Endothelial Transport Research Articles

Modeling serotonin uptake in the lung shows endothelial transporters dominate over cleft permeation

A four-region (capillary plasma, endothelium, interstitial fluid, cell) multipath model was configured to describe the kinetics of blood-tissue exchange for small solutes in the lung, accounting for regional flow heterogeneity, permeation of cell membranes and through interendothelial clefts, and intracellular reactions. Serotonin uptake data from the Multiple indicator dilution "bolus sweep" experiments of Rickaby and coworkers (Rickaby DA, Linehan JH, Bronikowski TA, Dawson CA. J Appl Physiol 51: 405-414, 1981; Rickaby DA, Dawson CA, and Linehan JH. J Appl Physiol 56: 1170-1177, 1984) and Malcorps et al. (Malcorps CM, Dawson CA, Linehan JH, Bronikowski TA, Rickaby DA, Herman AG, Will JA. J Appl Physiol 57: 720-730, 1984) were analyzed to distinguish facilitated transport into the endothelial cells (EC) and the inhibition of tracer transport by nontracer serotonin in the bolus of injectate from the free uninhibited permeation through the clefts into the interstitial fluid space. The permeability-surface area products (PS) for serotonin via the inter-EC clefts were ~0.3 ml·g⁻¹·min⁻¹, low compared with the transporter-mediated maximum PS of 13 ml·g⁻¹·min⁻¹ (with Km = ~0.3 μM and Vmax = ~4 nmol·g⁻¹·min⁻¹). The estimates of serotonin PS values for EC transporters from their multiple data sets were similar and were influenced only modestly by accounting for the cleft permeability in parallel. The cleft PS estimates in these Ringer-perfused lungs are less than half of those for anesthetized dogs (Yipintsoi T. Circ Res 39: 523-531, 1976) with normal hematocrits, but are compatible with passive noncarrier-mediated transport observed later in the same laboratory (Dawson CA, Linehan JH, Rickaby DA, Bronikowski TA. Ann Biomed Eng 15: 217-227, 1987; Peeters FAM, Bronikowski TA, Dawson CA, Linehan JH, Bult H, Herman AG. J Appl Physiol 66: 2328-2337, 1989) The identification and quantitation of the cleft pathway conductance from these studies affirms the importance of the cleft permeation.

Bispecific antibodies for delivery into the brain

The blood-brain barrier (BBB) is a formidable obstacle preventing drug delivery to the brain, particularly for large protein therapeutics. The utilization of endogenous brain endothelial transport pathways, however, represents a promising approach to cross the cellular barrier through receptor-mediated transcytosis. Therapeutics designed to take advantage of this approach require at least two functionalities, one that facilitates transport and the other to provide therapeutic benefit, and bispecific antibodies are ideally suited for this task.

Plasma protein protects endothelial glycocalyx mediated by sphingosine‐1‐phosphate

It has long been known that albumin reduction in endothelial cell (EC) media induces collapse or shedding of the surface glycocalyx layer. This leads to impairment of the endothelial transport barrier and other features of vessel homeostasis. The electrostatic interaction between arginine residues on albumin and negatively charged glycosaminoglycans in the glycocalyx has been hypothesized to stabilize the glycocalyx structure. But sphingosine‐1‐phosphate (S1P), which is bound to albumin, also plays important roles in maintaining vascular barrier integrity and homeostasis. Here, the role of S1P in maintaining glycocalyx stability was investigated in this study. Using confocal microscopy on EC in vitro, we observed that heparan sulfate (HS), chondroitin sulfate (CS) and the ectodomain of syndecan‐1 were shed from the surface after removal of plasma protein, but were retained in the presence of S1P or a general MMP inhibitor. S1P receptor 1 (S1P1) antagonism abolished the protection of the glycocalyx by S1P. GAGs released to culture media and the general MMP activity increased significantly after removal of plasma protein, but could be diminished by S1P. Thus, S1P plays a critical role in protecting the glycocalyx via S1P1, and inhibits the protease activity‐dependent shedding of CS, HS and the syndecan‐1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.Grants: Supported by NIH Grant HL094889 and HL 28607.

