Cerebral Capillary Endothelial Cells Research Articles

RGD-coated polymeric microbubbles promote ultrasound-mediated drug delivery in an inflamed endothelium-pericyte co-culture model of the blood-brain barrier.

Drug delivery to central nervous pathologies is compromised by the blood-brain barrier (BBB). A clinically explored strategy to promote drug delivery across the BBB is sonopermeation, which relies on the combined use of ultrasound (US) and microbubbles (MB) to induce temporally and spatially controlled opening of the BBB. We developed an advanced in vitro BBB model to study the impact of sonopermeation on the delivery of the prototypic polymeric drug carrier pHPMA as a larger molecule and the small molecule antiviral drug ribavirin. This was done under standard and under inflammatory conditions, employing both untargeted and RGD peptide-coated MB. The BBB model is based on human cerebral capillary endothelial cells and human placental pericytes, which are co-cultivated in transwell inserts and which present with proper transendothelial electrical resistance (TEER). Sonopermeation induced a significant decrease in TEER values and facilitated the trans-BBB delivery of fluorescently labeled pHPMA (Atto488-pHPMA). To study drug delivery under inflamed endothelial conditions, which are typical for e.g. tumors, neurodegenerative diseases and CNS infections, tumor necrosis factor (TNF) was employed to induce inflammation in the BBB model. RGD-coated MB bound to and permeabilized the inflamed endothelium-pericyte co-culture model, and potently improved Atto488-pHPMA and ribavirin delivery. Taken together, our work combines in vitro BBB bioengineering with MB-mediated drug delivery enhancement, thereby providing a framework for future studies on optimization of US-mediated drug delivery to the brain.

Functional role of adenosine via KATP channels in cerebral capillary endothelial cells and pericytes.

Functional role of adenosine via KATP channels in cerebral capillary endothelial cells and pericytes.

Tuning the signal: ATP-sensitive K+ channels direct blood flow to cerebral capillaries.

Cerebral blood flow must be exquisitely regulated to match the metabolic demands of neurons. In this issue of Science Signaling, Sancho et al. characterize functional ATP-sensitive K+ (KATP) channels in cerebral capillary endothelial cells and pericytes that can be activated by adenosine signaling, thereby leading to increases in capillary blood flow.

Aberrant Cerebral Iron Trafficking Co-morbid With Chronic Inflammation: Molecular Mechanisms and Pharmacologic Intervention

The redox properties that make iron an essential nutrient also make iron an efficient pro-oxidant. Given this nascent cytotoxicity, iron homeostasis relies on a combination of iron transporters, chaperones, and redox buffers to manage the non-physiologic aqueous chemistry of this first-row transition metal. Although a mechanistic understanding of the link between brain iron accumulation (BIA) and neurodegenerative diseases is lacking, BIA is co-morbid with the majority of cognitive and motor function disorders. The most prevalent neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple System Atrophy (MSA), and Multiple Sclerosis (MS), often present with increased deposition of iron into the brain. In addition, ataxias that are linked to mutations in mitochondrial-localized proteins (Friedreich's Ataxia, Spinocerebellar Ataxias) result in mitochondrial iron accumulation and degradation of proton-coupled ATP production leading to neuronal degeneration. A comorbidity common in the elderly is a chronic systemic inflammation mediated by primary cytokines released by macrophages, and acute phase proteins (APPs) released subsequently from the liver. Abluminal inflammation in the brain is found downstream as a result of activation of astrocytes and microglia. Reasonably, the iron that accumulates in the brain comes from the cerebral vasculature via the microvascular capillary endothelial cells whose tight junctions represent the blood-brain barrier. A premise amenable to experimental interrogation is that inflammatory stress alters both the trans- and para-cellular flux of iron at this barrier resulting in a net accumulation of abluminal iron over time. This review will summarize the evidence that lends support to this premise; indicate the mechanisms that merit delineation; and highlight possible therapeutic interventions based on this model.

