Fabrizio Montecucco

HUMAN MESENCHYMAL STEM CELLS INHIBIT NEUTROPHIL APOPTOSIS: A MODEL FOR NEUTROPHIL PRESERVATION IN THE BONE MARROW NICHE

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here, we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors, at very low MSC:neutrophil ratios (up to 1:500), significantly inhibited apoptosis of resting and interleukin (IL)‐8‐activated neutrophils and dampened N‐formyl‐l‐methionin‐l‐leucyl‐l‐phenylalanine (f‐MLP)‐induced respiratory burst. The antiapoptotic activity of MSC did not require cell‐to‐cell contact, as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC‐derived soluble factor responsible for neutrophil protection from apoptosis was IL‐6, which signaled by activating STAT‐3 transcription factor. Furthermore, IL‐6 expression was detected in MSC by real‐time reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Finally, recombinant IL‐6 was found to protect neutrophils from apoptosis in a dose‐dependent manner. MSC had no effect on neutrophil phagocytosis, expression of adhesion molecules, and chemotaxis in response to IL‐8, f‐MLP, or C5a. These results support the following conclusions: (a) in the bone marrow niche, MSC likely protect neutrophils of the storage pool from apoptosis, preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism, and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified.

Common inflammatory mediators orchestrate pathophysiological processes in rheumatoid arthritis and atherosclerosis

RA is characterized by a systemic inflammatory state, in which immune cells and soluble mediators play a crucial role. These inflammatory processes resemble those in other chronic inflammatory diseases, such as atherosclerosis. The chronic systemic inflammation in RA can be considered as an independent risk factor for the development of atherosclerosis, and represents an important field to investigate the reasons of the increase of acute cardiovascular events in RA. In the present review, we focused on several mediators of autoimmunity, inflammation and endothelial dysfunction, which can be considered the most promising targets to prevent atherogenesis in RA. Among several mediators, the pro-inflammatory cytokine TNF-alpha has been shown as a crucial factor to induce atherosclerosis in RA patients.

Role of cytokines and chemokines in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) includes a variety of histological conditions (ranging from liver steatosis and steatohepatitis, to fibrosis and hepatocarcinoma) that are characterized by an increased fat content within the liver. The accumulation/deposition of fat within the liver is essential for diagnosis of NAFLD and might be associated with alterations in the hepatic and systemic inflammatory state. Although it is still unclear if each histological entity represents a different disease or rather steps of the same disease, inflammatory processes in NAFLD might influence its pathophysiology and prognosis. In particular, non-alcoholic steatohepatitis (the most inflamed condition in NAFLDs, which more frequently evolves towards chronic and serious liver diseases) is characterized by a marked activation of inflammatory cells and the upregulation of several soluble inflammatory mediators. Among several mediators, cytokines and chemokines might play a pivotal active role in NAFLD and are considered as potential therapeutic targets. In this review, we will update evidence from both basic research and clinical studies on the potential role of cytokines and chemokines in the pathophysiology of NAFLD.

CB2 cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion

Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB(2) cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB(2) selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27%+/-1.91) as compared to vehicle-treated mice (31.77%+/-2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-alpha, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-alpha induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.

HIV increases markers of cardiovascular risk: results from a randomized, treatment interruption trial.

Objectives:Plasma soluble inflammatory molecules are associated with the risk of ischaemic cardiovascular events. We investigated whether HIV replication modified the levels of these proteins in a combination antiretroviral therapy (cART) interruption trial. Method and results:In 145 HIV-infected Thai patients (62% women, median CD4 cell count 271 cells/μl, median plasma HIV-RNA 4.66 log10 copies/ml) included in the Swiss–Thai–Australia Treatment Interruption Trial (STACCATO) trial, leptin, adiponectin, C-reactive protein, soluble vascular cell adhesion molecule-1 (s-VCAM-1), P-selectin, chemokine ligand 2, chemokine ligand 3, interleukin (IL)-6, IL-10, granulocyte macrophage colony-stimulating factor and D-dimer were measured before cART was initiated, after cART had suppressed HIV replication to less than 50 copies/ml plasma (median 8 months) and again 12 weeks after randomization to continued cART (n = 48) or interrupted cART (n = 97). Multiple linear regression and logistic regression were used to investigate the association between each cardiovascular marker and plasma HIV-RNA. Initiation of cART resulted in significant declines in s-VCAM-1, P-selectin, leptin and D-dimer, whereas mediators with anti-inflammatory properties, such as adiponectin and IL-10, increased. At 12 weeks after randomization, we found positive associations between levels of s-VCAM-1 and chemokine ligand 2 with an increase in plasma HIV-RNA (r = 0.271, P = 0.001 and r = 0.24, P = 0.005, respectively), whereas levels of adiponectin decreased for each 1 log increase in plasma HIV-RNA (r = −0.24, P = 0.002). Detectable IL-10 was less likely (odds ratio = 0.64, 95% confidence interval = 0.43–0.96) for each 1 log increase in plasma HIV-RNA. Conclusion:Plasma levels of several inflammatory, anti-inflammatory and endothelial activation markers of cardiovascular disease are associated with HIV-RNA replication.

CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways.

Recruitment of leukocytes to inflammatory sites is crucial in the pathogenesis of chronic inflammatory diseases. The aim of this study was to investigate if activation of CB2 cannabinoid receptors would modulate the chemotactic response of human monocytes. Human monocytes treated with the CB2 agonist JWH-015 for 12-18 h showed significantly reduced migration to chemokines CCL2 and CCL3, associated with reduced mRNA and surface expression of their receptors CCR2 and CCR1. The induction of ICAM-1 in response to IFN-gamma was inhibited by JWH-015. Moreover, JWH-015 cross-desensitized human monocytes for migration in response to CCL2 and CCL3 by its own chemoattractant properties. The CB2-selective antagonist SR-144528, but not the CB1 antagonist SR-147778, reversed JWH-015-induced actions, whereas the CB2 agonist JWH-133 mimicked the effects of JWH-015. The investigation of underlying pathways revealed the involvement of phosphatidylinositol 3-kinase/Akt and ERK1/2 but not p38 MAPK. In conclusion, selective activation of CB2 receptors modulates chemotaxis of human monocytes, which might have crucial effects in chronic inflammatory disorders such as atherosclerosis or rheumatoid arthritis.

Anti-Apolipoprotein A-1 auto-antibodies are active mediators of atherosclerotic plaque vulnerability

AIMS Anti-Apolipoprotein A-1 auto-antibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high cardiovascular risk. The potential relationship between anti-ApoA-1 IgG and plaque vulnerability remains elusive. Thus, we aimed to investigate the role of anti-ApoA-1 IgG in plaque vulnerability. METHODS AND RESULTS Potential relationship between anti-ApoA-1 IgG and features of cardiovascular vulnerability was explored both in vivo and in vitro. In vivo, we investigated anti-ApoA-1 IgG in patients with severe carotid stenosis (n = 102) and in ApoE-/- mice infused with polyclonal anti-ApoA-1 IgG. In vitro, anti-ApoA-1 IgG effects were assessed on human primary macrophages, monocytes, and neutrophils. Intraplaque collagen was decreased, while neutrophil and matrix metalloprotease (MMP)-9 content were increased in anti-ApoA-1 IgG-positive patients and anti-ApoA-1 IgG-treated mice when compared with corresponding controls. In mouse aortic roots (but not in abdominal aortas), treatment with anti-ApoA-1 IgG was associated with increased lesion size when compared with controls. In humans, serum anti-ApoA-1 IgG levels positively correlated with intraplaque macrophage, neutrophil, and MMP-9 content, and inversely with collagen. In vitro, anti-ApoA-1 IgG increased macrophage release of CCL2, CXCL8, and MMP-9, as well as neutrophil migration towards TNF-α or CXCL8. CONCLUSION These results suggest that anti-ApoA-1 IgG might be associated with increased atherosclerotic plaque vulnerability in humans and mice.

Tumor necrosis factor-alpha (TNF-α) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1α) on human neutrophils through defined signalling pathways

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.

At the heart of the matter: the endocannabinoid system in cardiovascular function and dysfunction

Starting from the well-documented effects of marijuana smoking on heart rate and blood pressure, the cardiovascular effects of Δ⁹-tetrahydrocannabinol (THC, the main psychotropic ingredient of Cannabis) and endocannabinoids [THC endogenous counterparts that activate cannabinoid receptor type 1 (CB₁) and 2 (CB₂)] have been thoroughly investigated. These studies were mostly aimed at establishing the molecular bases of the hypotensive actions of THC, endocannabinoids and related molecules, but also evaluated their therapeutic potential in cardiac injury protection, metabolic cardiovascular risk factors and atherosclerotic plaque vulnerability. The results of these investigations, reviewed here, also served to highlight some of the most peculiar aspects of endocannabinoid signaling, such as redundancy in endocannabinoid targets and the often dualistic role of CB₁ and CB₂ receptors during pathological conditions.

The Renin-Angiotensin System Modulates Inflammatory Processes in Atherosclerosis: Evidence from Basic Research and Clinical Studies

Recent evidence shows that the renin-angiotensin system is a crucial player in atherosclerotic processes. The regulation of arterial blood pressure was considered from its first description of the main mechanism involved. Vasoconstriction (mediated by angiotensin II) and salt and water retention (mainly due to aldosterone) were classically considered as pivotal proatherosclerotic activities. However, basic research and animal studies strongly support angiotensin II as a proinflammatory mediator, which directly induces atherosclerotic plaque development and heart remodeling. Furthermore, angiotensin II induces proatherosclerotic cytokine and chemokine secretion and increases endothelial dysfunction. Accordingly, the pharmacological inhibition of the renin-angiotensin system improves prognosis of patients with cardiovascular disease even in settings of normal baseline blood pressure. In the present review, we focused on angiotensin-convertingenzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), and renin inhibitors to update the direct activities of the renin-angiotensin system in inflammatory processes governing atherosclerosis.