Abstract TMP82: Increased Intracerebral Hemorrhage During Acute and Chronic Hypertension in Alzheimer's Transgenic Mice

Hypertension is a major risk factor for intracerebral hemorrhage (ICH), and the accumulation of amyloid-beta (Aβ) in the cerebrovascular system, cerebral amyloid angiopathy (CAA), is also a significant risk factor for intracerebral hemorrhage ICH. Currently, there are no animal studies demonstrating a direct involvement of hypertension in the accumulation of Alzheimer’s disease-like pathology. To address this issue we have developed several mouse models that combine hypertension protocols with amyloid precursor protein (APP) transgenic mice (Tg2576), which accumulate significant CAA in the large cerebral vessels and the meninges by 18 months of age. The goal of this study was to determine the effect of acute and chronic hypertension on ICH in wildtype and a transgenic mouse model overexpressing a mutant human amyloid precursor protein (Tg2576 mice) associated with early onset AD and CAA. Fifteen-month-old Tg2576 mice and non-transgenic (nTg) littermates were treated with an angiotensin II (AngII) infusion (1000 ng/kg/min) and L-NAME (100 mg/kg/day) in drinking water to produce chronic hypertension. One week later, transient acute hypertension was induced by daily AngII injections (0.5 μg/g, s.c., twice daily) to produce ICH. A similar increase in mean blood pressure was observed in Tg2576 and nTg mice when evaluated 2 weeks after initiation of treatment. However Tg2576 mice had a higher incidence of signs of stroke compared with nTg littermates (P > 0.05). These data suggest that the accumulation of Aβ in the brain has an important role in development of ICH. Moreover, there was robust glial activation and increase in CAA in the gray matter of Tg2576 mice showing that hypertension may affect gray as well as white matter in the brain. Further studies may provide insights into the hypertension-induced changes in the cerebral vascular system that initiated the increase in CAA. The accumulation of Aβ in the cerebrovascular system is a significant risk factor for intracerebral hemorrhage (ICH), and has been linked to endothelial transport failure and blockage of perivascular drainage. While management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA.

PENETRATION OF ANTIRETROVIRAL THERAPY INTO THE NERVOUS SYSTEM; COGNITIVE IMPAIRMENT IN HIV AND THE ROLE OF THE CNS AS A SANCTUARY SITE. A PROGRESS REPORT ON THE PARTITION STUDY

BackgroundCombination antiretroviral therapy has revolutionised the treatment of HIV, however as these patients live for longer cognitive impairment is becoming increasingly prevalent—occurring in approximately 50% despite treatment. Indeed HIV is...

Blood-Brain Barrier Alterations in the Cerebral Cortex in Experimental Autoimmune Encephalomyelitis

The pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We investigated cerebral cortex microvessels during immune-mediated demyelination in the MS model chronic murine experimental autoimmune encephalomyelitis (EAE) by immunolocalization of the endothelial cell tight junction (TJ) integral proteins claudin-5 and occludin, a structural protein of caveolae, caveolin-1, and the blood-brain barrier-specific endothelial transporter, Glut 1. In EAE-affected mice, there were areas of extensive subpial demyelination and well-demarcated lesions that extended to deeper cortical layers. Activation of microglia and absence of perivascular inflammatory infiltrates were common in these areas. Microvascular endothelial cells showed increased expression of caveolin-1 and a coincident loss of both claudin-5 and occludin normal junctional staining patterns. At a very early disease stage, claudin-5 molecules tended to cluster and form vacuoles that were also Glut 1 positive; the initially preserved occludin pattern became diffusely cytoplasmic at more advanced stages. Possible internalization of claudin-5 on TJ dismantling was suggested by its coexpression with the autophagosomal marker MAP1LC3A. Loss of TJ integrity was confirmed by fluorescein isothiocyanate-dextran experiments that showed leakage of the tracer into the perivascular neuropil. These observations indicate that, in the cerebral cortex of EAE-affected mice, there is a microvascular disease that differentially targets claudin-5 and occludin during ongoing demyelination despite only minimal inflammation.

Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

Brain microvascular endothelial cells (BEC) constitute the blood-brain barrier (BBB) which forms a dynamic interface between the blood and the central nervous system (CNS). This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local surroundings of BEC within the neurovascular unit are presented and discussed.

