Maria Bertolotto

Gabriele Gaggero, M.D., Maria Bertolotto, Ph.D., Daniela Verzola, Ph.D., Jacopo Ferro, M.D., Pietro Fiaschi, M.D., Ph.D.. J Rheum Dis 2024;31:130-2. https://doi.org/10.4078/jrd.2023.0078

Pharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

Background and Purpose: Non‐steroidal anti‐inflammatory drugs (NSAIDs) have been shown to induce PG‐independent anti‐inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a.Experimental Approach: Human neutrophils were isolated from healthy volunteers by dextran and Ficoll‐Hypaque density gradients. Neutrophils were pre‐incubated with different concentrations (1–100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F‐actin polymerization was determined by Alexa‐Fluor 488‐conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot.Key Results: Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F‐actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a‐induced integrin (CD11b) up‐regulation, while only ibuprofen reduced CXCL8‐induced CD11b up‐regulation. Pre‐incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt‐dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE₂ did not affect C5a‐ or CXCL8‐induced activities. Short‐term incubation with NSAIDs did not affect neutrophil PGE₂ release.Conclusion and Implications: Treatment with NSAIDs reduced C5a‐ and CXCL8‐induced neutrophil migration and F‐actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down‐regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE₂ release.

Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN), RANKL/osteoprotegerin (OPG) ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content) as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques.

Angiotensin (Ang)-(1–7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1–7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1–7) in ApoE^-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1–7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1–7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

Aims: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD ⁺ ) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. Results: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model ( ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro . The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD ⁺ -dependent deacetylase in the production of this chemokine. Innovation: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. Conclusions: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice. Antioxid. Redox Signal. 18, 630–641.

Summary: We aimed at challenging the prognostic accuracies of myeloperoxidase (MPO) and antibodies anti-apolipoprotein A-1 (anti-apoA-1 IgG), alone or in combination, for major adverse cardiovascular events (MACE) prediction, one year after carotid endarterectomy (CEA). In this prospective single centre study, 178 patients undergoing elective CEA were included. Serum anti-apoA-1 IgG and MPO were assessed by enzyme-linked immunosorbent assay prior to the surgery. Post-hoc determination of the MPO cut-off was performed by receiver operating characteristics (ROC) analyses. MACE was defined by the occurrence of fatal or non-fatal acute coronary syndromes or stroke during one year follow-up. Prognostic accuracy of anti-apoA-1 IgG was assessed by ROC curve analyses, survival analyses and reclassification statistics. During follow-up, 5% (9/178) of patients presented a MACE, and 29% (52/178) were positive for anti-apoA-1 IgG. Patients with MACE had higher median MPO and anti-apoA-1 IgG levels at admission (p=0.01), but no difference for the 10-year global Framingham risk score (FRS) was observed (p=0.22). ROC analyses indicated that both MPO and anti-apoA-1 IgG were significant predictors of subsequent MACE (area under the curve [AUC]: 0.75, 95% confidence interval [95%CI]: 0.61–0.89, p=0.01; and 0.74, 95%CI: 0.59–90; p=0.01), but combining anti-apoA-1 IgG positivity and MPO>857 ng/ml displayed the best predictive accuracy (AUC: 0.78, 95%CI: 0.65–0.91; p=0.007). It was associated with a poorer MACE-free survival (98.2% vs. 57.1%; p<0.001, LogRank), with a positive likelihood ratio of 13.67, and provided incremental predictive ability over FRS. In conclusion, combining the assessment of anti-apoA-1 IgG and MPO appears as a promising risk stratification tool in patients with severe carotid stenosis.