Michele D’Amico

Cannabinoid CB2 receptor activation reduces mouse myocardial ischemia‐reperfusion injury: involvement of cytokine/chemokines and PMN

In this study, we have assessed the activation of the cannabinoid CB2 receptor (CB2‐R) in a model of mouse myocardial ischemia/reperfusion (I/R). The results show that treatment of animals with WIN55212‐2, a CB1/CB2‐R agonist, given 30 min before induction of I/R, significantly reduced the extent of infarct size (IS) in the area at risk, as measured 2.5 h later, with almost a 51% inhibition observed at the dose tested of 3.5 mg/kg intraperitoneally (i.p.). The protective effect of WIN55212‐2 was almost abolished by the selective CB2‐R antagonist AM630 (1 mg/kg i.p.) and not affected by the selective CB1‐R antagonist AM251 (3 mg/kg i.p.). The CB2‐R antagonist administered alone produced a slight but significant (P<0.05) increase in IS compared with vehicle alone. The protection afforded by WIN55212‐2 was paralleled by lower values of myeloperoxidase activity and interleukin‐1β and of the CXC chemokine ligand 8 into the injured tissue. In conclusion, we demonstrate for the first time that exogenous and endogenous CB2‐R activation reduces the leukocyte‐dependent myocardial damage associated with an I/R procedure.

Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action

Myocardial reperfusion injury is associated with the infiltration of blood‐borne polymorphonuclear leukocytes. We have previous described the protection afforded by annexin 1 (ANXA1) in an experimental model of rat myocardial ischemia‐reperfu‐sion (IR) injury. We examined the 1) amino acid region of ANXA1 that retained the protective effect in a model of rat heart IR;2) changes in endogenous ANXA1 in relation to the IR induced damage and after pharmacological modulation;and 3) potential involvement of the formyl peptide receptor (FPR) in the protective action displayed by ANXA1 peptides. Administration of peptide Ac2–26 at 0, 30, and 60 min postreperfusion produced a significant protection against IR injury, and this was associated with reduced myeloperoxidase activity and IL‐1ß levels in the infarcted heart. Western blotting and electron microscopy analyses showed that IR heart had increased ANXA1 expression in the injured tissue, associated mainly with the infiltrated leukocytes. Finally, an antagonist to the FPR receptor selectively inhibited the protective action of peptide ANXA1 and its derived peptides against IR injury. Altogether, these data provide further insight into the protective effect of ANXA1 and its mimetics and a rationale for a clinical use for drugs developed from this line of research.—La, M., D’Amico, M., Bandiera, S., Di Filippo, C., Oliani, S. M., Gavins, F. N. E., Flower, R. J., Perretti, M. Annexin 1 peptides protect against experimental myocardial ischemia‐reperfusion: analysis of their mechanism of action. FASEB J. 15, 2247–2256 (2001)

Long-term inhibition of dipeptidyl peptidase-4 in Alzheimer’s prone mice

We tested here the impact of a long-term inhibition of dipeptidyl peptidase-4 (DPP-4) with sitagliptin on the deposition of amyloid-beta within the brain and deficits in memory-related behavioral paradigms in a model of Alzheimers disease (AD): double transgenic mice B6*Cg-Tg(APPswe,PSEN1dE9)85Dbo/J. Mice began to receive sitagliptin at 7 months of age. Three different dose of sitagliptin (5, 10 and 20 mg/kg), were administered daily for 12 weeks by gastric gavage. The treatments counteracted: (i) the memory impairment in the contextual fear conditioning test; (ii) increased the brain levels of GLP-1; (iii) produced significant reductions of nitrosative stress and inflammation hallmarks within the brain, as well as (iv) a significant diminution in the ultimate number and total area of betaAPP and Abeta deposits. All these effects much more evident for the dose of 20 mg/kg sitagliptin. The long-term inhibition of the endogenous DPP-4 enzymes with sitagliptin can significantly delay some forms of AD pathology, including amyloid deposition, when administered early in the disease course of a transgenic mouse model of AD.