BBB-endothelial cell response to cerebral cortex demyelination in a mouse model of chronic EAE

Changes in blood-brain barrier (BBB) function have been implicated in demyelinating diseases. This study aimed to investigate the response of cerebral cortex microvessels to nerve fibre demyelination in a chronic model of murine experimental autoimmune encephalomyelitis (EAE) characterized by areas of extensive subpial demyelination along with well-demarcated lesions extended to deeper cortex layers. These cortices showed activation of microglia and astrogliosis with absence of typical perivascular inflammatory infiltrates. On the basis of these data, we have analyzed the expression of two integral proteins of endothelial tight junctions, claudin-5 and occludin, a structural protein of caveolae, caveolin-1, as well as the BBB-specific endothelial transporter, Glut1 in the cerebral cortex of EAE-affected mice by immunofluorescence confocal microscopy. Microvascular endothelial cells showed an increased expression of caveolin-1 and a coincident decrease of both claudin-5 and occludin junctional staining pattern. At a very early disease stage, claudin-5 molecules formed aggregates and vacuoles that also stained for Glut 1, whereas occludin pattern became diffusely cytoplasmic at advanced stages of the disease. Internalization/dismantling and loss of tight junction proteins and impairment of BBB function were confirmed by coexpression of claudin-5 whit the autophagosomal marker MAP1LC3A and by FITC-dextran experiments that showed leakage of the tracer into the perivascular neuropil. Overall, these observations indicate that in the cerebral cortex of EAE mice, during demyelination and independently from the inflammatory involvement of the cortex, a ‘microvascular disease’ characterized by a differential involvement of claudin-5 and occludin occurs, thereby possibly contributing to demyelinating disease progression.

Abstract P132: Diabetes-Induced Cardiac Dysfunction Is Rescued by Endothelial Cationic Amino Acid Transporter 1 Overexpression in Mice

Nitric oxide (NO) bioavailability is markedly impaired in diabetes, contributing to endothelial and myocardial dysfunction. Our aim was to determine if endothelial-specific overexpression of Cationic Amino Acid Transporter 1 (CAT-1, to enhance L-arginine uptake and NO generation) prevents diabetes-induced endothelial and cardiac dysfunction in diabetic mice. Male CAT-1 transgenic and non-transgenic (Ntg) 6 week old mice were administered streptozotocin (STZ, 55mg/kg ip daily for 5 days). Sham mice received citrate buffer vehicle. Myocardial function was assessed in anaesthetised mice in vivo 12 weeks later, via echocardiography. Vascular reactivity was then assessed in vitro. Compared to their non-diabetic littermates (n=6), Ntg STZ mice (n=6) exhibited reduced early-to-late blood flow velocity ratios (E/A, 1.7±0.2 vs 2.4±0.1, p<0.01) and prolonged deceleration time (DT, 40±1 vs 31±2msec, p<0.002), indicative of diastolic dysfunction. Decreased E-wave velocity (109±4 vs 76±12msec, p<0.01) and increased A-wave velocity (45±3 vs 55±4msec, p<0.05) were also observed. Systolic dysfunction was observed in Ntg STZ mice compared to shams, on reduced fractional shortening (FS, 30±1 vs 39±1%, p<0.002). In CAT-1 mice, diabetes did not impair E/A ratio (2.1±0.2), FS (40±3%) or DT (33±1msec, n=8). Thoracic aortic responses to the endothelium-dependent vasodilator acetylcholine (ACh) were unchanged by diabetes in Ntg and CAT-1 Tg mice compared to sham Ntg mice (pEC50 7.0±0.1 vs 7.2±0.1 and 7.1±0.1). There was a non-significant trend for sham CAT-1 Tg mice to show greater sensitivity to ACh than sham Ntg mice (pEC50 7.5±0.1 vs 7.1±0.1). Responses to the endothelium-independent vasodilator sodium nitroprusside and the vasoconstrictor noradrenaline were unaffected by diabetes and/or genotype. In conclusion, our results suggest that endothelial-specific CAT-1 overexpression protects against cardiac dysfunction via a mechanism independent of the systemic vasculature. The involvement of the coronary vasculature remains to be determined. Endothelial CAT-1 up-regulation as a potential therapeutic target for diabetic cardiomyopathy is an exciting prospect.

The ATP-binding cassette transporters ABCB1 and ABCC1 are not regulated by hypoxia in immortalised human brain microvascular endothelial cells

