Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

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.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTION: Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated. AIM: Here, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. METHODS: Hypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips. MAIN OUTCOME MEASURES: ACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. RESULTS: ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile. CONCLUSION: ACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .

Treatment with Evasin-3 abrogates neutrophil-mediated inflammation in mouse acute pancreatitis.

BACKGROUND: Acute pancreatitis is characterized by inflammatory processes affecting not only the pancreas, but also the lung. Here, we investigated timing of leucocyte infiltration and chemokine expression within lung and pancreas during pancreatitis and whether treatments selectively inhibiting chemokines (using Evasins) could improve organ injury. MATERIAL AND METHODS: C57Bl/6 mice were submitted in vivo to 10-h intraperitoneal injections of cerulein and followed for up to 168 h. Five minutes after the first cerulein injection, a single intraperitoneal injection of 10 µg Evasin-3, 1 µg Evasin-4 or an equal volume of vehicle (PBS) was performed. Leucocytes, reactive oxygen species (ROS), necrosis and chemokine/cytokine mRNA expression were assessed in different organs by immunohistology and real-time RT-PCR, respectively. RESULTS: In the lung, neutrophil infiltration and macrophage infiltration peaked at 12 h and were accompanied by increased CXCL2 mRNA expression. CCL2, CXCL1 and TNF-alpha significantly increased after 24 h as compared to baseline. No increase in CCL3 and CCL5 was observed. In the pancreas, neutrophil infiltration peaked at 6 h, while macrophages increased only after 72 h. Treatment with Evasin-3 decreased neutrophil infiltration, ROS production and apoptosis in the lung and reduced neutrophils, macrophages apoptosis and necrosis in the pancreas. Evasin-4 only reduced macrophage content in the lung and did not provide any benefit at the pancreas level. CONCLUSION: Chemokine production and leucocyte infiltration are timely regulated in lung and pancreas during pancreatitis. CXC chemokine inhibition with Evasin-3 improved neutrophil inflammation and injury, potentially interfering with damages in acute pancreatitis and related pulmonary complications.

Fatty acid amide hydrolase deficiency enhances intraplaque neutrophil recruitment in atherosclerotic mice.

OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

Statin treatment is associated with reduction in serum levels of receptor activator of NF-κB ligand and neutrophil activation in patients with severe carotid stenosis.

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.

Pannexin1 deletion in lymphatic endothelium affects lymphatic function in a sex-dependent manner.

The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex-dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex-dependent differences therein, in Prox1-CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1-CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1-CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1-CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1-CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1-CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC-specific role for Panx1 in the functions of the lymphatic system.

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.

Endogenous cannabinoid receptor CB1 activation promotes vascular smooth-muscle cell proliferation and neointima formation.

Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.

Mas receptor deficiency is associated with worsening of lipid profile and severe hepatic steatosis in ApoE-knockout mice.

The classical renin-angiotensin system pathway has been recently updated with the identification of additional molecules [such as angiotensin converting enzyme 2, ANG-(1-7), and Mas receptor] that might improve some pathophysiological processes in chronic inflammatory diseases. In the present study, we focused on the potential protective role of Mas receptor activation on mouse lipid profile, liver steatosis, and atherogenesis. Mas/apolipoprotein E (ApoE)-double-knockout (DKO) mice (based on C57BL/6 strain of 20 wk of age) were fed under normal diet and compared with aged-matched Mas and ApoE-single-knockout (KO), as well as wild-type mice. Mas/ApoE double deficiency was associated with increased serum levels of atherogenic fractions of cholesterol, triglycerides, and fasting glucose compared with wild-type or single KO. Serum levels of HDL or leptin in DKO were lower than in other groups. Hepatic lipid content as well as alanine aminotransferase serum levels were increased in DKO compared with wild-type or single-KO animals. Accordingly, the hepatic protein content of mediators related to atherosclerotic inflammation, such as peroxisome proliferator-activated receptor-α and liver X receptor, was altered in an adverse way in DKO compared with ApoE-KO. On the other hand, DKO mice did not display increased atherogenesis and intraplaque inflammation compared with ApoE-KO group. In conclusion, Mas deletion in ApoE-KO mice was associated with development of severe liver steatosis and dyslipidemia without affecting concomitant atherosclerosis. Mas receptor activation might represent promising strategies for future treatments targeting both hepatic and metabolic alterations in chronic conditions clustering these disorders.

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.

Cannabinoid receptor CB2 protects against balloon-induced neointima formation.

Cannabinoid receptor CB(2) activation inhibits inflammatory proliferation and migration of vascular smooth muscle cells in vitro. The potential in vivo relevance of these findings is unclear. We performed carotid balloon distension injury in hypercholesterolemic apolipoprotein E knockout (ApoE(-/-)) mice receiving daily intraperitoneal injection of the CB(2) agonist JWH133 (5 mg/kg) or vehicle, with the first injection given 30 min before injury. Alternatively, we subjected CB(2)(-/-) and wild-type (WT) mice to balloon injury. We determined CB(2) mRNA and protein expression in dilated arteries of ApoE(-/-) mice. Neointima formation was assessed histologically. We used bone marrow-derived murine CB(2)(-/-) and WT macrophages to study adhesion to plastic, fibronectin, or collagen, and migration was assayed by modified Boyden chamber. Aortic smooth muscle cells were isolated to determine in vitro proliferation rates. We found increased vascular CB(2) expression in ApoE(-/-) mice in response to balloon injury. Seven to twenty-one days after dilatation, injured vessels of JWH133-treated mice had less intimal nuclei numbers as well as intimal and medial areas, associated with less staining for proliferating cells, smooth muscle cells, and macrophages. Complete endothelial repair was observed after 14 days in both JWH133- and vehicle-treated mice. CB(2) deficiency resulted in increased intima formation compared with WT, whereas JWH133 did not affect intimal formation in CB(2)(-/-) mice. Apoptosis rates assessed by in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining 1 h postballooning were significantly higher in the CB(2) knockouts. In vitro, bone marrow-derived CB(2)(-/-) macrophages showed enhanced adherence and migration compared with WT cells and elevated mRNA levels of adhesion molecules, chemokine receptors CCR1 and 5, and chemokine CCL2. Proliferation rates were significantly increased in CB(2)(-/-) smooth muscle cells compared with WT. In conclusion, pharmacological activation or genetic deletion of CB(2) receptors modulate neointima formation via protective effects in macrophages and smooth muscle cells.