Morning Blood Pressure Surge as a Destabilizing Factor of Atherosclerotic Plaque Role of Ubiquitin–Proteasome Activity

Whether morning blood pressure surge influences the molecular mechanisms of plaque progression toward instability is not known. Recently, we have demonstrated enhanced activity of the ubiquitin–proteasome system in human plaques and evidenced that it is associated with inflammatory-induced plaque rupture. We evaluated the inflammatory infiltration and ubiquitin–proteasome activity in asymptomatic carotid plaques of hypertensive patients with different patterns of morning blood pressure surge. Plaques were obtained from 32 hypertensive patients without morning blood pressure surge and 28 with morning blood pressure surge enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis. Plaques were analyzed for macrophages, T-lymphocytes, human leukocyte antigen–DR+cells, ubiquitin–proteasome activity, nuclear factor-&kgr;B, inhibitor kB-&bgr;, tumor necrosis factor-&agr;, nitrotyrosine, matrix metalloproteinase-9, and collagen content (immunohistochemistry and ELISA). Compared with plaques obtained from hypertensive patients without morning blood pressure surge, plaques from with morning blood pressure surge had more macrophages, T-lymphocytes, human leukocyte antigen–DR+cells (P<0.001), ubiquitin-proteasome activity, tumor necrosis factor-&agr;, nuclear factor-kB (P<0.001), nitrotyrosine, and matrix metalloproteinase-9 (P<0.01), along with a lesser collagen content and IkB-&bgr; levels (P<0.001). Enhanced ubiquitin–proteasome activity in atherosclerotic lesions of patients with morning blood pressure surge is associated with inflammatory-dependent unstable plaque phenotype. These data suggest a potential interplay between morning blood pressure surge and ubiquitin–proteasome activity in atherosclerosis pathophysiology.

Proresolving and Tissue-Protective Actions of Annexin A1–Based Cleavage-Resistant Peptides Are Mediated by Formyl Peptide Receptor 2/Lipoxin A4 Receptor

Endogenous mechanisms regulating the host response during inflammation resolution are critical in ensuring disposal of noxious stimuli and return to homeostasis. In this article, we engineered novel Annexin A1 (AnxA1)–based peptides, AnxA12–50, that displayed specific binding to the AnxA1 receptor (formyl peptide receptor 2/Lipoxin A4 receptor [FPR2/ALX]; IC50 ∼4 nM). Intravenous administration of AnxA12–50 markedly reduced (>60%) leukocyte adhesion to postcapillary venules in wild type and Fpr1−/−, but not Fpr2/Alx−/−, mice. Generation of a metabolically stable form of this peptide (CR-AnxA12–50), engineered by substituting a cleavage site shared by human proteinase 3 and neutrophil elastase, yielded an agonist that was resistant to neutrophil-mediated cleavage and displayed enhanced proresolving actions: accelerated resolution of self-limited inflammation and enhanced macrophage efferocytosis after sterile injury, when compared with AnxA12–50. These actions were retained with human primary leukocytes where CR-AnxA12–50 decreased neutrophil–endothelial interactions (∼25–45%), and stimulated neutrophil apoptosis and macrophage efferocytosis (∼45%). In murine cardiac ischemia/reperfusion injury, CR-AnxA12–50 elicited tissue-protective actions reducing infarct size (∼60%) and incidence of 24-h death. These results identify AnxA12–50 and CR-AnxA12–50 as FPR2/ALX agonists that harness the proresolving actions of AnxA1, and thus may represent therapeutic tools for treatment of inflammatory conditions.

Recruitment of classical monocytes can be inhibited by disturbing heteromers of neutrophil HNP1 and platelet CCL5

Disrupting the HNP1 and CCL5 heteromer between neutrophils and platelets blocks monocyte recruitment to inflammatory sites. Anti-inflammatory reaches for the SKY Inflammation aids the body’s response to infection or injury, but can cause damage if excessive or unresolved. Alard et al. now examine how two early inflammatory mediators—neutrophils and platelets—cooperate to enhance inflammation. They found that human neutrophil peptide 1 (HNP1), which is secreted from neutrophils, forms a heteromer with CCL5 on platelets, resulting in stimulated monocyte adhesion and an increase in inflammation. Disrupting this interaction with a peptide (SKY) decreased inflammation and blocked monocyte recruitment in a mouse model of myocardial infarction. If these results hold true in humans, they could form the basis for a new specific therapeutic in inflammation-associated diseases. In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.