BackgroundATP-binding cassette transporters at the blood-brain barrier are actively regulated upon ischemic stroke in a way that impedes the access of pharmacological compounds to the brain tissue. The luminal endothelial transporter ABCB1 was recently shown to be increased, whereas the abluminal transporter ABCC1 was decreased on ischemic brain capillaries. In vitro studies using epithelial cells suggested that ABCB1 is regulated during hypoxia in a hypoxia-inducible factor (HIF)-1α-dependent way.MethodsIn order to investigate whether hypoxia might be responsible for the expression changes of ABCB1 and ABCC1 in the ischemic brain, the immortalised human brain microvascular endothelial cell line hCMEC/D3 was exposed to hypoxia (1%) or anoxia (0%). Cell lysates were analysed by Western blot to detect the protein expression of ABCB1, ABCC1, HIF-1α and HIF-2α.ResultsDuring hypoxia, an accumulation of HIF-1α and HIF-2α was noticed in hCMEC/D3 cells that followed different time kinetics. Both HIF-1α and HIF-2α abundance increased within 4 h of hypoxia. HIF-1α levels decreased to below detection levels within 16 h of hypoxia, whereas HIF-2α remained elevated even after 48 h. No changes of ABCB1 and ABCC1 expression were detected, neither on the mRNA nor protein level.ConclusionOur data suggests that other factors than hypoxia may be responsible for the expression changes of ATP-binding cassette transporters in the ischemic brain.

Synthesis of cysteinyl leukotrienes in human endothelial cells: subcellular localization and autocrine signaling through the CysLT2receptor

The purpose of this study was to characterize enzyme, receptor, and signaling involved in the synthesis and the activity of cysteinyl leukotrienes (cys-LTs) in human umbilical vein endothelial cells (HUVECs). We used primary cultures of HUVECs and evaluated the formation of cys-LTs by RP-HPLC. Suicide inactivation and subcellular localization of the enzyme responsible for the conversion of leukotriene (LT) A(4) into LTC(4) were studied by repeated incubations with LTA(4) and immunogold electron microscopy. The CysLT(2) receptor in HUVECs was characterized by equilibrium binding studies, Western blot analysis, and immunohistochemistry. Concentration-response curves in HUVECs and in transfected COS-7 cells were used to characterize a novel specific CysLT(2) receptor antagonist (pA(2) of 8.33 and 6.79 against CysLT(2) and CysLT(1) receptors, respectively). The results obtained provide evidence that the mGST-II synthesizing LTC(4) in HUVECs is pharmacologically distinguishable from the LTC(4)-synthase (IC(50) of MK886 <5 μM for LTC(4)-synthase and >30 μM for mGST-II), is not suicide-inactivated and is strategically located on endothelial transport vesicles. The CysLT(2) receptor is responsible for the increase in intracellular Ca(2+) following exposure of HUVECs to cys-LTs and is coupled to a pertussis toxin-insensitive G(q) protein. The synthesis of cys-LTs from LTA(4) by endothelial cells is directly associated with the activation of the CysLT(2) receptor (EC(50) 0.64 μM) in a typical autocrine fashion.

Caveolae and caveolin-1 in reptilian liver

Caveolae are plasma-membrane invaginations that, by interacting with membrane-associated molecules such as endothelial nitric oxide synthase and tyrosine kinases, precisely regulate cell-signalling pathways responsible for cell structure and cell function. Indeed, there is widespread evidence that caveolae associate, structurally and functionally, with proteins, lipids and solutes to facilitate transcellular transport of these macromolecules. Caveolin-1, one of the family of membrane proteins that form caveolae, is most prominently expressed in endothelial cells of the vascular bed. Therefore, we have applied advanced electron microscopy as well as molecular biology techniques to study the presence of caveolae and caveolin-1 in the liver sinusoidal endothelium of reptiles. Reptiles are known to store excess lipid in the liver as an energy source for hibernation, and so offer a useful animal model in which to assess the structural and functional implications these subcellular compartments might have on liver sinusoidal endothelial transport. This study demonstrates that caveolae are indeed conserved across vertebrate species, whether mammalian or reptilian. It also presents as first novel data on the presence of caveolin-1-associated, tubular structures located within the cytoplasm of the lizard liver sinusoidal endothelium.

Carboxyl Terminus of Apolipoprotein A-I (ApoA-I) Is Necessary for the Transport of Lipid-free ApoA-I but Not Prelipidated ApoA-I Particles through Aortic Endothelial Cells

