Discovery of a novel cytokine signature for the diagnosis of autism spectrum disorder in young Arab children in Qatar.

Background: Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterized by impaired social interaction and communication and the occurrence of stereotyped and repetitive behaviors. Several studies have reported altered cytokine profiles in ASD and hence may serve as potential diagnostic biomarkers of the disorder. This study aims to identify diagnostic biomarkers for ASD in a well-defined study cohort in Qatar. Methods: We measured the protein levels of 45 cytokines in the plasma samples of age- and gender-matched children (2-4 years) with ASD (n = 100) and controls (n = 60) using a Luminex multiplex assay. We compared the differences in the levels of these cytokines between the two study groups and then fitted the significantly altered cytokines into a logistic regression model to examine their diagnostic potential for ASD. Results: We found elevated levels of IFN-γ, FGF-2, IL-1RA, and IL-13 and reduced levels of eotaxin, HGF, IL-1 alpha, IL-22, IL-9, MCP-1, SCF, SDF-1 alpha, VEGFA, and IP-10 in the plasma of children with ASD compared to controls. Furthermore, we observed that elevated levels of IFN-γ (odds ratio (OR) = 1.823; 95% (confidence interval) CI = 1.206, 2.755; p = 0.004) and FGF-2 (OR = 2.528; 95% CI = 1.457, 4.385; p < 0.001) were significantly associated with increased odds of ASD, whereas reduced levels of eotaxin (OR = 0.350; 95% CI = 0.160, 0.765; p = 0.008) and HGF (OR = 0.220; 95% CI = 0.070, 0.696; p = 0.010) were significantly associated with lower odds of ASD relative to controls. The combination of these four cytokines revealed an area under the curve (ROC-AUC) of 0.829 (95% CI = 0.767, 0.891; p < 0.001), which demonstrates the diagnostic accuracy of the four-cytokine signature. Conclusions: Our results identified a panel of cytokines that could discriminate between children with ASD and controls in Qatar. In addition, our findings support the predominance of a Th1 immune phenotype in ASD children and emphasize the need to validate these results in larger populations.

Validation of plasma protein glycation and oxidation biomarkers for the diagnosis of autism.

Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder in children. It is currently diagnosed by behaviour-based assessments made by observation and interview. In 2018 we reported a discovery study of a blood biomarker diagnostic test for ASD based on a combination of four plasma protein glycation and oxidation adducts. The test had 88% accuracy in children 5-12 years old. Herein, we present an international multicenter clinical validation study (N = 478) with application of similar biomarkers to a wider age range of 1.5-12 years old children. Three hundred and eleven children with ASD (247 male, 64 female; age 5.2 ± 3.0 years) and 167 children with typical development (94 male, 73 female; 4.9 ± 2.4 years) were recruited for this study at Sidra Medicine and Hamad Medical Corporation hospitals, Qatar, and Hospital Regional Universitario de Málaga, Spain. For subjects 5-12 years old, the diagnostic algorithm with features, advanced glycation endproducts (AGEs)-Nε-carboxymethyl-lysine (CML), Nω-carboxymethylarginine (CMA) and 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and oxidative damage marker, o,o'-dityrosine (DT), age and gender had accuracy 83% (CI 79 - 89%), sensitivity 94% (CI 90-98%), specificity 67% (CI 57-76%) and area-under-the-curve of receiver operating characteristic plot (AUROC) 0.87 (CI 0.84-0.90). Inclusion of additional plasma protein glycation and oxidation adducts increased the specificity to 74%. An algorithm with 12 plasma protein glycation and oxidation adduct features was optimum for children of 1.5-12 years old: accuracy 74% (CI 70-79%), sensitivity 75% (CI 63-87%), specificity 74% (CI 58-90%) and AUROC 0.79 (CI 0.74-0.84). We conclude that ASD diagnosis may be supported using an algorithm with features of plasma protein CML, CMA, 3DG-H and DT in 5-12 years-old children, and an algorithm with additional features applicable for ASD screening in younger children. ASD severity, as assessed by ADOS-2 score, correlated positively with plasma protein glycation adducts derived from methylglyoxal, hydroimidazolone MG-H1 and Nε(1-carboxyethyl)lysine (CEL). The successful validation herein may indicate that the algorithm modifiable features are mechanistic risk markers linking ASD to increased lipid peroxidation, neuronal plasticity and proteotoxic stress.