Proteasome Activity as a Target of Hormone Replacement Therapy–Dependent Plaque Stabilization in Postmenopausal Women

The molecular mechanisms of the atheroprotective effect evoked by hormone replacement therapy in postmenopausal women is not well known. Recently, we have demonstrated enhanced activity of the ubiquitin-proteasome system in human atherosclerotic plaques and evidenced that it is associated with inflammatory-induced plaque rupture. Therefore, we hypothesized that hormone replacement therapy may exert the cardioprotective effects modulating the ubiquitin-proteasome activity. To investigate this possibility, this study examined the differences in inflammatory infiltration, as well as ubiquitin-proteasome activity, between asymptomatic carotid plaques of postmenopausal women with and without concomitant hormone replacement therapy. Plaques were obtained from 20 postmenopausal women treated with hormone replacement therapy (current users) and 32 nontreated women (never-users) enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis. Plaques were analyzed for macrophages, T lymphocytes, human leukocyte antigen-DR+ cells, ubiquitin-proteasome system, nuclear factor &kgr;B, inhibitor of nuclear factor &kgr;B&bgr;, tumor necrosis factor-&agr;, nitrotyrosine, matrix metalloproteinase-9, and collagen content (immunohistochemistry and ELISA). Compared with plaques from current users, plaques from never-users had more macrophages, T lymphocytes, and human leukocyte antigen-DR+ cells (P<0.001); more ubiquitin-proteasome activity, tumor necrosis factor-&agr;, and nuclear factor &kgr;B (P<0.001); and more nitrotyrosine and matrix metalloproteinase-9 (P<0.001), along with a lesser collagen content and inhibitor of nuclear factor &kgr;B&bgr; levels (P<0.001). This study supports the hypothesis that hormone replacement therapy inhibits plaque ubiquitin-proteasome activity by decreasing oxidative stress generation in postmenopausal women. This effect, in turn, might contribute to plaque stabilization by inhibiting the activation of nuclear factor &kgr;B–dependent inflammation, responsible for plaque rupture.

Diabetes, Ubiquitin Proteasome System and Atherosclerotic Plaque Rupture

To the Editor: We read with interest the review of Kanter and coworkers about the role of glucose and lipids on atherosclerotic lesion initiation or progression to advanced plaques.1 He pointed out that hyperglycemia alone appears insufficient to accelerate atherosclerosis, at least in various animal models. He adds that little is still known about the role of diabetes in accelerating cardiovascular events merely by inducing an accelerated progression of atherosclerotic lesions. Finally, he concludes that studies on advanced lesions will be necessary to further our knowledge on the cellular and molecular mechanisms whereby diabetes leads to cardiovascular events. This gap in knowledge, reducing the role of the hyperglycemia on atherosclerosis progression toward plaque rupture, may limit the therapeutic strategy to reduce the impact of diabetes on cardiovascular events. In recent years, it has been firmly established that inflammation contributes to plaque rupture and cardiovascular events.2 Several inflammatory markers have …

Protection from Endotoxic Uveitis by Intravitreal Resolvin D1: Involvement of Lymphocytes, miRNAs, Ubiquitin-Proteasome, and M1/M2 Macrophages

This study investigated the protective effects of intravitreal Resolvin D1 (RvD1) against LPS-induced rat endotoxic uveitis (EIU). RvD1 was administered into the right eye at a single injection of 5 μL volume containing 10–100–1000 ng/kg RvD1 1 h post-LPS injection (200 μg, Salmonella minnesota) into thefootpad of Sprague-Dawley rats. 24 h later, the eye was enucleated and examined for clinical, biochemical, and immunohistochemical evaluations. RvD1 significantly and dose-dependently decreased the clinical score attributed to EIU, starting from the dose of 10 ng/kg and further decreased by 100 and 1000 ng/kg. These effects were accompanied by changes in four important determinants of the immune-inflammatory response within the eye: (i) the B and T lymphocytes, (ii) the miRNAs pattern, (iii) the ubiquitin-proteasome system (UPS), and (iv) the M1/M2 macrophage phenotype. LPS+RvD1 treated rats showed reduced presence of B and T lymphocytes and upregulation of miR-200c-3p, miR 203a-3p, miR 29b-3p, and miR 21-5p into the eye compared to the LPS alone. This was paralleled by decreases of the ubiquitin, 20S and 26S proteasome subunits, reduced presence of macrophage M1, and increased presence of macrophage M2 in the ocular tissues. Accordingly, the levels of the cytokine TNF-α, the chemokines MIP1-α and NF-κB were reduced.

The Cardio‐Protective Properties of Ncx‐6550, a Nitric Oxide Donating Pravastatin, in the Mouse

Please cite this paper as: Di Filippo, Monopoli, Ongini, Perretti and D’Amico (2010). The Cardio‐Protective Properties of Ncx‐6550, a Nitric Oxide Donating Pravastatin, in the Mouse. Microcirculation17(6), 417–426.