Treatment with anti-RANKL antibody reduces infarct size and attenuates dysfunction impacting on neutrophil-mediated injury.

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.

Macrophage-specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP-9 pathways.

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice.

Genetic deletion of the adaptor protein p66Shc increases susceptibility to short-term ischaemic myocardial injury via intracellular salvage pathways.

AIMS: Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion. METHODS AND RESULTS: Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT. CONCLUSIONS: Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients.

Role of NADPH oxidase isoforms NOX1, NOX2 and NOX4 in myocardial ischemia/reperfusion injury.

Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia-reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease.

CC chemokine CCL5 plays a central role impacting infarct size and post-infarction heart failure in mice.

AIMS: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS: C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION: Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques.

AIMS: The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS: The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION: Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Articular Cartilage Repair After Implantation of Hyaline Cartilage Beads Engineered From Adult Dedifferentiated Chondrocytes: Cartibeads Preclinical Efficacy Study in a Large Animal Model.

BACKGROUND: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. HYPOTHESIS: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. RESULTS: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). CONCLUSION: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. CLINICAL RELEVANCE: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

Manganese kinetics demonstrated double contrast in acute but not in chronic infarction in a mouse model of myocardial occlusion reperfusion.

Manganese (Mn(2+)) is considered as a specific MRI contrast agent that enters viable cardiomyocytes through calcium pathways. Compared to extracellular gadolinium based contrast agents, it has the potential to assess cell viability. To date, only information from the washout phase after recirculation has been used for the detection and characterization of myocardial infarct. This study showed for the first time that in a mouse model of coronary occlusion-reperfusion, Mn(2+) wash-in kinetics are different at 24 h after surgery (acute infarction) than at eight days after surgery (chronic infarction). A fast but transient entry of Mn(2+) into the acute infarct area led to a double contrast between infarct and remote areas, whereas entry of Mn(2+) into the chronic infarct area remained reduced compared to remote regions during both wash-in and washout phases. The main hypothesis is that extracellular space is largely enhanced in acute infarction due to cell membrane rupture and interstitial edema, whereas scar tissue is densely composed of collagen fibers that reduce the distribution volume of free Mn(2+) ions. In addition to its ability to accurately depict the infarct area during the redistribution phase, Mn(2+) is also able to discriminate acute versus chronic injury by the observation of double-contrast kinetics in a mouse model of ischemia reperfusion.

Pathophysiological role of inflammatory molecules in paediatric ischaemic brain injury.

Ischaemic stroke is one of the major causes of death and lifelong disability also in the paediatric population. Strong scientific effort has been put to clarify the pathophysiology of this disease in adults. However, only few studies have been performed in children. Preliminary results indicate that pathophysiological processes might differently affect the poststroke neuronal injury in neonates as compared to children. During the neural development, selective molecular mechanisms might be differently triggered by an ischaemic insult, thus potentially resulting in defined postischaemic clinical outcomes. Basic research studies in neonatal animal models of cerebral ischaemia have recently shown a potential role of soluble inflammatory molecules (such as cytokines, chemokines and oxidants) as pivotal players of neuronal injury in both perinatal and childhood ischaemic stroke. Although larger clinical trials are still needed to confirm these preliminary results, the potential benefits of selective treatments targeting inflammation in perinatal asphyxia encephalopathy might represent a promising investigation field in the near future. In this review, we will update evidence on the pathophysiological role of soluble inflammatory mediators in neonatal and childhood ischaemic stroke. Recent evidence on potential anti-inflammatory treatments to improve paediatric stroke prognosis will be discussed.

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.

MicroCT Can Characterize Clots Retrieved With Mechanical Thrombectomy From Acute Ischemic Stroke Patients-A Preliminary Report.

Background: Characterization of the clot occluding the arteries in acute ischemic stroke received ample attention, in terms of elucidating the relationship between the clot composition, its etiology and its amenability for pharmacological treatment and mechanical thrombectomy approaches. Traditional analytical techniques such as conventional 2D histopathology or electron microscopy sample only small parts of the clot. Visualization and analysis in 3D are necessary to depict and comprehend the overall organization of the clot. The aim of this study is to investigate the potential of microCT for characterizing the clot composition, structure, and organization. Methods: In a pilot study, we analyzed with microCT clots retrieved from 14 patients with acute ischemic stroke. The following parameters were analyzed: overall clot density, clot segmentation with various density thresholds, clot volume. Results: Our findings show that human clots are heterogeneous in terms of CT intra-clot density distribution. After fixation in formalin, the clots display a shift toward negative values. On average, we found the mean HU values of red clots retrieved from patients to be -153 HU, with SD = 23.8 HU, for the intermediate clots retrieved from patients -193 HU, SD = 23.7 HU, and for the white clots retrieved from patients -229 HU, SD = 64.8 HU. Conclusion: Our study shows that volumetric and density analysis of the clot opens new perspectives for clot characterization and for a better understanding of thrombus structure and composition.

Hyaline Cartilage Microtissues Engineered from Adult Dedifferentiated Chondrocytes: Safety and Role of WNT Signaling.

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions.

