Inhalation of Fine Particulate Matter Impairs Endothelial Progenitor Cell Function Via Pulmonary Oxidative Stress.
ABSTRACT: OBJECTIVE:Exposure to fine particulate matter (PM2.5) air pollution is associated with the depletion of circulating endothelial progenitor cells (EPCs), as well as vascular injury and dysfunction. Nevertheless, it remains unclear whether PM2.5 exposure leads to significant impairments in EPC function. Hence, we studied the effects of PM2.5 on EPC-mediated recovery of vascular perfusion after hindlimb ischemia and examined the mechanisms whereby PM2.5 exposure affects EPC abundance and function. APPROACH AND RESULTS:In comparison with EPCs isolated from mice breathing filtered air, EPCs from mice exposed for 9 consecutive days (6 hours per day) to concentrated ambient PM2.5 (CAP) had defects in both proliferation and tube formation. However, CAP exposure of mice overexpressing extracellular superoxide dismutase (ecSOD-Tg) in the lungs did not affect EPC tube formation. Exposure to CAP also suppressed circulating EPC levels, VEGF (vascular endothelial growth factor)-stimulated aortic Akt phosphorylation, and plasma NO levels in wild-type but not in ecSOD-Tg mice. EPCs from CAP-exposed wild-type mice failed to augment basal recovery of hindlimb perfusion when injected into unexposed mice subjected to hindlimb ischemia; however, these deficits in recovery of hindlimb perfusion were absent when using EPCs derived from CAP-exposed ecSOD-Tg mice. The improved reparative function of EPCs from CAP-exposed ecSOD-Tg mice was also reflected by greater expression of Mmp-9 and Nos3 when compared with EPCs from CAP-exposed wild-type mice. CONCLUSIONS:Exposure to PM2.5 impairs EPC abundance and function and prevents EPC-mediated vascular recovery after hindlimb ischemia. This defect is attributed, in part, to pulmonary oxidative stress and was associated with vascular VEGF resistance and a decrement in NO bioavailability.
Project description:Epidemiological evidence suggests that exposure to ambient air fine particulate matter (PM2.5) increases the risk of developing type 2 diabetes and cardiovascular disease. However, the mechanisms underlying these effects of PM2.5 remain unclear.We tested the hypothesis that PM2.5 exposure decreases vascular insulin sensitivity by inducing pulmonary oxidative stress.Mice fed control (10-13% kcal fat) and high-fat (60% kcal fat, HFD) diets, treated with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) or mice overexpressing lung-specific extracellular superoxide dismutase (ecSOD) were exposed to HEPA-filtered air or to concentrated PM2.5 (CAP) for 9 or 30 days, and changes in systemic and organ-specific insulin sensitivity and inflammation were measured.In control diet-fed mice, exposure to CAP for 30 days decreased insulin-stimulated Akt phosphorylation in lung, heart, and aorta but not in skeletal muscle, adipose tissue, and liver and did not affect adiposity or systemic glucose tolerance. In HFD-fed mice, 30-day CAP exposure suppressed insulin-stimulated endothelial nitric oxide synthase (eNOS) phosphorylation in skeletal muscle and increased adipose tissue inflammation and systemic glucose intolerance. In control diet-fed mice, a 9-day CAP exposure was sufficient to suppress insulin-stimulated Akt and eNOS phosphorylation and to decrease I?B? (inhibitor of the transcription factor NF-?B levels in the aorta. Treatment with the antioxidant TEMPOL or lung-specific overexpression of ecSOD prevented CAP-induced vascular insulin resistance and inflammation.Short-term exposure to PM2.5 induces vascular insulin resistance and inflammation triggered by a mechanism involving pulmonary oxidative stress. Suppression of vascular insulin signaling by PM2.5 may accelerate the progression to systemic insulin resistance, particularly in the context of diet-induced obesity. Citation: Haberzettl P, O'Toole TE, Bhatnagar A, Conklin DJ. 2016. Exposure to fine particulate air pollution causes vascular insulin resistance by inducing pulmonary oxidative stress. Environ Health Perspect 124:1830-1839;?http://dx.doi.org/10.1289/EHP212.
