Exposure to fine particulate matter (PM) is a significant risk for cardiovascular diseases largely due to increased reactive oxygen species (ROS) production and inflammation. Caspase recruitment domain (CARD)9 is critically involved in innate immunity and inflammation. The present study was designed to test the hypothesis that CARD9 signaling is critically involved in PM exposure-induced oxidative stress and impaired recovery of limb ischemia.Critical limb ischemia (CLI) was created in male wildtype C57BL/6 and age matched CARD9 deficient mice with or without PM (average diameter 2.8 μm) exposure. Mice received intranasal PM exposure for 1 month prior to creation of CLI and continued for the duration of the experiment. Blood flow and mechanical function were evaluated in vivo at baseline and days 3, 7, 14, and 21 post CLI. PM exposure significantly increased ROS production, macrophage infiltration, and CARD9 protein expression in ischemic limbs of C57BL/6 mice in association with decreased recovery of blood flow and mechanical function. CARD9 deficiency effectively prevented PM exposure-induced ROS production and macrophage infiltration and preserved the recovery of ischemic limb with increased capillary density. CARD9 deficiency also significantly attenuated PM exposure-induced increase of circulating CD11b+/F4/80+ macrophages.The data indicate that CARD9 signaling plays an important role in PM exposure-induced ROS production and impaired limb recovery following ischemia in mice.
Males have a higher risk for cardiovascular diseases (CVDs) than females. Ambient fine particulate matter (PM) exposure increases CVD risk with increased reactive oxygen species (ROS) production and oxidative stress. Endothelial progenitor cells (EPCs) are important to vascular structure and function and can contribute to the development of CVDs. The aims of the present study were to determine if sex differences exist in the effect of PM exposure on circulating EPCs in mice and, if so, whether oxidative stress plays a role. Male and female C57BL/6 mice (8-10 weeks old) were exposed to PM or a vehicle control for six weeks. ELISA analysis showed that PM exposure substantially increased the serum levels of IL-6 and IL-1β in both males and females, but the concentrations were significantly higher in males. PM exposure only increased the serum levels of TNF-α in males. Flow cytometry analysis demonstrated that ROS production was significantly increased by PM treatment in males but not in females. Similarly, the level of circulating EPCs (CD34+/CD133+ and Sca-1+/Flk-1+) was significantly decreased by PM treatment in males but not in females. Antioxidants N-acetylcysteine (NAC) effectively prevented PM exposure-induced ROS and inflammatory cytokine production and restored circulating EPC levels in male mice. In sharp contrast, circulating EPC levels remained unchanged in female mice with PM exposure, an effect that was not altered by ovariectomy. In conclusion, PM exposure selectively decreased the circulating EPC population in male mice via increased oxidative stress without a significant impact on circulating EPCs in females independent of estrogen.
Background/Aims: Endothelial progenitor cells (EPCs) play a critical role in angiogenesis and vascular repair. Some environmental insults, like fine particulate matter (PM) exposure, significantly impair EPCs through inflammation and ROS production. Cytosolic adaptor caspase recruitment domain 9 (CARD9) is important to the function of macrophages and involved in the innate immune response. The present study was to determine the role of macrophage-mediated immune response in PM-induced adverse effects on EPCs. Methods: PM was intranasal-distilled into male C57BL/6 mice for 1, 2, 7, 14 and 28 days. The level of EPCs (CD34 + /Flk-1 + , Sca-1 + /Flk-1 + , c-Kit + /CD31 + and CD34 + /CD133 + ) in the bone marrow (BM) and circulation was determined using flow cytometry along with the proliferation and apoptotic level of EPCs as well as intracellular reactive oxygen species (ROS) formation. Serum TNF-α, IL-1β, IL-6 and IL-10 were measured using ELISA. To determine the role of CARD9-mediated signaling in PM-induced detrimental effects on EPCs, CARD9 knock out (CARD9 -/- ) mice were used to repeat the experiment. The population of total macrophages as well as M1 and M2 subpopulations in both BM and blood were analyzed in the mice with and without PM exposure. To study the function of macrophages after PM exposure, the peritoneal macrophages were isolated and cultured for 48 hours to measure the production of TNF- α, IL-1β, IL-6 and IL-10 in the media. Results: PM exposure significantly decreased BM and circulating CD34 + /FLk-1 + as well as BM Scal-1 + /Flk-1 + population, promoted apoptosis and inhibited proliferation of EPCs in association with increased ROS production, serum TNF-α, IL-1β, IL-6 and decreased serum IL-10 level. These PM-induced effects on EPC population and cytokine production were largely prevented in CARD9 -/- mice along with increased M2 population in both BM and blood. The production of TNF-α, IL-1β and IL-6 was significantly decreased, while the IL-10 level increased, from cultured macrophage of CARD9 -/- mice compared with WT control. Conclusion: PM exposure significantly decreased BM and circulating EPCs population due to CARD9-mediated innate immune response.
