Aging decreases docosahexaenoic acid transport across the blood-brain barrier in C57BL/6J mice.

Nutrients are actively taken up by the brain via various transporters at the blood-brain barrier (BBB). A lack of specific nutrients in the aged brain, including decreased levels of docosahexaenoic acid (DHA), is associated with memory and cognitive dysfunction. To compensate for decreased brain DHA, orally supplied DHA must be transported from the circulating blood to the brain across the BBB through transport carriers, including major facilitator superfamily domain-containing protein 2a (MFSD2A) and fatty acid-binding protein 5 (FABP5) that transport esterified and non-esterified DHA, respectively. Although it is known that the integrity of the BBB is altered during aging, the impact of aging on DHA transport across the BBB has not been fully elucidated. We used 2-, 8-, 12-, and 24-month-old male C57BL/6 mice to evaluate brain uptake of [14C]DHA, as the non-esterified form, using an in situ transcardiac brain perfusion technique. Primary culture of rat brain endothelial cells (RBECs) was used to evaluate the effect of siRNA-mediated MFSD2A knockdown on cellular uptake of [14C]DHA. We observed that the 12- and 24-month-old mice exhibited significant reductions in brain uptake of [14C]DHA and decreased MFSD2A protein expression in the brain microvasculature compared with that of the 2-month-old mice; nevertheless, FABP5 protein expression was up-regulated with age. Brain uptake of [14C]DHA was inhibited by excess unlabeled DHA in 2-month-old mice. Transfection of MFSD2A siRNA into RBECs decreased the MFSD2A protein expression levels by 30% and reduced cellular uptake of [14C]DHA by 20%. These results suggest that MFSD2A is involved in non-esterified DHA transport at the BBB. Therefore, the decreased DHA transport across the BBB that occurs with aging could be due to age-related down-regulation of MFSD2A rather than FABP5.

Senescence in brain pericytes attenuates blood-brain barrier function in vitro: A comparison of serially passaged and isolated pericytes from aged rat brains.

Aging is associated with the dysfunction of the blood-brain barrier (BBB), which comprises brain microvessel endothelial cells (BMECs), astrocytes, and pericytes. Pericytes are present at intervals along the walls of the brain capillaries and play a key role in maintaining BBB integrity. Accumulation of senescent cells and the senescence-associated secretory phenotype (SASP) in the brain facilitate the development of age-related neurodegenerative diseases with BBB dysfunction. However, the ability of pericytes to support BBB integrity and their correlation with cellular senescence or aging remain unknown. Here, we investigated cellular senescence in pericytes focusing on its impact on BBB function using BBB models comprising intact BMECs co-cultured with senescent pericytes, which were obtained through a serial passage or isolated from 18-month-old rats. To assess BBB function, transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na-F) were studied. Both serially passaged pericytes (in passage 4, 7, and 10) and aged pericytes isolated from 18-month-old rats showed decreased TEER and enhanced permeability of BMECs to Na-F compared to that of normal pericytes (passage 2 or young). Furthermore, serially passaged and aged pericytes showed characteristic features of cellular senescence, including increased ß-galactosidase activity, cell cycle arrest, enhanced expression of mRNA, and SASP factors. However, the senescence-induced mRNA expression profile of pericyte markers varied between serially passaged and aged pericytes. Hence, in vitro serial passages and isolation from naturally aged rodents differently influenced genetic and biochemical features of senescent brain pericytes. We conclude that senescent brain pericytes can induce BBB dysfunction and those isolated from aged rodents retain the senescence-specific properties. Our findings provide an alternative tool to investigate the senescence in brain pericytes in vitro.

The Neuroinflammatory Role of Pericytes in Epilepsy.

