Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes.

In contrast to the endothelial cells in large vessels where LDL receptors are downregulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced threefold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL receptor expression. We have shown that the lipid requirement of astrocytes increases the expression of endothelial cell LDL receptors. Experiments with dialysis membranes of different pore size showed that this effect is mediated by a soluble factor(s) with relative molecular mass somewhere between 3,500 and 14,000. Substituting astrocytes with smooth muscle cells or brain endothelium with endothelium from the aorta or the adrenal cortex did not enhance the luminal LDL receptor expression on endothelial cells, demonstrating the specificity of the interactions. This factor(s) is exclusively secreted by astrocytes cocultured with brain capillary endothelial cells, but it also upregulates the LDL receptor on other cell types. This study confirms the notion that the final fine tuning of cell differentiation is under local control.

A new function for the LDL receptor: transcytosis of LDL across the blood-brain barrier.

Lipoprotein transport across the blood-brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.

[Quantitative assessment of carotid stenosis: comparison between Doppler ultrasound, spiral computed tomography angiography, magnetic resonance angiography and digital angiography]. / Quantification du degré de sténose carotidienne. Comparaison entre l'échographie Doppler, le scanner hélicoïdal, l'imagerie par résonance magnétique et l'angiographie numérise.

BACKGROUND: The North American Symptomatic Carotid Endarterectomy Trial has confirmed the benefit of carotid endarterectomy in comparison to medical treatment in stroke prevention in symptomatic patients having a carotid stenosis of 70% or more. The Asymptomatic Carotid Atherosclerosis Study has concluded that the benefit of surgical treatment remains significant in asymptomatic patients with 60% (or more) stenosis of the ipsilateral internal carotid artery, when mortality rate remains inferior to 3%. In these two trials, angiography has been used to quantify the stenosis. Though this test is carrying some neurological and renal risks, replacing the angiography stenosis grading for a non or less invasive test, seems to be permissible. METHODS: In our retroprospective study, the assessments of the carotid stenosis by several non-invasive tests findings were compared to the angiography results. Nineteen carotid arteries of fifteen patients, both symptomatic and asymptomatic, having a carotid stenosis at least 60% or more and being detected by the Doppler ultrasound were explored either by magnetic resonance angiography (MRA), spiral computed tomography angiography (SCTA) and angiography. RESULTS: The ultrasonography and angiography findings were well correlated (r=0,88; p<0.002) according to the Spearman test. The assessments of the MRA were better correlated to the angiography than to the SCTA (respectively r=0.91, p<0.0001 and r=0,68, p<0.001). Using both ultrasonography and MRA as a confirmatory test, the rate of injustified carotid endarterectomy was 25%. And this rate rose up to 33% when the ultrasonography was used with the SCTA. It is noteworthy that negative predictive value of ARM was 100%. To reduce the mortality rate, several surgical teams managed the carotid stenosis without angiography. CONCLUSION: MRA could replace angiography, on condition that the rate of unjustified carotid endarterectomy lowers and becomes acceptable. Far reaching complementary studies are necessary to confirm the fiability of those non-invasive tests. In order to raise the benefit to carotid endarterectomy, the research studies should turn to the predictive score determination of a surgical international risk and towards the "High benefit" patients groups after endarterectomy.

[Angiography of coronary arteries by nuclear magnetic resonance]. / Angiographie des artères coronaires par résonance magnétique nucléaire.

OBJECTIVES: Evaluate magnetic resonance imaging of the coronary arteries. METHODS: The study was conducted in 18 healthy volunteers (mean age 26 years, range 22-32). A superconducting magnet operating at 1.5 Tesla was used with an in-phase matrix surface coil. Images were obtained with 2D acquisition with flow compensation on ultra-rapid echo gradient sequences coupled with the electrocardiogram and segmentation of the Fourier plane. A coronary image was obtained during a 15 to 20 sec apnea. Frontal slices were used to identify cardiac structures before selecting axial slices starting from the origin of the aorta to the coronary ostia. Orthogonal, oblique and radial slices were then obtained starting from the axial slices. Slice thickness was 3 to 5 mm separated by 0.5 mm with a 30 degrees angle and a 320 mm field. Echo times were 5.8 and 10.3 msec with 2 excitations. Heart rate varied from 59 to 85 sec giving 15 to 18 ms repetition and 96 to 144 msec time resolution. RESULTS: The proximal, middle and disatal segments of the right coronary artery were seen in 100% of the subjects, the common left coronary artery in 100%, the proximal anterior interventricular artery in 83% and the proximal circumflex artery in 80%. All the coronary arteries were visualized on at least two slices with different incidences. The proximal measurements were: common trunk 3.75 +/- 0.66 mm; anterior interventricular 3.25 +/- 0.43 mm; circumflex 2.62 +/- 0.48; right coronary 3.37 +/- 0.51. CONCLUSION: Magnetic resonance angiography provides good visualization of the coronary arteries.

Evaluation of effect of charge and lipid coating on ability of 60-nm nanoparticles to cross an in vitro model of the blood-brain barrier.

A cell culture model of the blood-brain barrier (BBB) consisting of a coculture of bovine brain capillary endothelial cells and rat astrocytes has been used to examine the ability of 60-nm nanoparticles with different physicochemical characteristics to cross the BBB. Neutral, anionic, and cationic nanoparticles were made from crosslinked malto-dextrins derivatized or not (neutral) with phosphates (anionic), quaternary ammoniums (cationic) ligands. Then, these particles were coated or not with a lipid bilayer made of dipalmitoyl phosphatidyl choline and cholesterol. Lipid coating of ionically charged nanoparticles was able to increase BBB crossing 3- or 4-fold compared with uncoated particles, whereas coating of neutral particles did not significantly alter their permeation characteristics across the endothelial cell monolayer. Lipid-coated nanoparticles were nontoxic toward BBB integrity, and crossed the BBB by transcytosis without any degradation. Furthermore, a 27-fold increase in albumin transport was observed when albumin had previously been loaded in the cationic lipid-coated nanoparticles. The influence of red blood cells was studied; a marked inhibition of the transport was observed, probably due to strong interaction between nanoparticles and red blood cells.

Tumor necrosis factor-alpha increases lactoferrin transcytosis through the blood-brain barrier.

Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood-brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor-alpha (TNF-alpha). As TNF-alpha can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF-alpha, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune-mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up-regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.

Percutaneous recanalization of occlusion of central and proximal veins in chronic hemodialysis. Technical note.

Occlusion of the central and proximal veins in chronic hemodialysis patients results in considerable edema of the arm of the vascular access that is unable to drain normally. This is a formidable problem because it is very often necessary to close the vascular access, which is sometimes the last available one. To avoid resorting to this disastrous solution, recanalization of the occluded vein by percutaneous recanalization followed by endoluminal angioplasty was successfully performed in five patients (4 innominate veins and one axillary vein). Immediate failure occurred in a sixth patient, and delayed failure after two months of patency (innominate vein) in another patient for whom there had been no systematic stent placement. Recanalization was still patent in four other patients at 3, 6, 12 and 26 months. These results are an encouragement to attempt percutaneous recanalization by angioplasty of occluded central veins because, when successful, this technique makes it possible to preserve the vascular access and to avoid onerous surgery. We believe that this technique should therefore become better known.

In vitro model for evaluating drug transport across the blood-brain barrier.

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.