An optimized protocol for the preparation of blood immune cells for transmission electron microscopy.

Transmission electron microscopy (TEM) is a powerful technique that enables visualization of structural details inside cells. Prior to TEM imaging, biological samples must undergo several preparation steps that are optimized according to the sample type. Currently, there are limited protocols for the preparation of blood samples for TEM imaging. Here, we provide a detailed step-by-step method for preparing blood samples for TEM imaging. This protocol enables robust visualization of the ultrastructures of blood immune cells. In addition, we describe the typical cellular features that can be used to distinguish between different immune cells in the blood, such as neutrophils, eosinophils, monocytes, and lymphocytes. This protocol is useful for studying ultrastructural changes in blood immune cells under various physiological and disease conditions.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of ß-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

The Role of Oxidative Stress in the Progression of Secondary Brain Injury Following Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) can be a fatal condition responsible for the death of 1.7% of all neonates in the USA. The majority of GMH survivors develop long-term sequalae with debilitating comorbidities. Higher grade GMH is associated with higher mortality rates and higher prevalence of comorbidities. The pathophysiology of GMH can be broken down into two main titles: faulty hemodynamic autoregulation and structural weakness at the level of tissues and cells. Prematurity is the most significant risk factor for GMH, and it predisposes to both major pathophysiological mechanisms of the condition. Secondary brain injury is an important determinant of survival and comorbidities following GMH. Mechanisms of brain injury secondary to GMH include apoptosis, necrosis, neuroinflammation, and oxidative stress. This review will have a special focus on the mechanisms of oxidative stress following GMH, including but not limited to inflammation, mitochondrial reactive oxygen species, glutamate toxicity, and hemoglobin metabolic products. In addition, this review will explore treatment options of GMH, especially targeted therapy.

In search of immune cellular sources of abnormal cytokines in the blood in autism spectrum disorder: A systematic review of case-control studies.

Abnormal cytokine levels in circulating blood have been repeatedly reported in autism; however, the underlying cause remains unclear. This systematic review aimed to investigate cytokine levels in peripheral blood compartments and identify their potential immune cellular sources in subjects with autism through comparison with controls. We conducted an electronic database search (PubMed, Scopus, ProQuest Central, Ovid, SAGE Journals, and Wiley Online Library) from inception (no time limits) to July 9, 2020, and identified 75 relevant articles. Our qualitative data synthesis focused on results consistently described in at least three independent studies, and we reported the results according to the PRISMA protocol. We found that compared with controls, in subjects with autism, cytokines IL-6, IL-17, TNF-α, and IL-1ß increased in the plasma and serum. We also identified monocytes, neutrophils, and CD4+ T cells as potential sources of these elevated cytokines in autism. Cytokines IFN-γ, TGF-ß, RANTES, and IL-8 were increased in the plasma/serum of subjects with autism, and IFN-γ was likely produced by CD4+ T cells and natural killer (NK) cells, although conflicting evidence is present for IFN-γ and TGF-ß. Other cytokines-IL-13, IL-10, IL-5, and IL-4-were found to be unaltered in the plasma/serum and post-stimulated blood immune cells in autistic individuals as compared with controls. The frequencies of T cells, monocytes, B cells, and NK cells were unchanged in subjects with autism as opposed to controls, suggesting that abnormal cytokines were unlikely due to altered cell numbers but might be due to altered functioning of these cells in autism. Our results support existing studies of abnormal cytokines in autism and provide comprehensive evidence of potential cellular sources of these altered cytokines in the context of autism. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020205224, identifier [CRD42020205224].