Pericytes are a component of the blood-brain barrier (BBB) neurovascular unit, in which they play a crucial role in BBB integrity and are also implicated in neuroinflammation. The association between pericytes, BBB dysfunction, and the pathophysiology of epilepsy has been investigated, and links between epilepsy and pericytes have been identified. Here, we review current knowledge about the role of pericytes in epilepsy. Clinical evidence has shown an accumulation of pericytes with altered morphology in the cerebral vascular territories of patients with intractable epilepsy. In vitro, proinflammatory cytokines, including IL-1ß, TNFα, and IL-6, cause morphological changes in human-derived pericytes, where IL-6 leads to cell damage. Experimental studies using epileptic animal models have shown that cerebrovascular pericytes undergo redistribution and remodeling, potentially contributing to BBB permeability. These series of pericyte-related modifications are promoted by proinflammatory cytokines, of which the most pronounced alterations are caused by IL-1ß, a cytokine involved in the pathogenesis of epilepsy. Furthermore, the pericyte-glial scarring process in leaky capillaries was detected in the hippocampus during seizure progression. In addition, pericytes respond more sensitively to proinflammatory cytokines than microglia and can also activate microglia. Thus, pericytes may function as sensors of the inflammatory response. Finally, both in vitro and in vivo studies have highlighted the potential of pericytes as a therapeutic target for seizure disorders.

Reactive pericytes in early phase are involved in glial activation and late-onset hypersusceptibility to pilocarpine-induced seizures in traumatic brain injury model mice.

In this study, among neurovascular unit (NVU) cells, we focused on pericyte reactivity in mice subjected to controlled cortical impact (CCI) to understand how traumatic brain injury (TBI) causes uncoordinated crosstalk in the NVU and alters neuronal activity. Histological analyses of brain pericytes, microglia and astrocytes were performed for up to 28 days after CCI in the injured ipsilateral hippocampus. To evaluate altered neuronal activity caused by CCI, we measured seizure susceptibility to a sub-threshold dose of pilocarpine on postoperative day 7, 14, 21 and 28. Platelet-derived growth factor receptor (PDGFR) ß immunoreactivity in pericytes significantly increased from 1 h to 4 days after CCI. The expression of Iba1 and GFAP, as markers of microglia and astrocytes, respectively, increased from 4 to 28 days after CCI. The severity of seizure induced by pilocarpine gradually increased, becoming significant at 28 days after CCI. Then, we treated CCI mice with an inhibitor of PDGFR signaling, imatinib, during the postoperative day 0-4 period. Imatinib lowered seizure susceptibility to pilocarpine and suppressed microglial activation in the injured hippocampus at postoperative day 28. These findings indicate that brain pericytes with rapidly increased PDGFRß expression may drive TBI-induced dysregulation of NVU function and brain hyperexcitability.

Serum amyloid A-induced blood-brain barrier dysfunction associated with decreased claudin-5 expression in rat brain endothelial cells and its inhibition by high-density lipoprotein in vitro.

The blood-brain barrier (BBB) is the multicellular interface located between the peripheral circulation and the brain parenchyma. BBB dysfunction is reported in many CNS diseases, such cognitive impairment, depression, Alzheimer's disease (AD), and multiple sclerosis (MS). Emerging evidence indicates that liver-derived inflammatory mediators are upregulated in neurological diseases with BBB dysfunction. Serum amyloid A (SAA), an acute phase protein secreted by hepatocytes, could be a candidate inflammatory signaling molecule transmitted from the liver to the brain; however, its contribution to BBB dysfunction is poorly understood. The present study aimed to elucidate the involvement of SAA in BBB impairment in an in vitro BBB model using rat brain microvascular endothelial cells (RBECs). We demonstrated that Apo-SAA significantly decreased transendothelial electrical resistance (TEER) and increased sodium fluorescein (Na-F) permeability in RBEC monolayers. Apo-SAA also decreased claudin-5 expression levels in RBECs. Furthermore, the Apo-SAA-mediated impairment of the BBB with decreased claudin-5 expression was inhibited by the addition of a high-density lipoprotein (HDL) related to SAA in plasma. These findings suggest that HDL counteracts the effects of SAA on BBB function. Therefore, the functional imbalance between SAA and HDL may induce BBB impairment, thereby triggering development of neuroinflammation. SAA could be a significant endogenous mediator in the liver-to-brain inflammation axis.

Brain-transportable soy dipeptide, Tyr-Pro, attenuates amyloid ß peptide25-35-induced memory impairment in mice.

In this study, experiments on amyloid ß peptide25-35-induced mice were performed to provide in vivo evidence on the potential of the blood-brain barrier transportable soy dipeptide, Tyr-Pro, in combating memory impairment. We demonstrated for the first time that oral administration of Tyr-Pro (100 mg/kg, twice a day) in mice for 16 days significantly improved impaired memory by spontaneous alternation and shortened step-through latency in amyloid ß-induced mice.