High density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) must leave the circulation and pass the endothelium to exert their atheroprotective actions in the arterial wall. We previously demonstrated that the transendothelial transport of apoA-I involves ATP-binding cassette transporter (ABC) A1 and re-secretion of lipidated particles. Transendothelial transport of HDL is modulated by ABCG1 and the scavenger receptor BI (SR-BI). We hypothesize that apoA-I transport is started by the ABCA1-mediated generation of a lipidated particle which is then transported by ABCA1-independent pathways. To test this hypothesis we analyzed the endothelial binding and transport properties of initially lipid-free as well as prelipidated apoA-I mutants. Lipid-free apoA-I mutants with a defective carboxyl-terminal domain showed an 80% decreased specific binding and 90% decreased specific transport by aortic endothelial cells. After prior cell-free lipidation of the mutants, the resulting HDL-like particles were transported through endothelial cells by an ABCG1- and SR-BI-dependent process. ApoA-I mutants with deletions of either the amino terminus or both the amino and carboxyl termini showed dramatic increases in nonspecific binding but no specific binding or transport. Prior cell-free lipidation did not rescue these anomalies. Our findings of stringent structure-function relationships underline the specificity of transendothelial apoA-I transport and suggest that lipidation of initially lipid-free apoA-I is necessary but not sufficient for specific transendothelial transport. Our data also support the model of a two-step process for the transendothelial transport of apoA-I in which apoA-I is initially lipidated by ABCA1 and then further processed by ABCA1-independent mechanisms.

Hypoxia-associated protein expression in primary central nervous system diffuse large B-cell lymphoma: does it predict prognosis?

Hypoxia-associated proteins are commonly expressed as a consequence of disturbances in microcirculation. However, the clinical relevance of the proteins has never been studied in primary central nervous system lymphoma (PCNSL). The expression of hypoxia-inducible factor 1α (HIF-1α) and its downstream proteins, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), were evaluated in a central nervous system (CNS) lymphoma xenograft model and in human PCNSL tissue. In the CNS lymphoma xenograft model, the expression of HIF-1α, VEGF, and GLUT-1 co-localized in subsets of lymphoma cells adjacent to necrosis. In tumor specimens from 51 patients with PCNSL, positive HIF-1α staining was found in 26 patients (51.0%), positive VEGF in 30 (58.8%), and positive GLUT-1 in 17 (33.3%), and HIF-1α showed a significant correlation with VEGF (p < 0.05). However, no significant association was seen between hypoxia-associated protein positivity and unfavorable clinical characteristics. Thus, the results failed to show an association with shorter overall survival or time to progression, except that the percentage of lymphoma cells positive for GLUT-1 (>20%) was significantly associated with worse survival. In conclusion, hypoxia-associated proteins were expressed in PCNSL, suggesting a hypoxic microenvironment. However, the prognostic relevance of these proteins for PCNSL was not demonstrated in this study.

Diabetes-Induced Cardiac Dysfunction is Rescued by Endothelial Cationic Amino Acid Transporter 1 (CAT-1) Overexpression in Mice

Diabetes-Induced Cardiac Dysfunction is Rescued by Endothelial Cationic Amino Acid Transporter 1 (CAT-1) Overexpression in Mice

Aging and cerebrovascular dysfunction: contribution of hypertension, cerebral amyloid angiopathy, and immunotherapy

Age-related cerebrovascular dysfunction contributes to ischemic stroke, intracerebral hemorrhages (ICHs), microbleeds, cerebral amyloid angiopathy (CAA), and cognitive decline. Importantly, there is increasing recognition that this dysfunction plays a critical secondary role in many neurodegenerative diseases, including Alzheimer's disease (AD). Atherosclerosis, hypertension, and CAA are the most common causes of blood-brain barrier (BBB) lesions. The accumulation of amyloid beta (Aβ) in the cerebrovascular system is a significant risk factor for ICH and has been linked to endothelial transport failure and blockage of perivascular drainage. Moreover, recent anti-Aβ immunotherapy clinical trials demonstrated efficient clearance of parenchymal amyloid deposits but have been plagued by CAA-associated adverse events. Although management of hypertension and atherosclerosis can reduce the incidence of ICH, there are currently no approved therapies for attenuating CAA. Thus, there is a critical need for new strategies that improve BBB function and limit the development of β-amyloidosis in the cerebral vasculature.

Neuronal Activity Drives Localized Blood-Brain-Barrier Transport of Serum Insulin-like Growth Factor-I into the CNS

Upon entry into the central nervous system (CNS), serum insulin-like growth factor-1 (IGF-I) modulates neuronal growth, survival, and excitability. Yet mechanisms that trigger IGF-I entry across the blood-brain barrier remain unclear. We show that neuronal activity elicited by electrical, sensory, or behavioral stimulation increases IGF-I input in activated regions. Entrance of serum IGF-I is triggered by diffusible messengers (i.e., ATP, arachidonic acid derivatives) released during neurovascular coupling. These messengers stimulate matrix metalloproteinase-9, leading to cleavage of the IGF binding protein-3 (IGFBP-3). Cleavage of IGFBP-3 allows the passage of serum IGF-I into the CNS through aninteraction with the endothelial transporter lipoprotein related receptor 1. Activity-dependent entrance of serum IGF-I into the CNS may help to explain disparate observations such as proneurogenic effects of epilepsy, rehabilitatory effects of neural stimulation, and modulatory effects of blood flow on brain activity.

