The Blood-Brain Barrier

Microglial Cells and Astrocytes are capable of processing and presenting Antigens for efficient activation of T-Cells.

However, the Antigen-Presenting function and role of CerebroVascular Endothelial Cells (CVEs) in Central Nervous System Inflammatory Responses remain controversial.

We compared the expression of necessary Accessory Molecules and the functional Antigen-Presenting capacity of cloned SJL/J CVEs and primary Astrocytes in response to the Pro-Inflammatory Cytokines Interferon-gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-).

Astrocytes and CVEs up-regulated Major Histocompatibility Complex (MHC) Class II, and primarily B7-1 as opposed to B7-2, in response to IFN-γ.

TNF- inhibited the IFN-γ-induced up-regulation of MHC Class II on CVEs correlating to a decrease in the mRNA for the Class II TransActivator (CIITA).

Whereas CIITA expression in Astrocytes was unaffected. Unlike Astrocytes, CVEs did not elicit significant MHC Class II-restricted T-Cell responses.

Furthermore, we have found that CVE MonoLayers are altered following T-Cell contact, implicating CVE/T-Cell contact in the breakdown of the Blood-Brain Barrier during Neuro-Inflammatory Responses.

Endothelial Cells of Tumor vessels have well-documented alterations, but it is less clear whether Pericytes on these vessels are abnormal or even absent.

Here we report that alpha-smooth muscle actin (alpha-SMA) and Desmin-ImmunoReactive Pericytes were present on > 97% of blood vessels viewed by confocal microscopy in 100-microm-thick sections of three different spontaneous or implanted Tumors in mice.

However, the cells had multiple abnormalities. Unlike Pericytes on Capillaries in normal Pancreatic Islets, which had Desmin but not alpha-SMA ImmunoReactivity.

Pericytes on Capillary-size vessels in Insulinomas in RIP-Tag2 transgenic mice expressed both Desmin and alpha-SMA.

Furthermore, Pericytes in RIP-Tag2 Tumors, as well as those in MCa-IV Breast Carcinomas and Lewis Lung Carcinomas, had an abnormally loose association with Endothelial Cells and extended CytoPlasmic Processes deep into the Tumor tissue.

alpha-SMA-positive Pericytes also covered 73% of Endothelial Sprouts in RIP-Tag2 Tumors and 92% of Sprouts in the other Tumors.

Indeed, Pericyte sleeves were significantly longer than the CD31-ImmunoReactive Endothelial Cell Sprouts themselves in all three types of Tumors.

All three Tumors also contained alpha-SMA-positive MyoFibroblasts that resembled Pericytes but were not associated with blood vessels.

We conclude that Pericytes are present on most Tumor Vessels but have multiple abnormalities, including altered expression of Marker Proteins.

In contrast to some previous studies, the almost ubiquitous presence of Pericytes on Tumor Vessels found in the present study may be attributed to our use of both Desmin and alpha-SMA as markers and 100-microm-thick tissue sections.

The association of Pericytes with Endothelia Sprouts raises the possibility of an involvement in Sprout Growth or retraction in Tumors.

1. ATP-sensitive Potassium Channels (K(ATP)) are present in Vascular smooth muscle cells and play important roles in the Vascular responses to a variety of pharmacological and endogenous Vasodilators.

2. The K(ATP) channels are composed of four inwardly rectifying K⁺ Channel subunits and four Regulatory Sulphonylurea Receptors.

The K(ATP) channels are inhibited by IntraCellular ATP and by Sulphonylurea agents.

3. Pharmacological Vasodilators such as Cromakalim, Pinacidil and Diazoxide directly activate K(ATP) Channels.

The associated membrane HyperPolarization closes Voltage-dependent Ca2⁺ Channels, which leads to a reduction in IntraCellular Ca2⁺ and Vasodilation.

4. Endogenous Vasodilators such as Calcitonin gene-related peptide, Vasoactive Intestinal Polypeptide, Prostacylin and Adenosine activate K(ATP) by stimulating the formation of cAMP and increasing the activity of protein kinase A.

