For determining the implications of circulating endothelial progenitor cells (cEPCs) and cellular membrane microparticles (MPs) in diabetic stroke, levels of EPCs, EPC-MPs, and endothelium-derived MPs (EMPs) and their correlations with blood glucose concentration, cerebral microvascular density (cMVD), and ischemic damage were investigated in type 2 diabetic db/db and db/+ (wild-type control) mice. Therapeutic efficacy of EPC infusion (preincubated with MPs) was also explored. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Ischemic damage and cMVD were determined using histological analyses. The levels of cEPCs and MPs were determined using flow cytometric analyses. EPC generation and functions were evaluated by in vitro cell cultures. Results showed the following. 1) In db/db mice, the basal level of cEPCs was less and cMVDs were lower, but the levels of circulating EPC-MPs and EMPs were more; 2) MCAO induced a larger infarct volume and less of an increase in cEPCs in db/db mice; 3) the level of cEPCs correlated with blood glucose concentration (negatively), cMVD (positively), and ischemic damage (negatively), but the levels of EPC-MPs and EMPs correlated inversely with those parameters; 4) EPCs were reduced and dysfunctional in db/db mice, and preincubation with db/db MPs impaired EPC functions; and 5) infusion of EPCs preincubated with db/+ MPs increased the level of cEPCs and reduced ischemic damage, and these beneficial effects were reduced or lost in EPCs preincubated with db/db MPs. These data suggest that reduced cEPCs, impaired EPC generation/function, and increased production of MPs might be the mechanisms responsible for increased ischemic damage seen in db/db mice.

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1β, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1β concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1β levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1β production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4 and CD8 T cells in HNSCC mice. Notably, the numbers of exhausted PD-1 and Tim3 T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1β pathway may provide a novel approach for HNSCC therapy.

Oxytocinergic and vasopressinergic brain stem projections have been shown to play an important role in mediating cardiovascular adjustments during exercise training (ET). The aim of the present work was to determine whether the intrinsic excitability of hypothalamic neurons giving rise to brain stem peptidergic projections is altered as a consequence of ET. Whole cell patch-clamp recordings were obtained from nucleus of the solitarii tract (NTS)-projecting paraventricular nucleus of the hypothalamus (PVN) neurons and from supraoptic nucleus (SON) and PVN magnocellular cells (MNCs), in hypothalamic slices obtained from sedentary (S) and ET rats. Our results indicate that intrinsic excitability of PVN neurons that innervate the NTS (PVN-NTS) is enhanced by ET, resulting in a more efficient input-output function (increase number of evoked actions potentials, steeper frequency/current relationships and slower decaying frequency/time relationships). Changes in input-output function were accompanied by smaller hyperpolarizing afterpotentials (HAPs) and afterhyperpolarizing potentials (AHPs), during and after trains of spikes, respectively. On the other hand, a decreased efficacy in the input-output function was observed in SON/PVN MNCs during ET. Altogether, our results indicate that ET differentially affects the intrinsic excitability of autonomic and neurosecretory SON and PVN neurons. Increased excitability in PVN-NTS neurons may contribute to enhanced release of OT and VP peptides in the dorsal brain stem, and cardiovascular fine-tuning during exercise training.

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Y. Ischemia-induced brain damage is enhanced in human renin and angiotensinogen double-transgenic mice. Am J Physiol Regul Integr Comp Physiol 297: R1526 -R1531, 2009. First published September 16, 2009 doi:10.1152/ajpregu.91040.2008.-To investigate the role of brain angiotensin II (ANG II) in the pathogenesis of injury following ischemic stroke, mice overexpressing renin and angiotensinogen (RϩAϩ) and their wild-type control animals (RϪAϪ) were used for experimental ischemia studies. Focal brain ischemia was induced by middle cerebral artery occlusion (MCAO). The severity of ischemic injury was determined by measuring neurological deficits and histological damage at 24 and 48 h after MCAO, respectively. To exclude the influence of blood pressure and local collateral blood flow, brain slices were used for oxygen and glucose deprivation (OGD) studies. The severity of OGD-induced damage was determined by measuring indicators of tissue swelling and cell death, the intensity of the intrinsic optical signal (IOS), and the number of propidium iodide (PI) staining cells, respectively. Results showed 1) RϩAϩ mice showed higher neurological deficit score (3.8 Ϯ 0.5 and 2.5 Ϯ 0.3 for RϩAϩ and RϪAϪ, respectively, P Ͻ 0.01) and larger infarct volume (22.2 Ϯ 1.6% and 14.1 Ϯ 1.2% for RϩAϩ and RϪAϪ, respectively, P Ͻ 0.01); 2) The RϩAϩ brain slices showed more severe tissue swelling and cell death in the cortex (IOS: 140 Ϯ 6% and 114 Ϯ 10%; PI: 139 Ϯ 20 cells/field and 39 Ϯ 9 cells/field for RϩAϩ and RϪAϪ, respectively, P Ͻ 0.01); 3) treatment with losartan (20 mol/l) abolished OGD-induced exaggeration of cell injury seen in RϩAϩ mice. The data indicate that activation of ANG II/AT 1 signaling is harmful to brain exposed to ischemia. AT1 receptor; mouse; losartan; middle cerebral artery ISCHEMIC STROKE, THE THIRD leading cause of death in the United States, is a severe complication of hypertension and arteriosclerosis. There are limited avenues for prevention and treatment of ischemic stroke. Accumulating evidence suggests that the angiotensin II (ANG II)/AT 1 receptor pathway participates in the pathophysiology of ischemic stroke (8, 32). Blockade of the ANG II/AT 1 pathway has been shown to have beneficial effect on cerebral ischemia in animal studies (11,30) and clinical trials (10, 23). However, the mechanisms of ANG II/AT 1 signaling in conjunction with stroke are still elusive.The protective effect of blocking ANG II/AT 1 receptor pathway on ischemic stroke has been related to blood pressure control and improvement of cerebral blood supply. However, the benefits can go beyond those (10, 38). The ANG II/AT 1 pathway exists in neural/glial cells as well as cerebrovascular cells. AT 1 receptors located in the cerebral vasculature participate in the regulation of the cerebrovascular circulation (31) and platelet-leukocyte-endothelium interactions (19). The ANG II/AT 1 receptors located in the neural/glial cells participate in ischemic damage by inducing inflammation and oxidative stress (16,33,40).Genetically modified mice...