activation of Ang II type 1 receptors (AT1Rs) the present study was devised to investigate by means of real-time polymerase chain reaction (PCR) and flow cytometry techniques the expression of such receptors on circulating polymorphonuclear leukocytes (PMNs) from subjects at high risk for vascular events before and during treatment with simvastatin and in sex- and age-matched healthy controls. In vitro experiments were also performed to assess the ability of simvastatin to interfere with Ang II signaling in human PMNs. In comparison to controls, high-risk subjects had similar AT1R expression on the cell membranes but significantly higher AT1R messenger ribonucleic acid (mRNA) levels. Treatment of high-risk subjects with simvastatin for 30 days resulted in a reduction of AT1R mRNA down to the levels of cells from healthy subjects. In vitro, Ang II-induced activation of the guanosine triphosphate (GTP)-binding protein Rac 1 in human PMNs was inhibited by simvastatin. In conclusion, simvastatin induces downregulation of AT1R expression, interferes with Ang II activity in PMNs, and contributes to the antiinflammatory profile of statins that can explain the therapeutic effects of these drugs.

Simvastatin treatment in subjects at high cardiovascular risk modulates AT1R expression on circulating monocytes and T lymphocytes.

Objective Angiotensin II, through the activation of angiotensin II type 1 receptors, plays a crucial role in atherosclerosis. Statins may interfere with the effects of angiotensin II. Methods We have investigated the expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor and angiotensinogen on circulating monocytes and T-lymphocytes from subjects at high risk for vascular events before and during simvastatin treatment, and healthy controls. In-vitro experiments were also performed to assess the ability of simvastatin to interfere with angiotensin II signalling. Results In comparison with controls, high-risk subjects had similar angiotensin II type 1 receptor expression on the cell membranes but significantly higher angiotensin II type 1 receptor mRNA levels at least in monocyte subsets whereas their expression on T cells was similar. Angiotensin II type 2 receptor mRNA expression was higher than controls in both monocytes and T lymphocytes. No differences were observed in angiotensinogen expression on monocytes while T lymphocytes of high-risk subjects show higher expression. One-month treatment of high-risk subjects with simvastatin resulted in a reduction of angiotensin II type 1 receptor mRNA without affecting angiotensin II type 2 receptor whereas angiotensinogen mRNA expression was reduced at least in monocytes. Incubation in vitro with simvastatin reduces the expression of angiotensin II type 1 receptor mRNA levels on monocytes from untreated subjects. Conclusion Simvastatin induces down-regulation of the angiotensin II type 1 receptor, interferes with angiotensin II activity in immune cells and contributes to the anti-inflammatory profile of statins that can explain the therapeutic effects of these drugs.

Immunomodulatory activity of the lignan 7-hydroxymatairesinol potassium acetate (HMR/lignan) extracted from the heartwood of Norway spruce (Picea abies).

The pharmacological profile of the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) includes chemopreventive effects, antioxidant properties, and mild proestrogenic activity. The present study was devised to investigate the effects of HMR on THP-1 cells, an established model of human monocytes, and on human polymorphonuclear leukocytes (PMNs). In THP-1 cells, HMR concentration-dependently reduced LPS-stimulated tumor necrosis factor (TNF)-alpha secretion in the supernatant. HMR at low, sub-muM concentrations also reduced TNF-alpha mRNA, which was however enhanced by supra-muM concentrations of HMR. In human PMNs, HMR concentration-dependently reduced ROS production induced by either N-formyl-Met-Leu-Phe, phorbol myristate acetate or angiotensin II, as well as interleukin-8 production induced by either N-formyl-Met-Leu-Phe or angiotensin II. Results indicate that HMR is an effective inhibitor of both monocytic THP-1 cells and of human PMNs and warrant further studies to assess their relevance for the prevention and treatment of several conditions characterized by chronic systemic inflammation.