Preservation of transendothelial glucose transporter 1 and P-glycoprotein transporters in a cortical slice culture model of the blood–brain barrier

A variety of neurological diseases are characterized by disturbances of the blood–brain barrier (BBB) and its transporters. We recently introduced fibroblast growth factor treated cortical organotypic slice cultures of mice as a model for in vitro studies of the blood–brain barrier and have now further characterized the maintenance and function of transport-proteins typically expressed in the endothelium of cerebral blood vessels. The glucose transporter GLUT-1 is present in blood vessels of slice cultures derived from postnatal day 4 to 21 mice after 3 days in vitro. The endothelial multidrug resistance P-glycoprotein (P-gp) which is involved in the control of pharmacological substance transport across the blood–brain barrier is also maintained in blood vessels, most prominently in slice cultures derived from postnatal day 14 and 21 mice. To assess P-gp function, we tested rhodamine 123 transport in presence or absence of the P-gp inhibitor verapamil. Rhodamine 123-fluorescence accumulated rapidly in the vascular lumen both in acute slices and in slices cultured for 3 days in vitro. Our results provide evidence that endothelial transporters and their functional properties can be maintained in organotypic cortical slices cultures, thus making it an attractive model system for the study of the blood–brain barrier.

Shear stress and the endothelial transport barrier

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges.

Abstract 4510: Similarities in trans-Golgi localization of chlorotoxin in tumor and in normal vascular endothelial cells

Abstract Introduction: Chlorotoxin (TM601), a scorpion derived 36-amino acid peptide is a new experimental drug currently in Phase II clinical trials for adult recurrent glioma. Despite its known specificity for tumor cells, its exact mode of action is not known but anti-angiogenic effects have been observed. Here we provide evidence that TM601 localizes near the Golgi complex in human glioma (U373, U87), lung carcinoma (A549), and in normal human vascular endothelial cells (HUVEC) but not in normal human dermal fibroblasts (HDF) and astrocytes (NHA). TM601 enters glioma cells via clathrin-mediated transport and affects level of several Golgi associated peptides. Methods: Cells were treated with or without 10 µM TM601 or fluorescent TM601 for 1 or 24 hours. We evaluated: 1) TM601 cellular localization in relation to the Golgi (antibodies to TM601 and Golgin97 marker) and lysosomes (fluorescent trackers) using immunocytochemistry or direct immunofluorescence visually and quantitatively by confocal microscopy; 2) TM601 cellular entry in U373 and HDF cells using an inhibitor of clathrin-dependent endocytosis (chlorpromazine 10 µg/ml) or inhibitor of macropinocytosis (amiloride 300 µM). Cell lysates were prepared and proteins evaluated by Western blot; 3) TM601 effects on the U373 proteome in the sucrose fractionated Golgi-enriched fraction using liquid chromatography coupled to tandem mass spectrometry. Results: 1) In all tumor cells and HUVEC cells, TM601 was observed as an intense granular staining at the trans-Golgi. In normal fibroblasts and astrocytes it was found at a low level in the cytoplasm. In U373 glioma, but not in the normal HDF and NHA cells, TM601 showed a statistically significant colocalization with the LysoTracker (p=3.9E-05) that stained Golgi associated lysosomal vesicles. 2) A statistically significant effect on TM601 cellular level in U373 was observed after treatment with chlorpromazine and in HDF (but not U373) with amiloride. 3) Preliminary data (n=2) of Golgi-enriched fractions from TM601 treated U373 cells showed that TM601 treatment significantly affected levels of several peptides. Further evaluation by Western blots is planned. Summary: Our data show similarity between glioma, lung carcinoma and the normal vascular endothelial cells in the cellular uptake/transport of TM601 toward Golgi complexes. These cells differ drastically from other normal cells such as HDF and NHA which present low cytoplasmic level of TM601. It is expected that endothelial cell transport of TM601 may occur via receptor mediated clathrin dependent endocytosis pathway as in the glioma cells. In addition, our preliminary data showed TM601 effect on the level of several Golgi associated proteins in glioma cells. A similar study with endothelial cells is planned. The information obtained from these studies will assist in understanding how TM601 affects angiogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4510.