To understand how microglial cell function may change with aging, various protocols have been developed to isolate microglia from the young and aged central nervous system (CNS). Here we report modification of an existing protocol that is marked by less debris contamination and improved yields and demonstrates that microglial functions are varied and dependent on age. Specifically, we found that microglia from aged mice constitutively secrete greater amounts of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) relative to microglia from younger mice and are less responsive to stimulation. Also, microglia from aged mice have reduced glutathione levels and internalize less amyloid beta peptide (Aβ) while microglia from mice of all ages do not retain the amyloid beta peptide for a significant length of time. These studies offer further support for the idea that microglial cell function changes with aging. They suggest that microglial Aβ phagocytosis results in Aβ redistribution rather than biophysical degradation in vivo and thereby provide mechanistic insight to the lack of amyloid burden elimination by parenchymal microglia in aged adults and those suffering from Alzheimer’s disease.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Background-Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results-Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions-Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure. hypertension-induced target organ damage and hypertrophy, is a potent promoter of inflammation. 5 The signaling mechanisms that mediate these effects, however, remain largely obscure. In this study, we show that microRNA-155 (miR-155) expression by macrophages is a powerful mediator of cardiac hypertrophy and failure through the upregulation of proinflammatory paracrine signaling. Clinical Perspective on p 1432MicroRNAs are small noncoding RNAs that inhibit gene expression of complementary target genes at the posttranscriptional level. 6 Although others have studied the implication of cardiomyocyte-or fibroblast-derived microRNAs, 7-9 inflammatory microRNAs have hitherto remained unaddressed in pressure overload-induced heart disease. MiR-155 expression is upregulated in a multitude of inflammatory diseases, including rheumatoid arthritis and multiple sclerosis. MethodsAn expanded Methods section is available in the online-only Data Supplement. Animal StudiesAll mouse experiments were performed according to the local relevant guidelines; group sizes are summarized in the Table. Male miR-155 knockout (KO) and wild-type (WT) C57Bl/6J mice (10-12 weeks old) 13 were su...

Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Background— Abdominal aortic aneurysms (AAA) are characterized by extensive transmural inflammation and C-reactive protein (CRP) has emerged as an independent risk factor for the development of cardiovascular disease. Therefore, we evaluated a possible association between serum CRP and aneurysm dimension in patients with asymptomatic AAA. Furthermore, the possibility of CRP production by aneurysmal tissue has been examined. Methods and Results— Serum CRP was determined highly sensitive ( hs CRP) and aneurysmal size was measured in 39 patients with AAA. The presence of CRP mRNA was assessed in the aneurysmal tissue of 16 patients. Mean (SD) hs CRP was 3.23 (2.96) mg/L. After log-transformation, hs CRP correlated significantly with aneurysmal size ( r =0.477, P =0.002). When the patients were divided into 3 equally sized groups according to hs CRP level, aortic diameter increased from lowest to upper hs CRP-tertile (49 mm, 61 mm, and 67 mm, respectively; P <0.05 for 3rd versus 1st tertile). This association persisted after correction for risk factors. CRP mRNA was found in 25% of aneurysmal aortic tissues. Conclusions— This is the first report showing that serum hs CRP is associated with aneurysmal size and that—in at least some patients—CRP may be produced by aneurysmal tissue. These data underscore the inflammatory nature of AAA formation, suggesting that serum hs CRP may serve as a marker of AAA disease and that CRP produced in vascular tissue might contribute to aneurysm formation.

Serum hsCRP was related to the severity of PAD, showing a relation to future hemodynamic function and cardiovascular events in PAD patients. In addition to coronary plaques, aneurysmal aortas, and failed venous coronary bypasses, femoral plaques also produce CRP, thus illustrating that the production of CRP may represent a universal response to vascular injury and suggesting that vascular CRP may contribute to plaque development.

Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment.

Extracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1–CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.