Endothelial Ion Channels and Cell-Cell Communication in the Microcirculation

Endothelial cells in resistance arteries, arterioles, and capillaries express a diverse array of ion channels that contribute to Cell-Cell communication in the microcirculation. Endothelial cells are tightly electrically coupled to their neighboring endothelial cells by gap junctions allowing ion channel-induced changes in membrane potential to be conducted for considerable distances along the endothelial cell tube that lines arterioles and forms capillaries. In addition, endothelial cells may be electrically coupled to overlying smooth muscle cells in arterioles and to pericytes in capillaries via heterocellular gap junctions allowing electrical signals generated by endothelial cell ion channels to be transmitted to overlying mural cells to affect smooth muscle or pericyte contractile activity. Arteriolar endothelial cells express inositol 1,4,5 trisphosphate receptors (IP3Rs) and transient receptor vanilloid family member 4 (TRPV4) channels that contribute to agonist-induced endothelial Ca2+ signals. These Ca2+ signals then activate intermediate and small conductance Ca2+-activated K+ (IKCa and SKCa) channels causing vasodilator-induced endothelial hyperpolarization. This hyperpolarization can be conducted along the endothelium via homocellular gap junctions and transmitted to overlying smooth muscle cells through heterocellular gap junctions to control the activity of voltage-gated Ca2+ channels and smooth muscle or pericyte contraction. The IKCa- and SKCa-induced hyperpolarization may be amplified by activation of inward rectifier K+ (KIR) channels. Endothelial cell IP3R- and TRPV4-mediated Ca2+ signals also control the production of endothelial cell vasodilator autacoids, such as NO, PGI2, and epoxides of arachidonic acid contributing to control of overlying vascular smooth muscle contractile activity. Cerebral capillary endothelial cells lack IKCa and SKCa but express KIR channels, IP3R, TRPV4, and other Ca2+ permeable channels allowing capillary-to-arteriole signaling via hyperpolarization and Ca2+. This allows parenchymal cell signals to be detected in capillaries and signaled to upstream arterioles to control blood flow to capillaries by active parenchymal cells. Thus, endothelial cell ion channels importantly participate in several forms of Cell-Cell communication in the microcirculation that contribute to microcirculatory function and homeostasis.

Harmful Effects of COVID-19 on Major Human Body Organs: A Review

The world experienced the outbreak of a new pandemic disease in 2019, known as coronavirus (CoV) disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome-CoV-2 (SARS-CoV-2). The respiratory system is the organ system most commonly affected by COVID-19; however, several other organ systems have been reported to be affected. The SARS-CoV-2 RNA found in infected stub samples can cause lung contagion by binding to the angiotensin-converting enzyme-2 (ACE-2) receptor of the alveolar epithelial cells. The gut microbiota (GM) promote immunity, indicating that the alignment of the microbiota and corresponding metabolic processes in COVID-19 can help to identify novel biomarkers and new therapeutic targets for this disease. The cause of kidney damage in COVID-19 patients is possibly multifactorial, involving a complex mechanism that involves complement dysregulation and thrombotic microangiopathy, as well as the occurrence of a “cytokine storm” syndrome, which are immune responses that are abandoned and dysfunctional with unfavorable prognosis in severe COVID-19 cases. Furthermore, COVID-19 involves a continuous proliferation and activation of macrophages and lymphocytes. SARS-CoV-2 can also bind to the ACE-2 receptor expressed in the cerebral capillary endothelial cells that can invade the blood-brain wall, to penetrate the brain parenchyma. However, in the ongoing pandemic, there has been a surge in studies on a wide range of topics, including causes of respiratory failure, asymptomatic patients, intensive care patients, and survivors. This review briefly describes the damaging effects of COVID-19 on vital human organs and the inhibitory function of the ACE-2 receptor on the GM, which causes gut dysbiosis, and thus, this review discusses topics that have an opportunity for further investigation.

Reduction of mNAT1/hNAT2 Contributes to Cerebral Endothelial Necroptosis and Aβ Accumulation in Alzheimer’s Disease

The contribution and mechanism of cerebrovascular pathology in Alzheimer's disease (AD) pathogenesis are still unclear. Here, we show that venular and capillary cerebral endothelial cells (ECs) are selectively vulnerable to necroptosis in AD. We identify reduced cerebromicrovascular expression of murine N-acetyltransferase 1 (mNat1) in two AD mouse models and hNat2, the human ortholog of mNat1 and a genetic risk factor for type-2 diabetes and insulin resistance, in human AD. mNat1 deficiency in Nat1-/- mice and two AD mouse models promotes blood-brain barrier (BBB) damage and endothelial necroptosis. Decreased mNat1 expression induces lysosomal degradation of A20, an important regulator of necroptosis, and LRP1β, a key component of LRP1 complex that exports Aβ in cerebral ECs. Selective restoration of cerebral EC expression of mNAT1 delivered by adeno-associated virus (AAV) rescues cerebromicrovascular levels of A20 and LRP1β, inhibits endothelial necroptosis and activation, ameliorates mitochondrial fragmentation, reduces Aβ deposits, and improves cognitive function in the AD mouse model.