Project description:<h4>Background</h4>Exposure to ambient fine particulate matter air pollution (PM(2.5); < 2.5 µm in aerodynamic diameter) induces endothelial dysfunction and increases the risk for cardiovascular disease. Endothelial progenitor cells (EPCs) contribute to postnatal endothelial repair and regeneration. In humans and mice, EPC levels are decreased upon exposure to elevated levels of PM(2.5).<h4>Objective</h4>We examined the mechanism by which PM(2.5) exposure suppresses circulating levels of EPCs.<h4>Methods</h4>Mice were exposed to HEPA-filtered air or concentrated ambient fine particulate matter (CAP, 30-100 µg/m³) from downtown Louisville (Kentucky) air, and progenitor cells from peripheral blood or bone marrow were analyzed by flow cytometry or by culture ex vivo.<h4>Results</h4>Exposure of the mice to CAP (6 hr/day) for 4-30 days progressively decreased circulating levels of EPCs positive for both Flk-1 and Sca-1 (Flk-1(+)/Sca-1(+)) without affecting stem cells positive for Sca-1 alone (Sca-1(+)). After 9 days of exposure, a 7-day exposure-free period led to complete recovery of the circulating levels of Flk-1(+)/Sca-1(+) cells. CAP exposure decreased circulating levels of EPCs independent of apoptosis while simultaneously increasing Flk-1(+)/Sca-1(+) cells in the bone marrow. We observed no change in tissue deposition of these cells. CAP exposure suppressed vascular endothelial growth factor (VEGF)-induced Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in the aorta, and it prevented VEGF/AMD3100-induced mobilization of Flk-1(+)/Sca-1(+) cells into the peripheral blood. Treatment with stem cell factor/AMD3100 led to a greater increase in circulating Flk-1(+)/Sca-1(+) cells in CAP-exposed mice than in mice breathing filtered air.<h4>Conclusion</h4>Exposure to PM(2.5) increases EPC levels in the bone marrow by preventing their mobilization to the peripheral blood via inhibition of signaling events triggered by VEGF-receptor stimulation that are upstream of c-kit activation. Suppression of EPC mobilization by PM(2.5) could induce deficits in vascular repair or regeneration.
Project description:Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, are used to reduce cholesterol biosynthesis in the liver. Accordingly, statins regulate nitric oxide (NO) and glutamate metabolism, inflammation, angiogenesis, immunity and endothelial progenitor cells (EPCs) functions. The function of EPCs are regulated by stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGF), and transforming growth factor ? (TGF-?), etc. Even though the pharmacologic mechanisms by which statins affect the neovasculogenesis of circulating EPCs, it is still unknown whether statins affect the EPCs function through the regulation of CXCR4, a SDF-1 receptor expression. Therefore, we desired to explore the effects of statins on CXCR4 expression in EPC-mediated neovascularization by in vitro and in vivo analyses. In animal studies, we analyzed the effects of atorvastatin or rosuvastatin treatments in recovery of capillary density and blood flow, the expression of vWF and CXCR4 at ischemia sites in hindlimb ischemia ICR mice. Additionally, we analyzed whether the atorvastatin or rosuvastatin treatments increased the mobilization, homing, and CXCR4 expression of EPCs in hindlimb ischemia ICR mice that underwent bone marrow transplantation. The results indicated that statins treatment led to significantly more CXCR4-positive endothelial progenitor cells incorporated into ischemic sites and in the blood compared with control mice. In vivo, we isolated human EPCs and analyzed the effect of statins treatment on the vasculogenic ability of EPCs and the expression of CXCR4. Compared with the control groups, the neovascularization ability of EPCs was significantly improved in the atorvastatin or rosuvastatin group; this improvement was dependent on CXCR4 up-regulation. The efficacy of statins on improving EPC neovascularization was related to the SDF-1?/CXCR4 axis and might be regulated by the NO. In conclusion, atorvastatin and rosuvastatin improved neovascularization in hindlimb ischemia mice; this effect may have been mediated by increased CXCR4 expression in EPCs.
Project description:Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O??) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS(-/-)/GCH(+/-) hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O?? in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O?? levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.