Part of the mechanism of contraction of endogenous Vasoconstrictors is due to inhibition of K(ATP) Channels.

5. The K(ATP) Channels appear to be Tonically active in some Vascular beds and contribute to the physiological regulation of Vascular tone and blood flow.

These channels also are activated under PathoPhysiological conditions, such as Hypoxia, Ischemia, Acidosis and Septic Shock, and, in these disease states, may play an important role in the regulation of tissue perfusion.

Purpose
Pericytes are positioned on the Abluminal wall of Capillaries and are thought to play a role in regulating Retinal blood flow.

Although EndoThelin-1 (ET-1) is a putative EndoThelium-Pericyte signal, the mechanisms by which this molecule regulates Pericyte function remain unclear.

Because Ion Channels play a vital role in the response of Pericytes to ExtraCellular signals, this study was undertaken to assess the effects of ET-1 on Ionic Currents.

Methods
The perforated-patch configuration of the patch-clamp technique was used to monitor whole-cell currents of Pericytes located on MicroVessels freshly isolated from the rat Retina.

To assay cell-to-cell coupling within Retinal MicroVessels, a gap junction - permeant tracer was loaded through patch pipettes into Pericytes and the spreading of the tracer detected by ImmunoHistoChemistry.

Results
ET-1 acting through ET(A) receptors altered Pericyte currents and caused Depolarization of the Membrane Potential.

The effects on Pericyte currents were dynamic over time. Initially, the NonSpecific Cation (NSC) and Calcium-activated Chloride (Cl(Ca)) currents were activated and the Adenosine TriPhosphate (ATP)-sensitive Potassium (K(ATP)) current inhibited.

Subsequently, by a mechanism sensitive to a Protein Kinase C (PKC) inhibitor, the NSC, Cl(Ca), and Voltage-dependent Potassium currents diminished as Gap Junction Pathways closed within the MicroVessels.

Conclusions
ET-1 regulates Pericyte conductances by multiple mechanisms. One process involves a PKC-dependent closure of Gap Junction Pathways resulting in loss of electrotonic input from neighboring cells.

Thus, ET-1 not only affects individual MicroVascular Cells, but also regulates the effective size of the multicellular functional units that may serve to control Capillary blood flow.

This regulation of InterCellular communication within Pericyte-containing MicroVessels may be an important, previously unrecognized, action of ET-1.

Aims/Hypothesis
Thickening of the basement membrane and selective loss of Pericytes are early events in Diabetic Retinopathy.

We aimed at checking whether Pericyte interaction with ExtraCellular Matrix produced by Endothelial Cells is influenced by the Hexose concentrations in which Endothelial Cells are cultured.

Methods
Conditioned ExtraCellular Matrixes were obtained by growing human umbilical vein Endothelial Cells in media containing 28 mmol/l Hexoses (D-Glucose, D-Galactose, L-Glucose).

Which undergo different IntraCellular processing, before and after adding the inhibitors of protein Glycation Thiamine or Aminoguanidine.

Having removed the Endothelium, bovine Retinal Pericytes were grown on such Matrixes and, in separate experiments, on Laminin, Fibronectin or type IV Collagen.

Pericyte adhesion was determined by cell counts 18 h after seeding.

Results
Reduced adhesion was observed on Matrixes produced in high D-Glucose, high D-Galactose and high L-Glucose.

Both Thiamine and Aminoguanidine restored impaired Pericyte adhesion when added to high D-Glucose and high D-Galactose, but not L-Glucose.

Laminin, Fibronectin and type IV Collagen did not consistently modify Pericyte adhesion.

Conclusions/Interpretations
Pericyte adhesion is impaired on ExtraCellular Matrix produced by Endothelium in high Hexose concentrations.

This could result from excess protein Glycation, corrected by Aminoguanidine and Thiamine, rather than altered GlycoProtein composition.

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human Brains.

Microglia, as the resident Phagocyte, is a strong candidate for the clearance of Apoptotic Lymphocytes.