Ambient fine particulate matter (PM) exposure associates with an increased risk of cardiovascular diseases (CVDs). Major sex differences between males and females exist in epidemiology, pathophysiology, and outcome of CVDs. Endothelial progenitor cells (EPCs) play a vital role in the development and progression of CVDs. PM exposure-induced reduction of EPCs is observed in male, not female, mice with increased reactive oxygen species (ROS) production and oxidative stress. The lung is considered an important source of ROS in mice with PM exposure. The aim of the present study was to investigate the sex differences in pulmonary superoxide dismutase (SOD) expression and ROS production, and to test the effect of SOD mimic Tempol on the populations of EPCs in mice with PM exposure. Both male and female C57BL/6 mice (8-10 weeks) were exposed to intranasal PM or vehicle for 6 weeks. Flow cytometry analysis demonstrated that PM exposure significantly decreased the levels of EPCs (CD34+/CD133+) in both blood and bone marrow with increased ROS production in males, but not in females. ELISA analysis showed higher levels of serum IL-6 and IL-1βin males than in females. Pulmonary expression of the antioxidant enzyme SOD1 was significantly decreased in males after PM exposure, but not in females. Administration of the SOD mimic Tempol in male mice with PM exposure attenuated the production of ROS and inflammatory cytokines, and preserved EPC levels. These data indicated that PM exposure-induced reduction of EPC population in male mice may be due to decreased expression of pulmonary SOD1 in male mice.
Background Substantial sex differences exist in atherosclerosis. Excessive reactive oxygen species (ROS) formation could lead to endothelial dysfunction which is critical to atherosclerosis development and progression. Helicobacter pylori (H. pylori) infection has been shown to attenuate endothelial function via exosomes-mediated ROS formation. We have demonstrated that H. pylori infection selectively increases atherosclerosis risk in males with unknown mechanism(s). The present study was to test the hypothesis that H. pylori infection impaired endothelial function selectively in male mice through exosome-mediated ROS formation. Methods and results Age-matched male and female C57BL/6 mice were infected with CagA+ H. pylori to investigate sex differences in H. pylori infection-induced endothelial dysfunction. H. pylori infection attenuated acetylcholine (ACh)-induced endothelium-dependent aortic relaxation without changing nitroglycerine-induced endothelium-independent relaxation in male but not female mice, associated with increased ROS formation in aorta compared with controls, which could be reversed by N-acetylcysteine treatment. Treatment of cultured mouse brain microvascular endothelial cells with exosomes from H. pylori infected male, not female, mice significantly increased intracellular ROS production and impaired endothelial function with decreased migration, tube formation, and proliferation, which could be prevented with N-acetylcysteine treatment. Conclusions H. pylori infection selectively impairs endothelial function in male mice due to exosome-mediated ROS formation.
Oxidative stress and inflammation are considered an important mechanism for the development of cardiovascular diseases. Cytokines including interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) play an important role in oxidative stress and inflammation. It is known that ambient fine particulate matter ( PM ) exposure is closely associated with cardiovascular diseases and oxidative stress. Caspase-recruitment domain 9 ( Card9 ) signaling is critically involvement in the function of macrophages, neutrophils and monocytes that are important for oxidative stress and inflammation. The present study was designed to evaluate the role of CARD9-mediated signaling in cytokines production in mice with PM exposure. Both male wild-type (WT) C57BL/6 mice (8-10 weeks) and age-matched CARD9 knockout (KO) mice (with C57BL/6 background) were exposed to PM2.5 for 6 weeks via intranasal approach with PBS as the control. Serum concentrations of the cytokines including IL-6, IL-1β, and TNF-α were measured with ELISA in the mice before and after PM exposure. There was no difference in the serum levels of IL-6, IL-1β, or TNF-α between WT mice and CARD9 KO mice exposed to PBS. As expected, PM exposure substantially increased the serum levels of IL-6, IL-1β, and TNF-α in the WT mice (by up to 6 times). However, no significant increase in the serum concentrations for IL-6, IL-1β, and TNF-α was observed in CARD9 KO mice exposed to PM. Increased inflammatory infiltrations in the lungs were observed in the WT mice as compared to the CARD9 KO mice with PM exposure. In conclusion, the present study demonstrated that increased cytokines were produced in WT mice, but not in CARD9 KO mice with PM exposure. The data suggested that CARD9 signaling played a critical role in the production of inflammatory cytokines in the mice in response to PM exposure, and might contribute to the development of cardiovascular diseases related to PM exposure.
Inflammatory bowel disease (IBD) produces significant local and systemic inflammation with increased reactive oxygen species (ROS) formation. IBD Patients are at an increased risk for developing endothelial dysfunction and cardiovascular diseases. The present study tested the hypothesis that IBD impairs aortic endothelial function via ROS formation and investigate potential sex-related differences.
Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.
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