Serum resistin and inflammatory and endothelial activation markers in obese adolescents.

OBJECTIVES: To assess the level of serum resistin in obese and lean children and to establish a relationship with circulating inflammatory and vascular markers. STUDY DESIGN: This is a cross-sectional study including 67 obese and 62 lean children (mean age 10.9 ± 2.8 years, age range 5.4-16.6 years). We assessed circulating hormones (insulin, leptin, insulin-like growth factor 1), markers of inflammation (resistin, high sensitivity C-reactive protein, interleukin-6, chemokine ligand 2), and endothelial cell activation (vascular and intercellular adhesion molecules: vascular cell adhesion molecule 1 and intercellular adhesion molecule; E-selectin; P-selectin; endothelin 1). RESULTS: Body weight, body mass index (BMI), insulin, leptin, high-sensitivity C-reactive protein, vascular adhesion molecule 1, and E-selectin levels were significantly higher in obese than in lean subjects. Resistin was similar among the groups in the prepubertal period, but increased significantly in the obese adolescents (18.6 ± 24.9) compared with lean subjects (7.9 ± 10.7 ng/mL; P = .038). Resistin was not associated with BMI z score (P > .05). Subjects with resistin levels above 9 (ng/mL) had higher concentration of interleukin-6, chemokine ligand 2, endothelin-1, and insulin-like growth factor 1 but not of leptin, insulin, or BMI. CONCLUSION: Resistin was increased in obese adolescents independently of the quantity of the adipose tissue. In this population, increased resistin levels were related to inflammation and endothelial activation. We may hypothesize that interventions aiming to diminish resistin expression may slow down atherogenesis in adolescents.

Elevated E-selectin and diastolic blood pressure in diabetic children.

BACKGROUND: Cardiovascular risk markers are related to micro-angiopathy in children with type 1 diabetes (T1DM), but there is no information about their relationship with blood pressure (BP) and endothelial function. MATERIALS AND METHODS: This was a case-control study including 29 children with T1DM (mean age 10·5 ± 2·7 years, disease duration: 3·8 ± 2·2 years) and 39 healthy controls (mean age: 9·8 ± 2·7 years). We assessed 24-h ambulatory BP, vascular function and serum level of lipids, vascular cell adhesion molecule-1 (VCAM-1; ICAM) and selectins (E-selectin; P-selectin). RESULTS: The subject groups had similar physical characteristics and lipids level, except body mass index (BMI) which was higher in T1DM than in healthy children (18·6 ± 2·6 vs. 16·7 ± 2·5 kg/m(2), P = 0·003). Children with T1DM had increased 24 h diastolic BP z-score (0·62 ± 0·9 vs. -0·65 ± 0·8, P < 0·001), even after adjustment for BMI, as well as higher VCAM-1 concentration (492 ± 346 vs. 340 ± 225 ng/mL, P = 0·039) compared to healthy subjects. Diastolic BP z-scores were associated with disease duration, E-selectin and triglyceride levels in the T1DM group (P < 0·05). E-selectin was also related to triglycerides, otherwise there were no relationships between vascular function, markers and BP. CONCLUSION: E-selectin, an early atherosclerosis biomarker, is positively associated with diastolic BP values in children with T1DM, despite relatively short disease duration.

The inflammatory preatherosclerotic remodeling induced by intermittent hypoxia is attenuated by RANTES/CCL5 inhibition.

RATIONALE: The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients. OBJECTIVES: To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA. METHODS: We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure. MEASUREMENTS AND MAIN RESULTS: IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia. CONCLUSIONS: Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.

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.

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome.

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 µM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.

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.

Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease, and is the primary cause of heart disease and stroke in Western countries. Derivatives of cannabinoids such as delta-9-tetrahydrocannabinol (THC) modulate immune functions and therefore have potential for the treatment of inflammatory diseases. We investigated the effects of THC in a murine model of established atherosclerosis. Oral administration of THC (1 mg kg(-1) per day) resulted in significant inhibition of disease progression. This effective dose is lower than the dose usually associated with psychotropic effects of THC. Furthermore, we detected the CB2 receptor (the main cannabinoid receptor expressed on immune cells) in both human and mouse atherosclerotic plaques. Lymphoid cells isolated from THC-treated mice showed diminished proliferation capacity and decreased interferon-gamma secretion. Macrophage chemotaxis, which is a crucial step for the development of atherosclerosis, was also inhibited in vitro by THC. All these effects were completely blocked by a specific CB2 receptor antagonist. Our data demonstrate that oral treatment with a low dose of THC inhibits atherosclerosis progression in the apolipoprotein E knockout mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, THC or cannabinoids with activity at the CB2 receptor may be valuable targets for treating atherosclerosis.