Apoptosis was induced in cultured autologous Thymocytes and in Myelin Basic Protein (MBP)-specific, Encephalitogenic T-Cells from Lewis rats by the addition of 0.1 microg/ml MethylPrednisolone.

The amount of Phagocytosis of Apoptotic Cells was assessed using an in vitro Phagocytosis assay. Supernatants were collected to measure Microglial Cytokine secretion.

The state of Immune activation in Microglia was investigated by a T-Cell proliferation assay and by flow cytometric analysis of Microglial surface expression of Immune molecules.

Microglia ingested specifically Apoptotic Cells (Apoptotic Thymocytes as well as MBP-specific T-Cells) in contrast to NonApoptotic control cells (p < 0.0001).

Subsequent secretion of the ProInflammatory Cytokines TNF- and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-ß was not affected.

Furthermore, ingestion of Apoptotic Cells led to increased Microglial MHC Class II expression without concomitant increase in MHC Class I, CoStimulatory Molecules, and ICAM expression.

The Ag-specific activation of MBP-specific T-Cells in cocultures with Microglia that had ingested Apoptotic Cells was significantly less than that of identical T-Cells that interacted with NonPhagocytosing Microglia.

Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of Apoptotic Cells.

The uptake of Apoptotic Cells by Microglia is Tolerogenic and results in a reduced ProInflammatory Cytokine production and a reduced activation of Encephalitogenic T-Cells.

This might help to restrict an AutoImmune inflammation and minimize damage in the inflamed Brain.

Most in vitro studies of Capillary permeability focus on Endothelial Cell (MVEC) MonoLayers and ignore the second cell that forms the Capillary Wall: the MicroVascular Pericyte (PC).

We describe a model to study the permeability characteristics of MVEC, PC, and MVEC:PC cocultures.

Methods
Semipermeable culture inserts were coated with Collagen and then plated with early passage bovine pulmonary MVEC.

On Day 3, bovine pulmonary PC were added at concentrations to approximate MVEC:PC ratios of 1:1, 5:1, and 10:1.

Electrical resistance was measured on subsequent days and fluorescently labeled (FITC) Albumin was used in a permeability assay to calculate an Albumin clearance for each culture.

Results
The results for electrical resistance measurements and Albumin assays showed a similar pattern.

Resistance for Endothelial Cell MonoLayers was significantly higher and Albumin permeability was significantly lower than that of controls.

Addition of Pericytes at a 10:1 and 5:1 ratios increased the Permeability Barrier compared to Endothelial Cells alone, although these cultures were not significantly different from one another.

Cocultures at a 1:1 ratio had the best barrier, significantly better than all other cultures.

Conclusions
Endothelial Cell MonoLayers are an inadequate model of the MicroCirculation.

As PC form a key component of the Capillary Wall in vivo and as addition of PC to MVEC monolayers in vitro, optimally at a 1:1 ratio, increase their barrier effect to large and small molecules.

We believe it is necessary to include both cells in future in vitro studies.

Copyright 2001 Academic Press.

Evidence from a variety of sources suggests that Pericytes have contractile properties and may therefore function in the regulation of Capillary blood flow.

However, it has been suggested that contractility is not a ubiquitous function of Pericytes, and that Pericytes surrounding true Capillaries apparently lack the machinery for contraction.

The present study used a variety of techniques to investigate the expression of contractile proteins in the Pericytes of the CNS.

The results of ImmunoCytoChemistry on CryoSections of Brain and Retina, Retinal whole-mounts and ImmunoBlotting of isolated Brain Capillaries indicate strong expression of the smooth muscle isoform of Actin (alpha-SM Actin) in a significant number of Mid-Capillary Pericytes.

ImmunoGold labelling at the ultrastructural level showed that alpha-SM Actin expression in Capillaries was exclusive to Pericytes, and Endothelial Cells were negative. Compared to alpha-SM Actin, non-muscle Myosin was present in lower concentrations.

By contrast, smooth muscle Myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein Vimentin, but lacked Desmin which was consistently found in Vascular smooth muscle cells.