Systemic delivery of BACE1 siRNA through neuron-targeted nanocomplexes for treatment of Alzheimer's disease

β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key enzyme to cleave the amyloid precursor protein to develop Alzheimer's disease (AD). Reducing BACE1 expression in central neuron through RNA interference technology shows great promise to overcome AD. However, to obtain an efficient and neurons-specific delivery of siRNA against BACE1 through systemic administration remains challenging. Here, we design and prepare siRNA nano-carriers based on PEGylated poly(2-(N,N-dimethylamino) ethyl methacrylate) (PEG-PDMAEMA) modified with both the CGN peptide for blood-brain barrier (BBB) penetration and the Tet1 peptide for neuron-specific binding. The nanocomplexes CT/siRNA, composed of CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA at a weight ratio of 1:1, display a good stability in the blood and do not lead to hemolysis at N/P = 10. The internalization of nanocomplexes in neuron cells relies on clathrin-mediated endocytosis and micropinocytosis, while caveolae-mediated endocytosis plays a major role in entrance of CT/siRNA into cerebral capillary endothelial cell bEnd.3. The nanocomplexes successfully escape from lysosomes and enter in the cytoplasm of the neuron cells, inducing effective gene silence (about 50% decrease in BACE1 mRNA levels) and reversing Aβ25–35 oligomer-induced synaptic injury. After caudal vein injection in mice, CT/siRNA display higher brain accumulation than unmodified nanocomplexes (brain drug targeting index = 2.62), and colocalize with neurons or locate nearby. In APP/PS1 transgenic mice, the nanocomplexes significantly decrease BACE1 mRNA and the amyloid plaques, suppress phosphorylated tau protein levels, as well as promote hippocampal neurogenesis. Noticeably, administration of the nanocomplexes restores the cognitive performance of the AD transgenic mice to the level of wild-type control without significant adverse effects on myelination. Our results demonstrate the CT/siRNA nanocomplexes capable of specifically directing BACE1 siRNA to brain neurons with great potential for AD therapy.

Cerebral Capillary TRPA1 Channels Mediate Functional Hyperemia via Retrograde Conducted Vasodilation

Cerebral capillaries are the sites of nutrient exchange and removal of metabolic by‐products within the brain parenchyma. Recent studies showed that cerebral capillary endothelial cells (ECs) are central players in matching augmented neuronal metabolism to an increase in local blood perfusion, a process known as functional hyperemia. However, little is known about the molecular sensors present in capillary EC that orchestrate dilation of upstream parenchymal arterioles (PAs). We previously showed that the transient receptor potential ankyrin 1 (TRPA1) channel, a Ca2+‐permeable non‐selective cation channel, is present in ECs from cerebral arteries, but not in other peripheral vascular beds. We tested the hypothesis that TRPA1 is also present in cerebral capillary EC and its activation induces dilation of upstream PAs through a Ca2+‐dependent mechanism. Immunofluorescence staining of freshly isolated cerebral capillaries showed the presence of TRPA1, which was absent in EC‐specific TRPA1 knockout mice (eTRPA1−/−). Further, currents evoked by the TRPA1 agonist allyl isothyocyanate (AITC, 30 μM) in whole‐cell patch clamp electrophysiology experiments were blocked by the selective TRPA1 antagonist HC‐030031 (10 μM) and were absent from eTRPA1−/− mice. Using a recently‐described ex vivo capillary‐parenchymal arteriole preparation in which the capillary bed remains attached to pressurized PAs, we observed that picospritzing capillaries with AITC induced dilation of the upstream PA, an effect inhibited by HC‐030031 and absent in eTRPA1−/− mice. We then investigated the mechanisms underlying propagation of the vasodilatory signal from capillary to arteriolar EC. Using mice expressing the genetically‐encoded Ca2+ biosensor GCaMP6f in EC, we observed that picospritzing AITC onto capillary beds generated an intercellular Ca2+ wave that propagated from capillaries to the upstream PA. Pre‐incubation of preparations with the TRPA1 inhibitor HC‐030031 abolished the generation of Ca2+ waves. Moreover, the frequency of Ca2+ transients in arteriolar EC was increased immediately after dissipation of the Ca2+ wave, a process blocked by HC‐030031. Increases in arteriolar EC intracellular Ca2+ can activate intermediate and small conductance Ca2+‐activated K+ channels (IK and SK, respectively), leading to vasodilation. To test if this mechanism was responsible for PA dilation, preparations were incubated with the IK and SK channels inhibitors TRAM‐34 (1 μM) and apamin (1 μM), which blunted arteriolar dilation induced by picospritzing AITC onto capillaries. Somatosensory stimulation of the whisker barrel cortex, a model of functional hyperemia, caused a smaller increase in perfusion in eTRPA1−/− mice than in wildtype littermates. Taken together, these data suggest that functional TRPA1 channels are present in cerebral capillary EC and that activation of the channel elicits intercellular Ca2+ waves that propagate to upstream PAs, where it activates IK and SK channels to cause hyperpolarization and dilation, thus mediating functional hyperemia responses.Support or Funding InformationSupported by the National Institutes of Health (R01HL091095 to SE) and the American Heart Association (15POST2472002 to PWP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carrier-mediated cocaine transport at the blood-brain barrier as a putative mechanism in addiction liability.

