RESUMEN
BACKGROUND: Intermittent claudication (IC) is a common manifestation of peripheral arterial disease. Some patients with IC experience a rise in Urinary N-acetyl-ß-D-Glucosaminidase (NAG)/ Creatinine (Cr) ratio, a marker of renal injury, following exercise. In this study, we aim to investigate whether peripheral blood mononuclear cells (PBMC) from patients with IC who exhibit a rise in urinary NAG/ Cr ratio following exercise exhibit differential IL-10/ IL-12 ratio and gene expression compared to those who do not have a rise in NAG/ Cr ratio. METHODS: We conducted a single center observational cohort study of patients diagnosed with IC. Blood and urine samples were collected at rest and following a standardised treadmill exercise protocol. For comparative analysis patients were separated into those with any rise in NAG/Cr ratio (Group 1) and those with no rise in NAG/Cr ratio (Group 2) post exercise. Isolated PBMC from pre- and post-exercise blood samples were analysed using flow cytometry. PBMC were also cultured for 20 hours to perform further analysis of IL-10 and IL-12 cytokine levels. RNA-sequencing analysis was performed to identify differentially expressed genes between the groups. RESULTS: 20 patients were recruited (Group 1, n = 8; Group 2, n = 12). We observed a significantly higher IL-10/IL-12 ratio in cell supernatant from participants in Group 1, as compared to Group 2, on exercise at 20 hours incubation; 47.24 (IQR 9.70-65.83) vs 6.13 (4.88-12.24), p = 0.04. 328 genes were significantly differentially expressed between Group 1 and 2. The modulated genes had signatures encompassing hypoxia, metabolic adaptation to starvation, inflammatory activation, renal protection, and oxidative stress. DISCUSSION: Our results suggest that some patients with IC have an altered immune status making them 'vulnerable' to systemic inflammation and renal injury following exercise. We have identified a panel of genes which are differentially expressed in this group of patients.
Asunto(s)
Lesión Renal Aguda , Claudicación Intermitente , Acetilglucosaminidasa/orina , Lesión Renal Aguda/metabolismo , Biomarcadores/orina , Creatinina/orina , Citocinas/genética , Femenino , Expresión Génica , Humanos , Interleucina-10/genética , Interleucina-12/genética , Claudicación Intermitente/genética , Leucocitos Mononucleares/metabolismo , MasculinoRESUMEN
AIMS: Atherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis. METHODS AND RESULTS: Normoglycaemic Ldlr-/- hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10-11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 µM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects. CONCLUSION: Metformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Factor de Transcripción Activador 1/metabolismo , Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Macrófagos/efectos de los fármacos , Metformina/farmacología , Placa Aterosclerótica , Proteínas Quinasas Activadas por AMP/genética , Factor de Transcripción Activador 1/genética , Animales , Aorta/enzimología , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Macrófagos/enzimología , Macrófagos/patología , Ratones Noqueados , Fenotipo , Fosforilación , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción de SeñalRESUMEN
RATIONALE: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; HMOX1) and lipid homeostasis genes. OBJECTIVE: We asked whether this pathway had an in vivo role in mice. METHODS AND RESULTS: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene Spic. This response was lost in bone marrow-derived macrophages from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent. CONCLUSIONS: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.