Single administration of the CXC chemokine-binding protein Evasin-3 during ischemia prevents myocardial reperfusion injury in mice.

OBJECTIVE: Evasins (chemokine-binding proteins) have been shown to selectively neutralize chemokine bioactivity. We investigated the potential benefits of Evasin-3 on mouse myocardial ischemia/reperfusion injury. METHODS AND RESULTS: In vivo and ex vivo (Langendorff model) left coronary artery ligature was performed in C57Bl/6 mice. Coronary occlusion was maintained for 30 minutes, followed by different times (up to 24 hours) of reperfusion. Five minutes after coronary occlusion, mice received 1 intraperitoneal injection of Evasin-3 or vehicle. Infarct size was assessed histologically and by serum cardiac troponin I ELISA. In vitro neutrophil chemotaxis, immunohistology, oxidative stress quantification, real-time RT-PCR analysis of leukocyte chemoattractants, and Western blots for cardioprotective intracellular pathway activation were performed. Evasin-3 reduced infarct size and cardiac troponin I levels compared with vehicle. This effect was associated with the reduction of neutrophil infiltration and reactive oxygen species production within the infarcted myocardium. Evasin-3 did not reduce infarct size in the absence of circulating neutrophils (Langendorff model). Evasin-3 did not influence the activation of intracellular cardioprotective pathways or the expression of leukocyte chemoattractants during early phases of reperfusion. CONCLUSIONS: Single administration of Evasin-3 during myocardial ischemia significantly reduced infarct size by preventing CXC chemokine-induced neutrophil recruitment and reactive oxygen species production in myocardial ischemia/reperfusion.

Chemokine CCL5/RANTES inhibition reduces myocardial reperfusion injury in atherosclerotic mice.

Although beneficial for cardiomyocyte salvage and to limit myocardial damage and cardiac dysfunction, restoration of blood flow after prolonged ischemia exacerbates myocardial injuries. Several deleterious processes that contribute to cardiomyocyte death have been proposed, including massive release of reactive oxygen species, calcium overload and hypercontracture development or leukocyte infiltration within the damaged myocardium. Chemokines are known to enhance leukocyte diapedesis at inflammatory sites. The aim of the present study was to investigate the effect of chemokine CCL5/RANTES antagonism in an in vivo mouse model of ischemia and reperfusion. ApoE(-/-) mice were submitted to 30 min ischemia, by ligature of the left coronary artery, followed by 24 h reperfusion. Intraperitoneal injection of 10 mug of CCL5/RANTES antagonist [(44)AANA(47)]-RANTES, 5 min prior to reperfusion, reduced infarct size as well as Troponin I serum levels compared to PBS-treated mice. This beneficial effect of [(44)AANA(47)]-RANTES treatment was associated with reduced leukocyte infiltration into the reperfused myocardium, as well as decreased chemokines Ccl2/Mcp-1 and Ccl3/Mip-1alpha expression, oxidative stress, and apoptosis. However, mice deficient for the CCL5/RANTES receptor Ccr5 did not exhibit myocardium salvage in our model of ischemia-reperfusion. Furthermore, [(44)AANA(47)]-RANTES did not mediate cardioprotection in these ApoE(-/-) Ccr5(-/-) deficient mice, probably due to enhanced expression of compensatory chemokines. This study provides the first evidence that inhibition of CCL5/RANTES exerts cardioprotective effects during early myocardial reperfusion, through its anti-inflammatory properties. Our findings indicate that blocking chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce reperfusion injuries in patients during acute coronary syndromes.

Myocardial infarction quantification with Manganese-Enhanced MRI (MEMRI) in mice using a 3T clinical scanner.

Manganese (Mn(2+)) was recognized early as an efficient intracellular MR contrast agent to assess cardiomyocyte viability. It had previously been used for the assessment of myocardial infarction in various animal models from pig to mouse. However, whether Manganese-Enhanced MRI (MEMRI) is also able to assess infarction in the acute phase of a coronary occlusion reperfusion model in mice has not yet been demonstrated. This model is of particular interest as it is closer to the situation encountered in the clinical setting. This study aimed to measure infarction volume taking TTC staining as a gold standard, as well as global and regional function before and after Mn(2+) injection using a clinical 3T scanner. The first step of this study was to perform a dose-response curve in order to optimize the injection protocol. Infarction volume measured with MEMRI was strongly correlated to TTC staining. Ejection fraction (EF) and percent wall thickening measurements allowed evaluation of global and regional function. While EF must be measured before Mn(2+) injection to avoid bias introduced by the reduction of contrast in cine images, percent wall thickening can be measured either before or after Mn(2+) injection and depicts accurately infarct related contraction deficit. This study is the first step for further longitudinal studies of cardiac disease in mice on a clinical 3T scanner, a widely available platform.

CB(2) 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.