Acute cytotoxicity, genotoxicity, and apoptosis induced by petroleum VOC emissions in A549 cell line.

Gasoline is an essential petroleum-derived product powering the automotive economy worldwide. This research focused on the Volatile Organic Component (VOC) cocktail resulting from gasoline evaporation. Petroleum fugitive VOC inhalation by petrol station attendants have been widely associated with toxicological and health risks concerns. Another unusual practice in poor nations is gasoline sniffing to get high which can lead to intoxication and organ damages. In this study, a static air/liquid interface methodology was designed to emulate acute human lung-derived cell exposure to all the gasoline-derived generated VOCs. The research investigated the cytotoxic and genotoxic end points resulting from whole gasoline fumes in vitro exposure using A549 cells. Petroleum-derived VOCs were identified and characterized by GC-MS. VOCs exposure was emulated in a controlled environment by evaporating spiked crude gasoline (1 to 100 µl) in a closed exposure chamber. In the chamber, A549 cultured cells on snapwell inserts were exposed on their apical side to various concentrations of generated vapors for one hour at 37 °C to mimic lung exposure. The results indicated that acute gasoline whole VOCs exposure reduced cell viability (IC50 = 485 ppm immediately and IC50 = 516 ppm 24 h post-exposure), disrupted cell membrane integrity though LDH leakage and induced DNA damages. Furthermore, VOC exposure triggered caspase-independent apoptosis in exposed cells through upregulation of apoptotic pathways. Overall, the presented findings generated by the static exposure technique showed a practical and reproducible model that can be used to assess acute crude VOCs mixture toxicity endpoints and cell death pathways.

Gasoline fume inhalation induces apoptosis, inflammation, and favors Th2 polarization in C57BL/6 mice.

Gasoline exposure has been widely reported in the literature as being toxic to human health. However, the exact underlying molecular mechanisms triggered by its inhalation have not been thoroughly investigated. We herein present a model of sub-chronic, static gasoline vapor inhalation in adult female C57BL/6 mice. Animals were exposed daily to either gasoline vapors (0.86 g/animal/90 min) or ambient air for 5 days/week over 7 consecutive weeks. At the end of the study period, toxic and molecular mechanisms underlying the inflammatory, oxidative, and apoptotic effects triggered by gasoline vapors, were examined in the lungs and liver of gasoline-exposed (GE) mice. Static gasoline exposure induced a significant increase (+21%) in lungs/body weight (BW) ratio in GE versus control (CON) mice along with a pulmonary inflammation attested by histological staining. The latter was consistent with increases in the transcript levels of proinflammatory cytokines [Interleukins (ILs) 4 and 6], respectively by ~ 6- and 4-fold in the lungs of GE mice compared to CON. Interestingly, IL-10 expression was also increased by ~ 10-fold in the lungs of GE mice suggesting an attempt to counterbalance the established inflammation. Moreover, the pulmonary expression of IL-12 and TNF-α was downregulated by 2- and 4-fold, respectively, suggesting the skewing toward Th2 phenotype. Additionally, GE mice showed a significant upregulation in Bax/Bcl-2 ratio, caspases 3, 8, and 9 with no change in JNK expression in the lungs, suggesting the activation of both intrinsic and extrinsic apoptotic pathways. Static gasoline exposure over seven consecutive weeks had a minor hepatic portal inflammation attested by H&E staining along with an increase in the hepatic expression of the mitochondrial complexes in GE mice. Therefore, tissue damage biomarkers highlight the health risks associated with vapor exposure and may present potential therapeutic targets for recovery from gasoline intoxication.

Sulfated alginate/polycaprolactone double-emulsion nanoparticles for enhanced delivery of heparin-binding growth factors in wound healing applications.

