Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

AIM: Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS: Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION: Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy.

AIM/HYPOTHESIS: Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation. METHODS: We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68(+)CCR2(+)) and M2 (CX3CR1(+)CD206(+)/CD163(+)) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals. RESULTS: We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals. CONCLUSIONS/INTERPRETATION: We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

Role of human tissue kallikrein in gastrointestinal stromal tumour invasion.

BACKGROUND: Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST). METHODS: Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells. RESULTS: Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension. CONCLUSIONS: Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST.

Regular physical activity improves endothelial function in patients with coronary artery disease by increasing phosphorylation of endothelial nitric oxide synthase.

BACKGROUND: In stable coronary artery disease (CAD), exercise training has well-documented positive effects on arterial endothelial function. NO derived from endothelial NO synthase (eNOS) is regarded as a protective factor against atherosclerosis. The aim of the present study was to investigate the effects of exercise training on the endothelial function in relation to the expression of eNOS and Akt-dependent eNOS phosphorylation in the left internal mammary artery (LIMA) of patients with stable CAD. METHODS AND RESULTS: In 17 training patients (T) and 18 control patients (C), endothelium-dependent vasodilation and average peak flow velocity (APV) in response to acetylcholine were measured invasively at study beginning and after 4 weeks in the LIMA. In LIMA tissue sampled during bypass surgery, eNOS expression and content of pospho-eNOS-Ser1177, Akt, and phospho-Akt were determined by Western blot and quantitative reverse transcriptase-polymerase chain reaction. After exercise training, LIMA APV in response to acetylcholine was increased by 56+/-8% (from +48+/-8% at beginning to +104+/-11% after 4 weeks, P<0.001). Patients in T had a 2-fold higher eNOS protein expression (T 1.0+/-0.7 versus C 0.5+/-0.3 arbitrary units, P<0.05) and 4-fold higher eNOS Ser1177-phosphorylation levels in LIMA-endothelium (1.2+/-0.9 versus 0.3+/-0.2 arbitrary units, P<0.01). A linear correlation was confirmed between Akt phosphorylation and phospho-eNOS levels (R=0.80, P<0.05) and between phospho-eNOS and Delta APV (R=0.59, P<0.05). CONCLUSIONS: Exercise training in stable CAD leads to an improved agonist-mediated endothelium-dependent vasodilatory capacity. The change in acetylcholine-induced vasodilatation was closely related to a shear stress-induced/Akt-dependent phosphorylation of eNOS on Ser1177.

DPP-4 inhibition ameliorates atherosclerosis by priming monocytes into M2 macrophages.

OBJECTIVE: Glipitins are widely used for the treatment of type 2 diabetic patients. In addition to their improvement of glycemic control, animal studies have suggested an independent anti-atherosclerotic effect of gliptins. Nevertheless, recent clinical trials regarding long-term effects of gliptin therapy on vascular events have been disappointing. This discrepancy led us to better dissect the functional role of SDF-1/CXCR4 signaling as a potential mechanism underlying gliptin action. The study should give improved understanding of the potential of gliptin therapy in the prevention and treatment of atherosclerosis. METHODS AND RESULTS: In an ApoE-/- mouse model on high cholesterol diet, long-term treatment with the DPP-4 inhibitor Sitagliptin significantly reduced atherosclerosic plaque load in the aorta. Flow cytometry analyses showed an enrichment of M2 macrophages in the aortic wall under gliptin therapy. Importantly, the number of recruited CD206+ macrophages was inversely correlated with total plaque area while no correlation was found for the overall macrophage population or M1 macrophages. Blockade of CXCR4/SDF-1 signaling by AMD3100 inhibited aortic M2 accumulation and the therapeutic effect of Sitagliptin. Correspondingly, Sitagliptin shifted the polarization profile of macrophages towards a M2-like phenotype. CONCLUSION: Sitagliptin-mediated inhibition of early atherosclerosis is based on M2-polarization during monocyte differentiation via the SDF-1/CXCR4 signaling. In contrast to earlier assumptions gliptin treatment might be especially effective in prevention of atherosclerosis.