Background:The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been considered the unique mechanism known by which cocaine crosses the blood-brain barrier.Methods:We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion.Results:Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter.Conclusions:Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms.

Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5days and repeated 4 times with 1–4month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

Receptor-Mediated Delivery of Magnetic Nanoparticles across the Blood–Brain Barrier

A brain delivery probe was prepared by covalently conjugating lactoferrin (Lf) to a poly(ethylene glycol) (PEG)-coated Fe(3)O(4) nanoparticle in order to facilitate the transport of the nanoparticles across the blood-brain barrier (BBB) by receptor-mediated transcytosis via the Lf receptor present on cerebral endothelial cells. The efficacy of the Fe(3)O(4)-Lf conjugate to cross the BBB was evaluated in vitro using a cell culture model for the blood-brain barrier as well as in vivo in SD rats. For an in vitro experiment, a well-established porcine BBB model was used based on the primary culture of cerebral capillary endothelial cells grown on filter supports, thus allowing one to follow the transfer of nanoparticles from the apical (blood) to the basolateral (brain) side. For in vivo experiments, SD rats were used as animal model to detect the passage of the nanoparticles through the BBB by MRI techniques. The results of both in vitro and in vivo experiments revealed that the Fe(3)O(4)-Lf probe exhibited an enhanced ability to cross the BBB in comparison to the PEG-coated Fe(3)O(4) nanoparticles and further suggested that the Lf-receptor-mediated transcytosis was an effective measure for delivering the nanoparticles across the BBB.

Ammonia increases paracellular permeability of rat brain endothelial cells by a mechanism encompassing oxidative/nitrosative stress and activation of matrix metalloproteinases

Ammonia is responsible for cerebral edema associated with acute liver failure, but the role of the vasogenic mechanism has been a matter of dispute. Here, we tested the hypothesis that ammonia induces changes in blood-brain barrier (BBB) permeability by a mechanism coupled to oxidative/nitrosative stress (ONS) evoked in the BBB-forming cerebral capillary endothelial cells. Treatment of a rat brain endothelial cell line with ammonia (5 mmol/L, 24 h) caused accumulation of ONS markers: reactive oxygen species, nitric oxide and peroxidation products of phospholipid-bound arachidonic acid, F2-isoprostanes. Concurrently, ammonia increased the activity of extracellular matrix metalloproteinases (MMP-2/MMP-9), increased cell permeability to fluorescein isothiocyanate-dextran (40 kDa), and increased the expression of y+LAT2, a transporter that mediates the uptake to the cells of the nitric oxide precursor, arginine. The increase of cell permeability was ameliorated upon co-treatment with a MMP inhibitor, SB-3CT and with an antioxidant, glutathione diethyl ester, which also reduced F2-isoprostanes. Ammonia-induced ONS was attenuated by cytoprotective agents l-ornithine, phenylbutyrate, and their conjugate l-ornithine phenylbutyrate, an ammonia-trapping drug used to treat hyperammonemia. The results support the concept that ONS and ONS-related activation of MMPs in cerebral capillary endothelial cells contribute to the alterations in BBB permeability and to the vasogenic component of cerebral edema associated with acute liver failure.