Hesperidin blocks varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout mice by downregulating net uptake of oxidized low-density lipoprotein in macrophages.

Varenicline is a widely used and effective drug for smoking cessation. We have previously reported experimental evidence suggesting that varenicline increases the risk of cardiovascular events. Varenicline progresses atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. This adverse effect is likely due to enhanced net uptake of oxidized low-density lipoprotein (oxLDL) in macrophages as a result of increased scavenger receptors and decreased cholesterol efflux transporters. However, a regimen has not yet been presented for avoidance or amelioration of the risk for varenicline-induced cardiovascular events. The aim of this study was to examine the effect of hesperidin, a citrus flavonoid, on varenicline-aggravated atherosclerotic plaque formation in apolipoprotein E knockout (ApoE KO) mice. Hesperidin inhibited the aggravating effect of varenicline in the whole aorta, aortic arch, and aortic root of ApoE KO mice. In addition, hesperidin protected against varenicline-enhanced oxLDL net uptake by blocking the increased expression of CD36 and LOX-1 scavenger receptors and decreased expression of ABCA1 and ABCG1 cholesterol efflux transporters in RAW 264.7 cells. Our findings suggest that hesperidin can avoid or ameliorate the risk for cardiovascular events induced by varenicline treatment.

Varenicline aggravates atherosclerotic plaque formation in nicotine-pretreated ApoE knockout mice due to enhanced oxLDL uptake by macrophages through downregulation of ABCA1 and ABCG1 expression.

Varenicline is a widely used and effective drug for smoking cessation. We previously reported that varenicline aggravates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. However, it remains unknown whether varenicline affects cardiovascular events in patients with nicotine addiction. Here, we examined the effect of varenicline on atherosclerotic plaque formation in nicotine-pretreated ApoE KO mice and oxidized low-density lipoprotein (oxLDL) uptake in nicotine-treated peritoneal macrophages. Varenicline caused significant progression of plaque formation in the whole aorta and aortic root and further accelerated the increased formation of a macrophage-rich plaque area in the aortic root in nicotine-pretreated ApoE KO mice. Varenicline (10 µM) enhanced oxLDL uptake in peritoneal macrophages. Furthermore, this treatment significantly further lowered the decreased protein levels of ATP-binding cassette (ABC) transporter without affecting the expression of scavenger receptors LOX-1 and CD36 in RAW264.7 cells treated with 100 nM nicotine. Varenicline enhanced nicotine-induced oxLDL uptake in macrophages through decreased expression of cholesterol efflux transporters ABCA1 and ABCG1 and thereby progressed atherosclerotic plaque formation. Taken together, we tentatively conclude that nicotine exposure before and/or during varenicline treatment can aggravate varenicline-increased atherosclerotic plaque formation and progression. Therefore, this enhanced risk requires special consideration when prescribing varenicline to smoker patients.

Oligodendrocytes upregulate blood-brain barrier function through mechanisms other than the PDGF-BB/PDGFRα pathway in the barrier-tightening effect of oligodendrocyte progenitor cells.

White matter lesions are associated with impairment of the blood-brain barrier (BBB), an essential component of the cerebrovasculature. The BBB allows the brain to maintain its highly specialized microenvironment by restricting entry of blood-borne substances including molecules that induce myelin damage. Accumulating evidence suggests that interactions between brain endothelial cells and neighboring cells, including oligodendrocyte progenitor cells (OPCs), are required for the induction and maintenance of BBB function. Here, we compared the ability of OPCs and oligodendrocytes to modulate BBB integrity using co-cultures of rat brain endothelial cells with OPCs or oligodendrocytes. We found that OPCs lowered the brain endothelial permeability to sodium fluorescein, and this enhancement of BBB function was prevented by treatment with AG1296 (a PDGFRα inhibitor). Oligodendrocytes also enhanced BBB integrity. Pharmacological inhibition of PDGFRα did not affect the oligodendrocyte-induced BBB facilitation. These data indicate that oligodendrocytes enhance BBB integrity through pathways other than PDGF-BB/PDGFRα signaling triggered by the brain endothelial cell-derived PDGF-BB. Therefore, our findings suggest that oligodendrocytes constitutively support BBB integrity through soluble factors. Crosstalk between brain endothelial cells and oligodendrocytes could play a facilitatory role in maintaining BBB integrity in the white matter.