A novel RANTES antagonist prevents progression of established atherosclerotic lesions in mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease that represents the primary cause of death through coronary disease and stroke. Chemokines are known to play a crucial role in this disease by recruiting inflammatory leukocytes to the endothelium. Recently, the chemokine variant [44AANA47]-RANTES was shown to impair inflammatory cell recruitment in vivo by interfering with heparin binding and oligomerization. METHODS AND RESULTS: In this study we report that curative treatment with [44AANA47]-RANTES limits atherosclerotic plaque formation in LDLr-/- mice. This was associated with reduced infiltration of T cells and macrophages and reduced production of matrix metalloproteinase (MMP)-9. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. In addition, we provide evidence for direct inhibition of leukocyte recruitment into aortic root lesions, attenuated leukocyte rolling and arrest in mesenteric vessels, as well as a reduced proinflammatory response following Con A stimulation in vitro. CONCLUSIONS: Interference with chemokine oligomerization and chemokine/heparin interactions is a powerful novel approach that inhibits progression of established atherosclerosis in mice. By inhibiting leukocyte recruitment into plaques, [44AANA47]-RANTES mediates a less inflammatory plaque phenotype and thus reduced systemic inflammatory state.

PARP1 is required for adhesion molecule expression in atherogenesis.

AIMS: Atherosclerosis is the leading cause of death in Western societies and a chronic inflammatory disease. However, the key mediators linking recruitment of inflammatory cells to atherogenesis remain poorly defined. Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme, which plays a role in acute inflammatory diseases. METHODS AND RESULTS: In order to test the role of PARP in atherogenesis, we applied chronic pharmacological PARP inhibition or genetic PARP1 deletion in atherosclerosis-prone apolipoprotein E-deficient mice and measured plaque formation, adhesion molecules, and features of plaque vulnerability. After 12 weeks of high-cholesterol diet, plaque formation in male apolipoprotein E-deficient mice was decreased by chronic inhibition of enzymatic PARP activity or genetic deletion of PARP1 by 46 or 51%, respectively (P < 0.05, n >or= 9). PARP inhibition or PARP1 deletion reduced PARP activity and diminished expression of inducible nitric oxide synthase, vascular cell adhesion molecule-1, and P- and E-selectin. Furthermore, chronic PARP inhibition reduced plaque macrophage (CD68) and T-cell infiltration (CD3), increased fibrous cap thickness, and decreased necrotic core size and cell death (P < 0.05, n >or= 6). CONCLUSION: Our data provide pharmacological and genetic evidence that endogenous PARP1 is required for atherogenesis in vivo by increasing adhesion molecules with endothelial activation, enhancing inflammation, and inducing features of plaque vulnerability. Thus, inhibition of PARP1 may represent a promising therapeutic target in atherosclerosis.

Sex-related differences in wall remodeling and intraluminal thrombus resolution in a rat saccular aneurysm model.

OBJECTIVE: Intracranial aneurysms (IAs) are more often diagnosed in women. Hormones and vessel geometry, which influences wall shear stress, may affect pathophysiological processes of the arterial wall. Here, the authors investigated sex-related differences in the remodeling of the aneurysm wall and in intraluminal thrombus resolution. METHODS: A well-characterized surgical side-wall aneurysm model was used in female, male, and ovariectomized rats. Decellularized grafts were used to model highly degenerated and decellularized IA walls and native grafts to model healthy IA walls. Aneurysm growth and thrombus composition were analyzed at 1, 7, 14, and 28 days. Sex-related differences in vessel wall remodeling were compared with human IA dome samples of men and pre- and postmenopausal women. RESULTS: At 28 days, more aneurysm growth was observed in ovariectomized rats than in males or non-ovariectomized female rats. The parent artery size was larger in male rats than in female or ovariectomized rats, as expected. Wall inflammation increased over time in all groups and was most severe in the decellularized female and ovariectomized groups at 28 days compared with the male group. Likewise, in these groups the most elastin fragmentation was seen at 28 days. In female rats, on days 1, 7, and 14, the intraluminal thrombus was mainly composed of red blood cells and fibrin. On days 14 and 28, macrophage and smooth muscle cell invasion inside the thrombus was shown, leading to the removal of red blood cells and deposition of collagen and elastin. On days 14 and 28, similar profiles of thrombus reorganization were observed in male and ovariectomized female rats. However, collagen content in thrombi and vessel wall macrophage content were higher in aneurysms of male rats at 28 days than in those of female rats. On day 28, thrombus coverage by endothelial cells was lower in ovariectomized than in female or male rats. Finally, analysis of human IA domes showed that endothelial cell coverage was lower in men and postmenopausal women than in younger women. CONCLUSIONS: Aneurysm growth and intraluminal thrombus resolution show sex-dependent differences. While certain processes (endothelial cell coverage and collagen deposition) point to a strong hormonal dependence, others (wall inflammation and aneurysm growth) seem to be influenced by both hormones and parent artery size.

Ccr5 but not Ccr1 deficiency reduces development of diet-induced atherosclerosis in mice.

OBJECTIVE: Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined. METHODS AND RESULTS: We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E-deficient (ApoE-/-) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE-/- Ccr5-/- mice, ApoE-/- Ccr1-/- mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE-/- Ccr5-/- mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-gamma content were increased in ApoE-/- Ccr1-/- mice. CONCLUSIONS: Genetic deletion of Ccr5 but not Ccr1 in ApoE-/- mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.