Angiotensin type 1 receptor expression and interleukin-8 production in polymorphonuclear leukocytes of patients with peripheral arterial disease.

We investigated angiotensin type 1 receptor (AT1R) expression and interleukin-8 (IL-8) productions in polymorphonuclear leukocytes obtained from patients with peripheral arterial disease (PAD) undergoing femoral endarterectomy. Subjects at high cardiovascular risk (high-risk subjects, HRS) and healthy controls (HC) were also enrolled. To this end, patients with PAD were studied 1 month before surgery, at the time of surgery, and 3 and 6 months after surgery. Polymorphonuclear leukocytes were obtained from venous blood and evaluated for AT1R expression at messenger RNA (mRNA) and protein level and IL-8 production (by means of enzyme-linked immunosorbent assay). At baseline, AT1R membrane expression was similar in cells from patients with PAD, HRS, and HC, whereas AT1R mRNA was similar in patients with PAD and HC and higher in HRS. During the follow-up period, AT1R expression progressively decreased both on the cell membrane and at the mRNA level. Both resting and stimulated production of IL-8 was lower in patients with PAD in comparison to HC and HRS and did not change during the follow up period. In PAD patients, femoral endarterectomy is associated with reduction of AT1R expression however with no apparent effect on IL-8 production. The relevance of such effects for cardiovascular protection deserves consideration.

Angiotensin II type 1 and type 2 receptor expression in circulating monocytes of diabetic and hypercholesterolemic patients over 3-month rosuvastatin treatment

BackgroundIn diabetes, a variety of pro-inflammatory cellular changes has been found in various cell types, including monocytes which are known to be involved in all the phases of atherogenesis. Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the pro-atherogenic effects of Ang II whereas the type 2 receptor (AT2R) seems associated with atheroprotection. We sought to investigate the potential changes of AT1R-AT2R expression in human monocytes of type 2 diabetic- hypercholesterolemic patients and in hypercholesterolemic subjects, upon clinical treatment with rosuvastatin.MethodsThe AT1R membrane protein and mRNA AT1R and AT2R expression in monocytes were investigated in 10 type 2 diabetic-hypercholesterolemic patients and in 10 hypercholesterolemic subjects, before and after 3-month rosuvastatin treatment. Moreover, the serum cytokine levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) were detected.ResultsAs expected, rosuvastatin was associated with a change in the lipid profile in the two groups. Both the membrane protein (P = 0.008) and the AT1R mRNA expression (P = 0.038) were significantly reduced during treatment in the absence of AT2R expression change in diabetic-hypercholesterolemic patients whereas no significant difference was observed in hypercholesterolemic subjects. The serum IL-4 levels were increased during treatment whereas no change was observed in IFN-γ in diabetic-hypercholesterolemic patients. No cytokine change was observed in hypercholesterolemic subjects.ConclusionsOur study on monocytes of diabetic-hypercholesterolemic patients, showing a reduced AT1R but not AT2R expression during rosuvastatin treatment, suggests that statin therapy may modulate favorably the AT1-AT2 receptor balance in subjects with coexistent type 2 diabetes.

Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes

CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 μg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease.

Laminar Pattern of Mineral Calcium-Phosphorus Deposits in a Human Carotid Plaque Nanoscale Ultrastructure and Elemental Analysis

Atherosclerotic calcifications are related to poor prognosis and all-cause mortality in large population studies.1 Moreover, vascular calcifications are inversely associated with the potential reduction of plaque volume in regression studies during statin treatment, which suggests that more calcified lesions are less likely to undergo positive remodeling.2 Recently, it has been suggested that calcification is a tightly regulated process of mineralization akin to bone formation.3 In plaques, the deposits consist of a nonhomogeneous composite that contains hydroxyapatite mineral nanocrystals embedded in a collagenous organic matrix, whereas at a nanoscale level, apatite crystals interact with cholesterol crystallites. In this context, mineralization may result from a basic template pattern generated by the organic …