Clara Di Filippo

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)

Myocardial lipid accumulation in patients with pressure-overloaded heart and metabolic syndrome

We evaluated the role of sterol-regulatory element binding protein (SREBP)-1c/peroxisome proliferator activated receptor-&ggr; (PPAR&ggr;) pathway on heart lipotoxicity in patients with metabolic syndrome (MS) and aortic stenosis (AS). Echocardiographic parameters of heart function and structural alterations of LV specimens were studied in patients with (n = 56) and without (n = 61) MS undergoing aortic valve replacement. Tissues were stained with hematoxylin-eosin (H and E) and oil red O for evidence of intramyocyte lipid accumulation. The specimens were also analyzed with PCR, Western blot, and immunohistochemical analysis for SREBP-1c and PPAR&ggr;. Ejection fraction (EF) was lower in MS compared with patients without MS (P < 0.001); no difference was found in aortic orifice surface among the groups. H and E and oil red O staining of specimens from MS patients revealed several myocytes with intracellular accumulation of lipid, whereas these alterations were not detected in biopsies from patients without MS. Patients without MS have low levels and weak immunostaining of SREBP-1c and PPAR&ggr; in heart specimens. In contrast, strong immunostaining and higher levels of SREBP-1c and PPAR&ggr; were seen in biopsies from the MS patients. Moreover, we evidenced a significative correlation between both SREBP-1c and PPAR&ggr; and EF and intramyocyte lipid accumulation (P < 0.001). SREBP-1c may contribute to heart dysfunction by promoting lipid accumulation within myocytes in MS patients with AS; SREBP-1c may do it by increasing the levels of PPAR&ggr; protein.

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.

Tight Glycemic Control Reduces Heart Inflammation and Remodeling During Acute Myocardial Infarction in Hyperglycemic Patients

OBJECTIVES We analyzed the molecular mechanisms evoked by tight glycemic control during post-infarction remodeling in human hearts. BACKGROUND The molecular mechanisms by which tight glycemic control improves heart remodeling during acute myocardial infarction (AMI) are still largely unknown. METHODS Eighty-eight patients with first AMI undergoing coronary bypass surgery were studied: 38 normoglycemic patients served as the control group; hyperglycemic patients (glucose >or=140 mg/dl) were randomized to intensive glycemic control (IGC) (n = 25; glucose 80 to 140 mg/dl) or conventional glycemic control (CGC) (n = 25; glucose 180 to 200 mg/dl) for almost 3 days before surgery, with insulin infusion followed by subcutaneous insulin treatment. Echocardiographic parameters were investigated at admission and after treatment period. During surgery, oxidative stress (nitrotyrosine, superoxide anion [O(2)(-)] production, inducible nitric oxide synthase [iNOS]), inflammation (nuclear factor kappa B [NFkappaB], tumor necrosis factor [TNF]-alpha, and apoptosis (caspase-3) were analyzed in biopsy specimens taken from the peri-infarcted area. RESULTS Compared with normoglycemic patients, hyperglycemic patients had higher myocardial performance index (MPI) (p < 0.05), reduced ejection fraction (p < 0.05), more nitrotyrosine, iNOS, and O(2)(-) production, more macrophages, T-lymphocytes, and HLA-DR (Dako, Milan, Italy) cells, and more NFkappaB-activity, TNF-alpha, and caspase-3 levels (p < 0.01) in peri-infarcted specimens. After the treatment period, plasma glucose reduction was greater in the IGC than in the CGC group (p < 0.001). Compared with IGC patients, CGC patients had higher MPI (p < 0.02), had lower ejection fraction (p < 0.05), and had more markers of oxidative stress, more inflammation and apoptosis (p < 0.01) in peri-infarcted specimens. CONCLUSIONS Tight glycemic control, by reducing oxidative stress and inflammation, might reduce apoptosis in peri-infarcted areas and remodeling in AMI patients.

Lipocortin 1 reduces myocardial ischemia-reperfusion injury by affecting local leukocyte recruitment

We assessed here the effect of the glucocorticoid‐regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25‐min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with ~50% inhibition at the highest dose tested of 50 per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose‐dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor α, and macrophage inflammatory protein‐1α. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 μg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte‐dependent myocardial damage associated with an ischemia‐reperfusion procedure.

Circulating microRNA changes in heart failure patients treated with cardiac resynchronization therapy: responders vs. non-responders

MicroRNAs (miRNAs) play an important role in the pathogenesis of structural alterations of the failing heart through their ability to regulate negatively the expression levels of genes that govern the process of adaptive and maladaptive cardiac remodelling. We studied whether LV reverse remodelling after CRT was associated with changes of circulating miRNAs in patients with heart failure (HF) and dyssynchrony.

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.

The ubiquitin-proteasome system contributes to the inflammatory injury in ischemic diabetic myocardium: the role of glycemic control.

BACKGROUND Because the ubiquitin-proteasome pathway (UPS) is required for activation of nuclear factor kappa beta (NFkB), a transcription factor that regulates inflammatory genes, we evaluated the UPS activity, NFkB activation, and tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, in ischemic specimens of diabetic myocardium and relate them to the glycemic control (HbA(1c)), oxidative stress (nitrotyrosine, a modified amino acid produced by reactive O(2)), and cardiac outcome (echocardiographic parameters). Moreover, the role of UPS, NFkB, and TNF-alpha in the cardiac tissue injury of acute ischemia/reperfusion (I/R) was evaluated in streptozotocin (STZ)-hyperglycemic rats. Finally, this study aimed to elucidate whether an intervention on UPS with bortezomib, an inhibitor of UPS, may counteract the extensive myocardial infarction and increased inflammatory reaction into the hyperglycemic myocardium. METHODS Ventricular biopsy specimens from 16 nondiabetic and 18 type 2 diabetic patients presenting with unstable angina who underwent coronary artery bypass were collected during coronary bypass surgery. Ejection fraction (EF); myocardial performance index (MPI), which measures both systolic and diastolic function, immunostaining, and cardiac levels of nitrotyrosine; UPS activity; NFkB; and TNF-alpha were investigated in both ischemic human myocardium and heart tissue from STZ-hyperglycemic rats subject to a myocardial ischemia/reperfusion procedure. RESULTS We found that diabetic patients had higher MPI (P<.041) and reduced EF (P<.008) compared with nondiabetic patients. Diabetic specimens had higher nitrotyrosine, UPS activity, NFkB, and TNF-alpha levels compared with nondiabetic patients (P<.001). This was mirrored by consistently high levels of UPS and inflammatory markers in STZ-infarcted hearts, associated with high myocardial damage. In contrast, lesions from normoglycemic animals as well as from hyperglycemic rats treated with bortezomib showed low levels of ubiquitin-proteasome activity, inflammation, and myocardial damage (P<.01). CONCLUSIONS By contributing to the increased inflammation, the UPS overactivity may enhance the risk of complication during myocardial ischemia in diabetic patients.