Brain capillary pericytes are metabolic sentinels that control blood flow through a KATP channel-dependent energy switch.

Despite the abundance of capillary thin-strand pericytes and their proximity to neurons and glia, little is known of the contributions of these cells to the control of brain hemodynamics. We demonstrate that the pharmacological activation of thin-strand pericyte KATP channels profoundly hyperpolarizes these cells, dilates upstream penetrating arterioles and arteriole-proximate capillaries, and increases capillary blood flow. Focal stimulation of pericytes with a KATP channel agonist is sufficient to evoke this response, mediated via KIR2.1 channel-dependent retrograde propagation of hyperpolarizing signals, whereas genetic inactivation of pericyte KATP channels eliminates these effects. Critically, we show that decreasing extracellular glucose to less than 1 mM or inhibiting glucose uptake by blocking GLUT1 transporters in vivo flips a mechanistic energy switch driving rapid KATP-mediated pericyte hyperpolarization to increase local blood flow. Together, our findings recast capillary pericytes as metabolic sentinels that respond to local energy deficits by increasing blood flow to neurons to prevent energetic shortfalls.

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.

Ion channels in capillary endothelium.

Vascular beds are anatomically and functionally compartmentalized into arteries, capillaries, and veins. The bulk of the vasculature consists of the dense, anastomosing capillary network, composed of capillary endothelial cells (cECs) that are intimately associated with the parenchyma. Despite their abundance, the ion channel expression and function and Ca2+ signaling behaviors of capillaries have only recently begun to be explored in detail. Here, we discuss the established and emerging roles of ion channels and Ca2+ signaling in cECs. By mining a publicly available RNA-seq dataset, we outline the wide variety of ion channel genes that are expressed in these cells, which potentially imbue capillaries with a broad range of sensing and signal transduction capabilities. We also underscore subtle but critical differences between cEC and arteriolar EC ion channel expression that likely underlie key functional differences in ECs at these different levels of the vascular tree. We focus our discussion on the cerebral vasculature, but the findings and principles being elucidated in this area likely generalize to other vascular beds.

Decreased expression of neuronal nitric oxide synthase contributes to the endothelial dysfunction associated with cigarette smoking in human.

Endothelial nitric oxide synthase (eNOS) malfunctioning has been proposed to contribute to the endothelial damage produced by cigarette. Besides eNOS, neuronal NOS (nNOS) is also expressed in most vascular tissues and plays an important role in the endothelium-dependent vascular relaxation. We hypothesize that nNOS may contribute to the endothelium dysfunction produced by cigarette in smokers. Vascular function was assessed in human resistance mesenteric arteries using a wire myograph, the level of protein expression by Western blot, eNOS and nNOS localization by immunofluorescence. Measurement of NO was assessed by fluorescence microscopy. Arteries of smokers showed impaired endothelium-dependent vascular relaxation in response to acetylcholine. Pharmacological nonselective blockade of NOS with l-NAME and selective nNOS blockade with inhibitor 1 reduced the relaxation of the mesenteric artery of both smokers and nonsmokers. Interestingly, the inhibitory effect of NOS inhibitors was greater in nonsmokers than in smokers. The expression of total nNOS and eNOS and the level of phosphorylation at eNOS-pSer1177 were reduced in arteries of smokers as compared with nonsmokers. No differences between groups were observed in the expression of total COX-1, COX-2, catalase and SOD-1. Immunofluorescence analysis showed the presence of nNOS in the vascular endothelium in both groups. Acetylcholine-induced NO production was impaired in arteries from smokers as compared to nonsmokers. Selective inhibition of nNOS caused a decreased in NO production, which was greater in nonsmokers than in smokers. Our data show that a decrease in nNOS expression contributes to the endothelial dysfunction caused by cigarette smoking in human.

Activation of Cav1.2 and BKCa is involved in the downregulation of caffeine-induced contraction in mice mesenteric arteries.

AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 µM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 µM) and ruthenium red (RuR; 10 µM), transient receptor potential (TRPs) channels blockers; capsazepine (10 µM) and HC067047 (10 µM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 µM), a selective BKCa blocker; and SKF-96365 (30 µM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.

Neuronal nitric oxide synthase contributes to the normalization of blood pressure in medicated hypertensive patients.

Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and besides NO, generates H2O2. nNOS has been proposed to contribute to the control of blood pressure in healthy humans. The aim of this study was to verify the hypothesis that nNOS can contribute to the control of vascular relaxation and blood pressure in hypertensive patients undergoing drug treatment. The study was conducted in resistance mesenteric arteries from 63 individuals, as follows: 1) normotensive patients; 2) controlled hypertensive patients (patients on antihypertensive treatment with blood pressure normalized); 3) uncontrolled hypertensive patients (patients on antihypertensive treatment that remained hypertensive). Only mesenteric arteries from uncontrolled hypertensive patients showed impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh). Selective nNOS blockade with inhibitor 1 and catalase, which decomposes H2O2, decreased vasorelaxation in the three groups. However, the inhibitory effect was greater in controlled hypertensive patients. Decreased eNOS expression was detected in both uncontrolled and controlled hypertensive groups. Interestingly nNOS expression and ACh-stimulated H2O2 production were greater in controlled hypertensive patients, than in the other groups. ACh-stimulated NO production was lower in controlled hypertensive when compared to normotensive patients, while uncontrolled hypertensive patients showed the lowest levels. Catalase and nNOS blockade inhibited ACh-induced H2O2 production. In conclusion, nNOS-derived H2O2 contributes to the endothelium-dependent vascular relaxation in human resistance mesenteric arteries. The endothelial dysfunction observed in uncontrolled hypertensive patients involves decreased eNOS expression and NO production. The normalization of vascular relaxation and blood pressure in controlled hypertensive patients involves increased nNOS-derived H2O2 and NO production.

The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Genistein affects proliferation and migration of bovine oviductal epithelial cells.

Genistein is one of the most abundant isoflavones in soybean. This molecule induces cell cycle arrest and apoptosis in different normal and cancer cells. Genistein has been of considerable interest due to its adverse effects on bovine reproduction, altering estrous cycle, implantation and fetal development and producing subfertility or infertility. The objective of this work was to study the effects of genistein on the expression of selected genes involved in the regulation of cell cycle and apoptosis. Primary cultures of bovine oviductal epithelial cells (BOEC) were treated with different genistein concentrations (0.2, 2 and 10µM) to analyze CYCLIN B1, BCL-2 and BAX gene expression by Real-time RT-PCR. Results showed that genistein down-regulated CYCLIN B1 expression, affecting cell cycle progression, and caused a decrease in the BCL-2/BAX ratio starting at 2µM of genistein. In addition, in order to determine if genistein affects BOEC migration, in vitro wound healing assays were performed. A significant reduction in cell migration after 12h of culture was observed at both 0.2 and 10µM genistein concentrations. Also, in the presence of genistein the percentage of mitotic cells decreased, although apoptotic cells percentages were not affected. These findings indicate that genistein has an inhibitory effect on BOEC proliferation and migration, suggesting that it could influence the normal physiology of the oviductal epithelium.

Structure-related blockage of calcium channels by vasodilator alkamides in mice mesenteric artery.

The development of new calcium channel blockers is still relevant for the understanding of their physiological role and pharmacological and therapeutic purposes. For this task, natural products represent a relevant source of new drugs. The present work investigated the mechanism and the structural relationship of the vasodilator effect of riparins I, II and III in mouse small mesenteric artery. Riparins I, II and III induced an endothelium-independent and concentration-dependent vasodilator effect in mesenteric arteries. Riparins II and III were more potent than riparin I, suggesting a structural relationship of the effect of these drugs. All riparins inhibited the contractile effect of KCl, similarly to nifedipine. However, the inhibitory profile was different for the contractile responses to phenylephrine and caffeine, passing from similar to nifedipine with riparin I, for similar to SKF-96365 with riparin III. A comparable effect was observed for the increase in the intracellular calcium concentration induced by caffeine and phenylephrine. These results suggest that the higher hydroxylation provides the alkamides the ability to inhibit non-selective cation channels in addition to the inhibition of L-type calcium channels in mouse mesenteric arteries. These observations may give support to the development of new selective inhibitors of non-selective cation channels using alkamides as leading compounds.

Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

In vivo and in vitro expression of the plasminogen activators and urokinase type plasminogen activator receptor (u-PAR) in the pig oviduct.

Plasminogen activator activities have previously been reported in oviductal fluid. At present the question was whether the source of these activities is molecules come from blood plasma or if these activators are synthesized by the oviduct. Gene expression and protein synthesis of urokinase type (u-PA) and tissue type (t-PA) occur in different regions of the pig oviduct. Their relative concentrations do not vary between the ampulla and isthmus regions and are similar throughout the estrous cycle. However, while relative amounts of t-PA mRNA were not different between the different stages of the estrous cycle, u-PA mRNA was greater after ovulation (P<0.05). Regarding the function of u-PA, its receptor (u-PAR) was distinguished by immunohistochemistry at the apical region of the epithelial cells and was more noticeable in the isthmus. Expression of u-PA, t-PA, u-PAR and PAI-1 genes in primary oviductal epithelial cell cultures was studied under 17-ß-estradiol (100 pg/ml) and progesterone (100 ng/ml). u-PA mRNA increased in the presence of progesterone (P<0.05), but not by action of 17-ß-estradiol. t-PA, PAI-1 and u-PAR were similar when cultured with the hormones. These results suggest that u-PA could be regulated by progesterone at a transcriptional level, by the balance of their activity for PAI-1 or at the epithelial surface through the binding of u-PAR. In conclusion, plasminogen activation system components might cooperate in the oviductal lumen to control plasmin generation.