Inhibition of Intercellular Adhesion in Herpex Simplex Virus Infection by Glycyrrhizin

Herpes simplex virus (HSV) is one of the most common viruses infecting humans and animals. Cellular adhesion is increased in HSV and plays a role in pathogenesis of inflammatory response during this viral infection. In our study, we studied a potential role of glycyrrhizin in disrupting cellular adhesion in HSV. We isolated rat cerebral capillary vessel endothelial cells (CCECs) and polymorphonuclear leukocytes (PMN) and evaluated intercellular adhesion between these cells by micropipette aspiration technique. The adhesion force and stress between CCEC and PMN were significantly (P < 0.01) increased in HSV infection. Glycyrrhizin perfusion significantly (P < 0.01) reduced adhesion force and stress between CCEC and PMN. In conclusion, glycyrrhizin may attenuate inflammatory responses in HSV by inhibition of adhesion between CCEC and PMN.

Alterations of Blood Brain Barrier Function in Hyperammonemia: An Overview

Ammonia is a neurotoxin involved in the pathogenesis of neurological conditions associated with hyperammonemia, including hepatic encephalopathy, a condition associated with acute—(ALF) or chronic liver failure. This article reviews evidence that apart from directly affecting the metabolism and function of the central nervous system cells, ammonia influences the passage of different molecules across the blood brain barrier (BBB). A brief description is provided of the tight junctions, which couple adjacent cerebral capillary endothelial cells to each other to form the barrier. Ammonia modulates the transcellular passage of low-to medium-size molecules, by affecting their carriers located at the BBB. Ammonia induces interrelated aberrations of the transport of the large neutral amino acids and aromatic amino acids (AAA), whose influx is augmented by exchange with glutamine produced in the course of ammonia detoxification, and maybe also modulated by the extracellularly acting gamma-glutamyl moiety transferring enzyme, gamma-glutamyl-transpeptidase. Impaired AAA transport affects neurotransmission by altering intracerebral synthesis of catecholamines (serotonin and dopamine), and producing “false neurotransmitters” (octopamine and phenylethylamine). Ammonia also modulates BBB transport of the cationic amino acids: the nitric oxide precursor, arginine, and ornithine, which is an ammonia trap, and affects the transport of energy metabolites glucose and creatine. Moreover, ammonia acting either directly or in synergy with liver injury-derived inflammatory cytokines also evokes subtle increases of the transcellular passage of molecules of different size (BBB “leakage”), which appears to be responsible for the vasogenic component of cerebral edema associated with ALF.

Cerebral capillary endothelial cells are covered by the VEGF-expressing foot processes of astrocytes

Molecules that have crucial functions in both nervous and vascular systems have attracted keen attention recently, and the name “angioneurins” has been proposed. The most striking example of angioneurins is vascular endothelial growth factor A (VEGF), which was originally identified as a key regulator of angiogenesis and has only recently been found to have important functions in the nervous system. In this study, we compared VEGF expression in the vasculature in the brain with that in the aorta and the vasculature in the kidney in mice. In larger vessels containing smooth muscle cells, VEGF was expressed by smooth muscle cells covering the lining of endothelial cells, both in and outside the brain. In cerebral capillaries lacking smooth muscle cells, endothelial cells were closely covered by VEGF-expressing foot processes of astrocytes, whereas capillaries were surrounded by VEGF-expressing processes of podocytes in the renal glomeruli. We also found that cultured cerebral microvessel endothelial cells do not express VEGF, whereas cultured cortical astrocytes do express VEGF.