Oncostatin-M-Reactive Pericytes Aggravate Blood-Brain Barrier Dysfunction by Activating JAK/STAT3 Signaling In Vitro.

Oncostatin M (OSM) is a cytokine of the interleukin (IL)-6 family members. It induces blood-brain barrier (BBB) dysfunction by activating Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) 3 pathways in brain endothelial cells. Brain pericytes located around microvessels are one of the BBB constituents. Pericytes work as a boundary surface between the blood circulation and brain parenchyma, and their functions are altered under pathophysiological conditions, leading to BBB dysregulation. However, it remains unknown whether pericytes are associated with OSM-induced BBB dysfunction. We demonstrated that pericyte exposure to OSM (100 ng/mL) elevated phosphorylation of STAT3, a main OSM signaling pathway, and that pericytes expressed OSM receptors (OSMRs) including OSMRß and glycoprotein 130. These results suggest that pericytes are able to respond to OSM. To determine the effects of OSM-reactive pericytes on BBB functions, rat brain endothelial cell (RBEC) monolayers were cultured with OSM-treated pericytes. The presence of pericytes exposed to 100 ng/mL of OSM for 48 h aggravated both the elevated permeability to sodium fluorescein and the lowered transendothelial electrical resistance which were induced by OSM in RBECs. This OSM-reactive pericyte-induced aggravation of lowered RBEC barrier function was reversed by ruxolitinib, a JAK inhibitor. These findings suggest that activated JAK/STAT3 signaling in pericytes contributes to OSM-produced BBB breakdown. Thus, OSM-reactive pericytes may have to be considered a characteristic machinery in the formation and progression of BBB breakdown under pathological conditions associated with increased OSM levels.

Increased Plasma VEGF Levels in Patients with Cerebral Large Artery Disease Are Associated with Cerebral Microbleeds.

BACKGROUND/PURPOSE: Because atherosclerotic factors and antithrombotic agents sometimes induce cerebral microbleeds (CMBs), patients with cerebral large artery disease (CLAD) tend to have more CMBs than control subjects. On the other hand, VEGF contributes to the disruption of the blood-brain barrier, and it may induce parenchymal edema and bleeding. We conducted a study to evaluate the role of vascular endothelial growth factor (VEGF) in the occurrence of CMBs in patients with CLAD. METHODS: CLAD is defined as stenosis or occlusion of either the carotid artery or the middle cerebral artery of 50% or more. We prospectively registered patients with CLAD who were hospitalized in our neurocenter. Biological backgrounds, atherosclerotic risk factors, administration of antithrombotics before hospitalization, and levels of cytokines and chemokines were evaluated. Susceptibility-weighted imaging or T2*-weighted MR angiography was used to evaluate CMBs. The Brain Observer MicroBleed Scale (BOMBS) was used for CMB assessments. Images were analyzed with regard to the presence or absence of CMBs. We also examined plasma VEGF concentrations using a commercial ELISA kit. Because more than half showed plasma VEGF levels below assay detection limits (3.2 pg/mL), the patients were dichotomized by plasma VEGF levels into two groups (above and below the detection limit). After univariate analyses, logistic regression analysis was conducted to determine the factors associated with the CMBs after adjustment for age, sex, the presence of hypertension, and administration of antithrombotic agents. A similar analysis with CMBs separated by location (cortex, subcortex, or posterior circulation) was also conducted. RESULTS: Sixty-six patients (71.1 ± 8.9 years, 53 males and 13 females) were included in this study. Plasma VEGF levels were not correlated with age, sex, and atherosclerotic risk factors; however, patients with VEGF levels >3.2 pg/mL tended toward more frequent CMBs (60.0 vs. 32.6%, in the presence and absence of CMBs, p = 0.056). With regard to the location of CMBs, those in the cortex and/or at the gray-white junction were observed more frequently in the patients with VEGF levels >3.2 pg/mL after multivariable analyses (odds ratio: 3.80; 95% confidence interval: 1.07-13.5; p = 0.039). CONCLUSIONS: In patients with CLAD, elevated plasma VEGF might be associated with CMBs, especially those located in the cortex and/or at the gray-white junction.