Project description:Little is known about the role of endothelial progenitor cells (EPCs) in atherosclerosis. Accordingly, we performed a series of assessments with hypercholesterolemic (apolipoprotein E-null [ApoE(-/-)]) and wild-type (WT) mice to evaluate how cholesterol influences reendothelialization, atherosclerosis, and EPC function after arterial injury.Unexpectedly, reendothelialization (assessed by resistance to Evans blue staining) and circulating EPC counts (EPC culture assay) were greater in ApoE(-/-) mice than in WT mice, and transplantation of ApoE(-/-) bone marrow in WT mice accelerated endothelial recovery and increased recruitment of bone marrow-derived EPCs to the neoendothelium. Cholesterol concentration-dependently promoted the proliferation (MTS assay) of both ApoE(-/-) and WT EPCs, and the concentration dependence of EPC adhesion (to vitronectin-, collagen type I-, fibronectin-, and laminin-coated plates), migration (modified Boyden chamber assay), and antiapoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling stain) activity was biphasic. Cholesterol enhanced the messenger RNA expression (quantitative, real-time reverse-transcription polymerase chain reaction) of vascular endothelial growth factor and inhibited Notch1 messenger RNA expression in both ApoE(-/-) and WT EPCs, whereas endothelial nitric oxide synthase messenger RNA expression increased in ApoE(-/-) EPCs and declined in WT EPCs after cholesterol exposure. EPC activity was greater in Notch1(+/-) EPCs than in WT EPCs, and transplantation of Notch1(+/-) bone marrow accelerated endothelial recovery after arterial injury in WT mice.The results presented here provide novel insights into the role of EPCs during atherosclerosis and suggest that cholesterol and Notch1 may be involved in the regulation of EPC activity.
Project description:Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133+ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis.
Project description:Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.
Project description:Diabetic retinopathy is characterized by pathological retinal neovascularization. Accumulating evidence has indicated that high levels of circulating endothelial progenitor cells (EPCs) are an important risk factor for neovascularization. Paradoxically, the reduction and dysfunction of circulating EPCs has been extensively reported in diabetic patients. We hypothesized that EPCs are differentially altered in the various vasculopathic complications of diabetes mellitus, exhibiting distinct behaviors in terms of angiogenic response to ischemia and growth factors and potentially playing a potent role in motivating vascular precursors to induce pathological neovascularization. Circulating levels of EPCs from diabetic retinopathy patients were analyzed by flow cytometry and by counting EPC colony-forming units, and serum levels of neurotrophic factors were measured by enzyme-linked immunosorbent assay. We found increased levels of nerve growth factor and brain-derived neurotrophic factor in the blood of diabetic retinopathy patients; this increase was correlated with the levels of circulating EPCs. In addition, we demonstrated that retinal cells released neurotrophic factors under hypoxic conditions to enhance EPC activity in vitro and to increase angiogenesis in a mouse ischemic hindlimb model. These results suggest that neurotrophic factors may induce neoangiogenesis through EPC activation, leading to the pathological retinal neovascularization. Thus, we propose that neovascularization in the ischemic retina might be regulated by overexpression of neurotrophic factors.
Project description:Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury.To examine the mechanism of IGFBP-3-mediated repair following vascular injury.We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway.These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.
Project description:Multiple organ dysfunction syndrome (MODS) is a detrimental clinical complication in critically ill patients with high mortality. Emerging evidence suggests that oxidative stress and endothelial activation (induced expression of adhesion molecules) of vital organ vasculatures are key, early steps in the pathogenesis. We aimed to ascertain the role and mechanism(s) of enhanced extracellular superoxide dismutase (EcSOD) expression in skeletal muscle in protection against MODS induced by endotoxemia. We showed that EcSOD overexpressed in skeletal muscle-specific transgenic mice (TG) redistributes to other peripheral organs through the circulation and enriches at the endothelium of the vasculatures. TG mice are resistant to endotoxemia (induced by lipopolysaccharide [LPS] injection) in developing MODS with significantly reduced mortality and organ damages compared with the wild type littermates (WT). Heterogenic parabiosis between TG and WT mice conferred a significant protection to WT mice, whereas mice with R213G knock-in mutation, a human single nucleotide polymorphism leading to reduced binding EcSOD in peripheral organs, exacerbated the organ damages. Mechanistically, EcSOD inhibits vascular cell adhesion molecule 1 expression and inflammatory leukocyte adhesion to the vascular wall of vital organs, blocking an early step of the pathology in organ damage under endotoxemia. Therefore, enhanced expression of EcSOD in skeletal muscle profoundly protects against MODS by inhibiting endothelial activation and inflammatory cell adhesion, which could be a promising therapy for MODS.