Diabetic foot ulcers (DFUs) that are not effectively treated could lead to partial or complete lower limb amputations. The lack of connective tissue growth factor (CTGF) and insulin-like growth factor (IGF-I) in DFUs results in limited matrix deposition and poor tissue repair. To enhance growth factor (GF) availability in DFUs, heparin (HN)-mimetic alginate sulfate/polycaprolactone (AlgSulf/PCL) double emulsion nanoparticles (NPs) with high affinity and sustained release of CTGF and IGF-I were synthesized. The NPs size, encapsulation efficiency (EE), cytotoxicity, cellular uptake and wound healing capacity in immortalized primary human adult epidermal cells (HaCaT) were assessed. The sonication time and amplitude used for NPs synthesis enabled the production of particles with a minimum of 236 ± 25 nm diameter. Treatment of HaCaT cells with up to 50 µg mL-1 of NPs showed no cytotoxic effects after 72 h. The highest bovine serum albumin EE (94.6 %, P = 0.028) and lowest burst release were attained with AlgSulf/PCL. Moreover, cells treated with AlgSulf/CTGF (250 ng mL-1) exhibited the most rapid wound closure compared to controls while maintaining fibronectin synthesis. Double-emulsion NPs based on HN-mimetic AlgSulf represent a novel approach which can significantly enhance diabetic wound healing and can be expanded for applications requiring the delivery of other HN-binding GFs.

Cardiac Phosphodiesterases Are Differentially Increased in Diabetic Cardiomyopathy.

AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

A glimpse into the history of description of the antiphospholipid syndrome.

Prior to 1983, several landmark reports prepared the stage for a detailed description of the Antiphospholipid (Hughes) syndrome (APS). Formerly depicted as lupus-like, APS exhibits a wide spectrum of symptoms that overlap with Sjogren's, Hashimoto, and other autoimmune diseases. In this review, we take a glimpse into the history of description of APS, discussing the events that led to its recognition as one of the most common autoimmune diseases and the enormous impact of that recognition in the rheumatology field.

SARS, MERS and COVID-19: clinical manifestations and organ-system complications: a mini review.

Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) are caused by three distinct coronaviruses belonging to the same genus. COVID-19 and its two predecessors share many important features in their clinical presentations, and in their propensity for progression to severe disease which is marked by high rates of morbidity and mortality. However, comparison of the three viral illnesses also reveals a number of specific differences in clinical manifestations and complications, which suggest variability in the disease process. This narrative review delineates the pulmonary, cardiac, renal, gastrointestinal, hepatic, neurological and hematologic complications associated with these three respiratory coronaviruses. It further describes the mechanisms of immune hyperactivation-particularly cytokine release syndrome-implicated in the multi-organ system injury seen in severe cases of MERS, SARS and COVID-19.

Tobacco cigarette smoking exacerbates aortic calcification in an early stage of myocardial infarction in a female mouse model.

Despite increased social awareness, marketing restraints, tobacco taxation, and available smoking cessation rehab programs, active and passive smoking remain a worldwide challenging epidemic and a key risk factor for cardiovascular diseases development. Although cardiovascular (CV) protection is more pronounced in women than in men due to estrogenic effects, tobacco cigarette smoking exposure seems to alter this protection by modulating estrogen actions via undefined mechanisms. Premenopausal cigarette smoking women are at higher risk of adverse CV effects than non-smokers. In this study, we investigated the impact of cigarette smoking on early CV injury after myocardial infarction (MI) in non-menopausal female mice. Aortic arch calcification, fibrosis, reactive oxygen species, and gene expression of inflammatory and calcification genes were exaggerated in mice exposed to cigarette smoke (CS). These findings suggest that aortic injury following MI, characterized by vascular smooth muscle cells transdifferentiation, calcification, inflammation, and collagen deposition but not cardiac dysfunction is exacerbated with CS exposure. The novel findings of this study highlight the importance of aortic injury on short and long-term prognosis in CS-exposed MI females. Linking those findings to estrogen alteration is probable and entails investigation.

Thalassemia and malignancy: An emerging concern?