Brain-transportable dipeptides across the blood-brain barrier in mice.

Apart from nutrients required for the brain, there has been no report that naturally occurring peptides can cross the blood-brain barrier (BBB). The aim of this study was to identify the BBB-transportable peptides using in situ mouse perfusion experiments. Based on the structural features of Gly-N-methylated Gly (Gly-Sar), a reported BBB-transportable compound, 18 dipeptides were synthesized, and were perfused in the mouse brain for two minutes. Among the synthesized dipeptides, Gly-Sar, Gly-Pro, and Tyr-Pro were transported across the BBB with Ki values of 7.60 ± 1.29, 3.49 ± 0.66, and 3.53 ± 0.74 µL/g·min, respectively, and accumulated in the mouse brain parenchyma. Additionally, using MALDI-MS/MS imaging analysis of Tyr-Pro-perfused brain, we provide evidence for Tyr-Pro accumulation in the hippocampus, hypothalamus, striatum, cerebral cortex, and cerebellum of mouse brain.

Monomeric α-synuclein induces blood-brain barrier dysfunction through activated brain pericytes releasing inflammatory mediators in vitro.

Blood-brain barrier (BBB) disruption is often mediated by neuroinflammation, and occurs during various neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by loss of dopaminergic neurons and aggregated α-synuclein protein in inclusions known as Lewy bodies. Misfolded α-synuclein has been implicated in neurodegeneration and neuroinflammation through activation of microglia and astrocytes. Pericytes are a key cellular regulator of the BBB, although it is not known if they participate in α-synuclein-associated PD pathology. Here, we investigated the impact of pericytes on BBB integrity in response to α-synuclein using rat brain endothelial cells (RBECs) co-cultured with rat brain pericytes (RBEC/pericyte co-culture). In RBEC/pericyte co-cultures, α-synuclein added to the abluminal chamber (where pericytes were grown) significantly increased RBEC permeability to sodium fluorescein. In contrast, it had no marked effect when added to the luminal chamber. In the absence of pericytes, both luminal and abluminal addition of α-synuclein failed to affect permeability of the RBEC monolayer. α-Synuclein did not self-assemble in culture media within 24 h, suggesting that monomeric α-synuclein can disrupt the BBB by interacting with pericytes. We found that in response to α-synuclein, pericytes, but not RBECs, released interleukin (IL)-1ß, IL-6, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, and matrix metalloproteinase-9 (MMP-9). α-Synuclein did not affect platelet-derived growth factor (PDGF)-BB release from RBECs and PDGF receptor-ß expression in pericytes. These results suggest that pericytes are more sensitive to monomeric α-synuclein than RBECs regarding release of various inflammatory cytokines/chemokines and MMP-9. Thus, monomeric α-synuclein-activated pericytes may contribute to BBB breakdown in patients with PD.

Activation of the α7 nicotinic acetylcholine receptor upregulates blood-brain barrier function through increased claudin-5 and occludin expression in rat brain endothelial cells.

The blood-brain barrier (BBB) is formed by brain endothelial cells (BECs) and regulates brain homeostasis by restricting the entry of blood-borne substances into the brain. Recent in vivo studies have shown that administration of nicotinic acetylcholine receptor (nAChR) agonists protects against BBB disruption and neuroinflammation induced by stroke and traumatic brain injury through the systemic cholinergic anti-inflammatory pathway. In the present study, we focused on the nAChRs expressed on BECs rather than those widely expressed in the central nervous system and peripheral tissues, and examined whether activation of the nAChRs on BECs facilitates BBB function. We used primary cultures of rat brain endothelial cells to evaluate brain endothelial permeability and tight junction (TJ)-related protein expression after a 24-h exposure to PHA543613 (a selective α7 nAChR agonist) or 5-iodo-A-85380 (a selective α4ß2 nAChR agonist). We found that PHA543613 decreased sodium fluorescein permeability and increased the expression levels of claudin-5 and occludin, key TJ components. In contrast, 5-iodo-A-85380 had no effect on brain endothelial permeability or TJ protein expression. These findings suggest that the selective activation of α7 nAChRs on BECs has a specific role in upregulating BBB properties through increased claudin-5 and occludin expression.