Insights into the putative catechin and epicatechin transport across blood-brain barrier

The identification of mechanisms associated with phenolic neuroprotection is delayed due to a lack of information regarding the ability of phenolic compounds to enter the central nervous system (CNS). The aim of this work was to evaluate the transmembrane transport of catechin and epicatechin across blood-brain barrier (BBB). Two BBB cell lines, RBE-4 cells (immortalized cell line of rat capillary cerebral endothelial cells) and hCMEC/D3 (immortalized human cerebral microvessel endothelial cell line), were used. HPLC-DAD/MS was used to detect these compounds and their metabolites in the studied samples. The metabolites of the tested flavan-3-ols were synthesized to be used as standards. Catechin and epicatechin could cross both cells in a time-dependent manner. This transport was stereoselective (epicatechin ≫ catechin), involving one or more stereoselective entities. Additionally, these cells were capable of metabolizing these compounds, particularly by conjugation with glucuronic acid, since this metabolite was detected in the basolateral media. Several studies suggest that blood levels of catechin and epicatechin are far below the levels used in this study and that these compounds appeared mainly as methyl, sulfate and glucuronide metabolites. Nevertheless, the information obtained by this study is valuable for the new insights about flavan-3-ols transport. (i) catechin and epicatechin are capable of crossing the BBB; (ii) a stereoselective process was involved in the passage of these compounds across BBB cells; (iii) these endothelial cells have enzymes capable of metabolizing these compounds.

Flavonoid transport across RBE4 cells: A blood-brain barrier model.

There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.

The blood-brain barrier and glutamate

Glutamate concentrations in plasma are 50–100 μmol/L; in whole brain, they are 10,000–12,000 μmol/L but only 0.5–2 μmol/L in extracellular fluids (ECFs). The low ECF concentrations, which are essential for optimal brain function, are maintained by neurons, astrocytes, and the blood-brain barrier (BBB). Cerebral capillary endothelial cells form the BBB that surrounds the entire central nervous system. Tight junctions connect endothelial cells and separate the BBB into luminal and abluminal domains. Molecules entering or leaving the brain thus must pass 2 membranes, and each membrane has distinct properties. Facilitative carriers exist only in luminal membranes, and Na+-dependent glutamate cotransporters (excitatory amino acid transporters; EAATs) exist exclusively in abluminal membranes. The EAATs are secondary transporters that couple the Na+ gradient between the ECF and the endothelial cell to move glutamate against the existing electrochemical gradient. Thus, the EAATs in the abluminal membrane shift glutamate from the ECF to the endothelial cell where glutamate is free to diffuse into blood on facilitative carriers. This organization does not allow net glutamate entry to the brain; rather, it promotes the removal of glutamate and the maintenance of low glutamate concentrations in the ECF. This explains studies that show that the BBB is impermeable to glutamate, even at high concentrations, except in a few small areas that have fenestrated capillaries (circumventricular organs). Recently, the question of whether the BBB becomes permeable in diabetes has arisen. This issue was tested in rats with diet-induced obesity and insulin resistance or with streptozotocin-induced diabetes. Neither condition produced any detectable effect on BBB glutamate transport.

Glucocorticoids regulate the human occludin gene through a single imperfect palindromic glucocorticoid response element

The 65 kDa protein occludin is an essential element of the blood-brain barrier. This integral membrane protein represents an important part of the tight junctions, which seal and protect the blood brain barrier against paracellular diffusion of solutes to the brain parenchyme and are therefore responsible for the high resistance and low permeability between cerebral capillary endothelial cells. However, the molecular basis for the regulation of occludin gene expression is only incompletely understood. In former projects we showed that treatment of a brain microvascular cell line, cEND, with glucocorticoids resulted in increased occludin expression in cell-cell-contacts Induction of occludin expression by glucocorticoids was shown to be dependent on the glucocorticoid receptor. This study aims to identify the underlying molecular mechanism of gene expression and to identify potential glucocorticoid receptor binding sites within the occludin promoter, the glucocorticoid response elements (GRE). We identified one candidate GRE within the distal part of the occludin promoter that differs from the consensus GRE by the presence of a 4-basepair instead of a 3-basepair spacer between two highly degenerate halfsites (5'-ACATGTGTTTACAAAT-3'). Chromatin immunoprecipitation assay and site-directed mutagenesis confirmed binding of the glucocorticoid receptor to this site. We synthesized plasmid containing four copies of this GRE in the vector pGL-3 basic. After stimulation of the cells with hydrocortisone we could observe up to 8-fold increased activity in luciferase reporter assay. The need for glucocorticoid receptor dimerization to induce gene expression was further confirmed by transfection studies using wild type and glucocorticoid receptor dimerization-deficient expression vectors, indicating that transactivation of occludin occur through the GRE.