The thalassemias constitute a variable group of anemias that result from autosomal recessive inherited defects in the production of hemoglobin. The life expectancy of thalassemia patients has been extended over the last decades as a result of key milestones being achieved in optimizing management with transfusion and iron chelation therapy. Such advances have prolonged the survival of thalassemia patients and improved their overall quality of life. However, this increase in life expectancy has led to the manifestation of several morbidities, including multiple types of solid and hematologic malignancies. In this review we report the different types of solid and hematological malignancies that can develop in thalassemia patients, in addition to the possible predisposing factors and mechanisms behind their development.

Insights into the diagnosis and pathogenesis of the antiphospholipid syndrome.

The Antiphospholipid syndrome (APS), formerly known as Anticardiolipin or Hughes syndrome, is a systemic autoimmune disorder characterized by obstetrical complications and thrombotic events affecting almost every organ-system in patients persistently testing positive for antiphospholipid antibodies (aPL). The contribution of the extra-criteria aPL to the pathogenesis of APS have exceeded the expectations of a simple, direct pathologic 'hit' leading to thrombogenesis or obstetrical complications, and more pathologic pathways are being linked directly or indirectly to aPL. The value of extra-criteria aPL is on the rise, and these antibodies are nowadays evaluated as markers for risk assessment in the diagnostic approach to APS. A diagnosis of APS should be considered in pediatric patients with suggestive clinical and laboratory picture. Management of APS remains mostly based on anticoagulation, while other drugs are being tested for efficacy and side effects. Low-dose aspirin may have a role in the management of thrombotic and obstetric APS. Due to the high variability in disease severity and complication recurrence outcomes, new tools are being developed and validated to assess the damage index and quality of life of APS patients.

Arterial stenosis in antiphospholipid syndrome: Update on the unrevealed mechanisms of an endothelial disease.

First described in 1983, antiphospholipid syndrome (APS) is an autoimmune condition characterized by the occurrence of recurrent arterial and/or venous thrombosis, and/or pregnancy morbidity, in the setting of persistent presence of antiphospholipid antibodies (aPL). While thrombosis is the most well-known pathogenic mechanism in this disorder, the relevance of some other mechanisms such as arterial stenosis is being increasingly recognized. Arterial stenosis has been first described in the renal arteries in patients with APS, however intracranial and coeliac arteries can also be involved with various and treatable clinical manifestations. The underlying pathophysiology of this stenotic arterial vasculopathy is not fully understood but some recent studies revealed new insights into the molecular mechanism behind this endothelial cell activation in APS. In this review, we discuss these newly discovered mechanisms and highlight the diagnostic and therapeutic modalities of the APS related arterial stenosis.

Selective EGF-Receptor Inhibition in CD4+ T Cells Induces Anergy and Limits Atherosclerosis.

BACKGROUND: Several epidermal growth factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer, limiting tumor growth and metastasis. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response. OBJECTIVES: The aim of this study was to determine whether EGFR expressed on CD4+ T cells is functional and to address the consequences of EGFR inhibition in atherosclerosis, a T cell-mediated vascular chronic inflammatory disease. METHODS: The authors used EGFR tyrosine kinase inhibitors (AG-1478, erlotinib) and chimeric Ldlr-/-Cd4-Cre/Egfrlox/lox mouse with a specific deletion of EGFR in CD4+ T cells. RESULTS: Mouse CD4+ T cells expressed EGFR, and the EGFR tyrosine kinase inhibitor AG-1478 blocked in vitro T cell proliferation and Th1/Th2 cytokine production. In vivo, treatment of Ldlr-/- mice with the EGFR inhibitor erlotinib induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions, and protected against atherosclerosis development and progression. Selective deletion of EGFR in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced interferon-γ, interleukin-4, and interleukin-2 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated Ldlr-/- mice reconstituted with bone marrow from Cd4-Cre/Egfrlox/lox mice, compared to Cd4-Cre/Egfr+/+ chimeric mice, after 4, 6, and 12 weeks of high-fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vitro and in vivo. CONCLUSIONS: EGFR blockade induced T cell anergy in vitro and in vivo and reduced atherosclerosis development. Targeting EGFR may be a novel strategy to combat atherosclerosis.