Feeding-produced subchronic high plasma levels of uric acid improve behavioral dysfunction in 6-hydroxydopamine-induced mouse model of Parkinson's disease.

The development of Parkinson's disease (PD) involves the degeneration of dopaminergic neurons caused by oxidative stress. Accumulating clinical evidence indicates that high blood levels of uric acid (UA), an intrinsic antioxidative substance, are associated with reduced risk of PD. However, this hypothesis has not been confirmed by in-vivo experiments. The present study investigated the effects of UA on behavioral abnormalities in the development of PD. We used unilateral 6-hydroxydopamine-lesioned mice, which were fed on a diet containing 1% UA and 2.5% potassium oxonate (an uricase inhibitor) to induce hyperuricemia. A significant elevation in UA levels was found in groups that were fed a UA diet. The 6-hydroxydopamine-lesioned mice showed impaired rotarod performance and increased apomorphine-induced contralateral rotations. These behavioral abnormalities were significantly reversed by feeding a UA diet for 1 week before and 5 weeks after surgery (subchronic hyperuricemia). These behavioral improvements occurred in parallel with recovery of tyrosine hydroxylase protein levels in the lesioned striatal side. The present study with a dietary hyperuricemia mice model confirms that UA exerts a neuroprotective effect on dopaminergic neuronal loss, improving motor dysfunction and ameliorating PD development.

Oncostatin M-induced blood-brain barrier impairment is due to prolonged activation of STAT3 signaling in vitro.

Oncostatin M (OSM) is a member of the interleukin (IL)-6 family cytokines. We previously demonstrated that OSM induces blood-brain barrier (BBB) impairment. However, functional characterization of IL-6 family cytokines in BBB regulation and the cytokine-related intracellular signaling pathway remain unclear. In this study, we demonstrate that among IL-6 family cytokines, including IL-6 and leukemia inhibitory factor (LIF), OSM is the most potent molecule for inducing BBB dysfunction via prolonged activation of signal transducer and activator of transcription (STAT) 3 following Janus-activated kinase (JAK) activation. OSM but not IL-6 and LIF (100 ng/mL for 24 hours) markedly produced increased sodium fluorescein permeability and decreased transendothelial electrical resistance in rat brain endothelial cell (RBEC) monolayers. This OSM-induced BBB dysfunction was accompanied by decreased levels of claudin-5 expression in RBECs, which were ameliorated by JAK inhibitor. We examined the time-course of STAT3 phosphorylation in RBECs treated with OSM, IL-6, and LIF. OSM upregulated STAT3 phosphorylation levels during a 24 hours period with a peak at 10 minutes. While IL-6 and LIF transiently increased phosphorylated STAT3 at 10 minutes after addition, this phosphorylation decreased during the period from 1 to 24 hours after addition. These findings suggest that OSM-induced sustained increases in STAT3 phosphorylation levels largely contribute to BBB impairment. Thus, elevated OSM levels and activation of brain endothelial JAK/STAT3 signaling pathway under pathological conditions should be considered as a possible hallmark for induction and development of BBB impairment.

Varenicline is a smoking cessation drug that blocks alveolar expansion in mice intratracheally administrated porcine pancreatic elastase.

Smoking cessation is the most effective treatment in patients with emphysema and lung inflammation. The aim of the present study was to examine the effect of varenicline, a smoking cessation drug, on emphysema in porcine pancreatic elastase (PPE)-inhaled mice. PPE-inhaled mice were treated with varenicline and an α7 nicotinic acetylcholine receptor (nAChR) antagonist, methyllycaconitine (MLA) for 5 and 21 days. Varenicline markedly ameliorated alveolar expansion and inflammatory response in bronchoalveolar lavage fluid in PPE-inhaled mice. These blocking effects were inhibited by MLA. Our findings demonstrate that varenicline likely has an anti-inflammatory property including reduced inflammatory cell recruitment in lung tissue to protect PPE-induced alveolar expansion via α7 nAChR.

TNF-α-sensitive brain pericytes activate microglia by releasing IL-6 through cooperation between IκB-NFκB and JAK-STAT3 pathways.

Interleukin (IL)-6 is an important mediator of neurovascular dysfunction, neurodegeneration and/or neuroinflammation. We previously reported that brain pericytes released higher levels of IL-6 than did glial cells (astrocytes and microglia) in response to tumor necrosis factor (TNF)-α. Moreover, pericytes stimulated with TNF-α enhanced activation of BV-2 microglia. In this study, we investigated the mechanisms of TNF-α mediated induction of IL-6 release from brain pericytes and astrocytes and whether pericyte-derived IL-6 would facilitate activation of BV-2 microglia. Using rat brain pericyte and astrocyte primary cultures and pharmacological inhibitors, we found that, TNF-α induced the highest levels of IL-6 release from pericytes by activating the inhibitor kappa B (IκB)-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and Janus family of tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT)3 pathways. STAT3 contributed to TNF-α induced nuclear translocation of phospho-NFκB in pericytes. TNF-α-induced IL-6 release in astrocytes was mediated by NFκB but not by STAT3. The presence of pericytes amplified TNF-α-induced iNOS mRNA expression in BV-2 microglia. This effect was blocked by a neutralizing antibody for IL-6. These findings indicated that crosstalk between the IκB-NFκB and JAK-STAT3 pathways is a pericyte specific mechanism, not occurring in astrocytes, for TNF-α-induced IL-6 release. IL-6 derived from pericytes enhanced microglial activation. Our findings increase understanding of the role of pericyte-microglia crosstalk in the brain under neuroinflammatory conditions and suggest a potentially attractive therapeutic target for brain inflammation.

Effect of varenicline on behavioral deficits in a rat model of Parkinson's disease induced by unilateral 6-hydroxydopamine lesion of substantia nigra.

Nicotinic acetylcholine receptors (nAChRs) are implicated in the pathogenesis of Parkinson's disease (PD). Varenicline tartrate is a partial agonist at α4ß2 and full agonist at α7 neuronal nAChR subunits. A unilateral lesion of the substantia nigra (SN) has been used as a reliable model of PD. This study aimed to investigate the effect of varenicline on locomotor and nonlocomotor behavioral deficits induced by a unilateral lesion of the SN induced by 6-hydroxydopamine (6-OHDA) (8 µg/4 µl). Varenicline (1 mg/kg) was administered to the lesioned rats daily for 2 weeks, which commenced 3 weeks after 6-OHDA administration. The results showed that varenicline improved motor deficits induced by 6-OHDA. It improved locomotor and nonlocomotor activities such as forelimb use, rotarod performance, and forelimb asymmetry. Varenicline did not change rearing or vibrissae-elicited forelimb placing but did increase apomorphine-induced rotation. In conclusion, the present results suggest that drugs with specific partial/full agonistic activity on nAChR subunits could be of value in the treatment of neurodegenerative disorders such as PD.

Varenicline promotes endothelial cell migration by lowering vascular endothelial-cadherin levels via the activated α7 nicotinic acetylcholine receptor-mitogen activated protein kinase axis.

Varenicline is a widely used and effective drug for smoking cessation. Despite its efficacy, varenicline increases the risk of cardiovascular disease. We previously demonstrated that varenicline aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice. However, little is known about its effects in vascular endothelial cells. Therefore, we examined whether varenicline promotes migration of human umbilical vein endothelial cells (HUVECs) using the Boyden chamber assay. Varenicline (100µM) markedly promoted migration of HUVECs and decreased expression of vascular endothelial (VE)-cadherin, an endothelial adhesion molecule. Extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) signaling were markedly activated by varenicline. Methyllycaconitine (MLA; 100nM), an α7 nicotinic acetylcholine receptor (nAChR) antagonist, but not dihydro-ß-erythroidine hydrobromide (DHßE; 20µM) blocked varenicline-stimulated migration and varenicline-activated ERK, p38 and JNK signaling in HUVECs. MLA (100nM), PD98059 (an ERK inhibitor; 20µM), SB203580 (a p38 inhibitor; 20µM) and SP600125 (a JNK inhibitor; 20µM) also blocked cell migration and varenicline-induced downregulation of VE-cadherin expression in HUVECs. These findings suggest that varenicline promotes HUVEC migration by lowering VE-cadherin expression due to activated ERK/p38/JNK signaling through α7 nAChR. These processes probably contribute to varenicline-aggravated atherosclerotic plaque. Hence, an increased risk of cardiovascular events upon varenicline treatment might occur and must be considered in patients with cardiovascular diseases.