Genetic Depletion or Hyperresponsiveness of Natural Killer Cells Do Not Affect Atherosclerosis Development.

RATIONALE: Chronic inflammation is central in the development of atherosclerosis. Both innate and adaptive immunities are involved. Although several studies have evaluated the functions of natural killer (NK) cells in experimental animal models of atherosclerosis, it is not yet clear whether NK cells behave as protective or proatherogenic effectors. One of the main caveats of previous studies was the lack of specificity in targeting loss or gain of function of NK cells. OBJECTIVES: We used 2 selective genetic approaches to investigate the role of NK cells in atherosclerosis: (1) Ncr1iCre/+R26lsl-DTA/+ mice in which NK cells were depleted and (2) Noé mice in which NK cells are hyperresponsive. METHODS AND RESULTS: No difference in atherosclerotic lesion size was found in Ldlr-/- (low-density lipoprotein receptor null) mice transplanted with bone marrow (BM) cells from Ncr1iCreR26Rlsl-DTA , Noé, or wild-type mice. Also, no difference was observed in plaque composition in terms of collagen content, macrophage infiltration, or the immune profile, although Noé chimera had more IFN (interferon)-γ-producing NK cells, compared with wild-type mice. Then, we investigated the NK-cell selectivity of anti-asialoganglioside M1 antiserum, which was previously used to conclude the proatherogenicity of NK cells. Anti-asialoganglioside M1 treatment decreased atherosclerosis in both Ldlr-/- mice transplanted with Ncr1iCreR26Rlsl-DTA or wild-type bone marrow, indicating that its antiatherogenic effects are unrelated to NK-cell depletion, but to CD8+ T and NKT cells. Finally, to determine whether NK cells could contribute to the disease in conditions of pathological NK-cell overactivation, we treated irradiated Ldlr-/- mice reconstituted with either wild-type or Ncr1iCreR26Rlsl-DTA bone marrow with the viral mimic polyinosinic:polycytidylic acid and found a significant reduction of plaque size in NK-cell-deficient chimeric mice. CONCLUSIONS: Our findings, using state-of-the-art mouse models, demonstrate that NK cells have no direct effect on the natural development of hypercholesterolemia-induced atherosclerosis, but may play a role when an additional systemic NK-cell overactivation occurs.

Adiponectin Attenuates Angiotensin II-Induced Vascular Smooth Muscle Cell Remodeling through Nitric Oxide and the RhoA/ROCK Pathway.

INTRODUCTION: Adiponectin (APN), an adipocytokine, exerts protective effects on cardiac remodeling, while angiotensin II (Ang II) induces hypertension and vascular remodeling. The potential protective role of APN on the vasculature during hypertension has not been fully elucidated yet. Here, we evaluate the molecular mechanisms of the protective role of APN in the physiological response of the vascular wall to Ang II. METHODS AND RESULTS: Rat aortic tissues were used to investigate the effect of APN on Ang II-induced vascular remodeling and hypertrophy. We investigated whether nitric oxide (NO), the RhoA/ROCK pathway, actin cytoskeleton remodeling, and reactive oxygen species (ROS) mediate the anti-hypertrophic effect of APN. Ang II-induced protein synthesis was attenuated by pre-treatment with APN, NO donor S-nitroso-N-acetylpenicillamine (SNAP), or cGMP. The hypertrophic response to Ang II was associated with a significant increase in RhoA activation and vascular force production, which were prevented by APN and SNAP. NO was also associated with inhibition of Ang II-induced phosphorylation of cofilin. In addition, immunohistochemistry revealed that 24 h Ang II treatment increased the F- to G-actin ratio, an effect that was inhibited by SNAP. Ang II-induced ROS formation and upregulation of p22(phox) mRNA expression were inhibited by APN and NO. Both compounds failed to inhibit Nox1 and p47(phox) expression. CONCLUSION: Our results suggest that the anti-hypertrophic effects of APN are due, in part, to NO-dependent inhibition of the RhoA/ROCK pathway and ROS formation.