These results add support for a contractile role in Pericytes of the CNS MicroVasculature, similar to that of Vascular smooth muscle cells.

Any perturbation of the Blood-Brain Barrier, whether from changes in cell physiology or from direct injury, may result in MicroVascular Dysfunction and disease.

We examined, at the ultrastructural level, MicroVascular Pericyte responses in a well-defined model of Traumatic Brain Injury in the rat.

In areas close to the site of impact Cortical Pericytes underwent a number of changes within the first hour. Approximately 40% of Pericytes migrated from their MicroVascular location.

Migration occurred concomitant with a thinning of the Abluminal surface of the Basal Lamina and an accumulation of the Receptor for the Urokinase PlasMinogen Activator on the leading surface of the migrating cell.

Migrated Pericytes appeared viable and remained in a PeriVascular location in the adjacent Neuropil.

Nonmigrating Pericytes in the same section displayed Cytoplasmic alterations and Nuclear Chromatin changes consistent with a rapid degenerative process.

Copyright 2000 Academic Press.

Three major functional roles have been ascribed to Pericytes associated with Central Nervous System MicroVasculature: Contractility, regulation of Endothelial Cell activity, and Macrophage activity.

A host of different cell factors and signalling agents appear to be involved with these cellular functions, some effecting the Pericyte and others produced by this cell.

These include NeuroModulators, Vasoactive Peptides, Metabolic Factors, Growth Factors and Cytokines.

The specific compounds and their actions are collectively viewed in an effort to provide an overall picture of the regulation of Pericyte functional activity.

This small Vascular Cell is emerging as a significant entity in several Physiological processes through the functions of above.

These processes include control of blood flow, regulation of Vascular development and Immune Responses. Defining the regulatory agents and their mechanisms is key to understanding the role that Pericytes play in these processes.

Because these cells have begun to receive increasing attention in NeuroBiological studies, an overview of signalling properties should be timely and beneficial.

The cellular constituents of the Blood-Brain Barrier (BBB) must make finely tuned, regulatory responses to maintain MicroVascular Homeostasis.

The mechanisms by which this task is accomplished are largely unknown.

However, it is thought they involve a series of cross-talk mechanisms among Endothelial Cells (EC), Pericytes (PC), and Astrocytes.

During inflammation, the BBB is exposed to a number of biological response modifiers including Cytokines released by infiltrating Leukocytes.

The response to Inflammatory Cytokine may alter the normal regulatory function of EC and PC.

These changes may account for some of the pathological findings in Central Nervous System (CNS) Inflammatory Disease.

Previous studies have shown that PC and EC may have Immune potential. We have investigated the response of the PC to a variety of Inflammatory Cytokines.

Primary rat PC constitutively express low levels of InterCellular Adhesion Molecule-1 (ICAM-1) and Major Histocompatibility Complex (MHC) Class I molecule.

Which can be upregulated in response to the Cytokine Interferon-gamma (IFN-γ). IFN-γ also induced the expression of MHC Class II molecule.

After induction of MHC Class II molecule, CNS PC acquired the capacity to present Antigen to primed syngeneic rat T-Lymphocytes.

Antigen presentation by PC was comparable to that seen with classic Antigen-Presenting Cells.

A small number of primary PC constitutively express low levels of Vascular Cell Adhesion Molecule-1 (VCAM-1), which was increased on exposure to Tumor Necrosis Factor-alpha (TNF-).

Results suggest that CNS PC respond to Inflammatory Cytokines, are involved in T-Lymphocyte activation, and express cell surface Adhesion Molecules (VCAM-1, ICAM-1).

That may provide costimulatory activity. It is likely that CNS PC are important in NeuroImmune Networks at the BBB.

CNS Pericytes are an integral part of the Blood-Brain Barrier (BBB), but their function is not well understood. We questioned whether primary cultured CNS Pericytes have Immune potential.

Primary cultured Pericytes exhibit Phagocytic activity when exposed to FluoroChrome-conjugated polystyrene beads and AntiBody-coated Zymosan. Maximum Phagocytic activity occurred by 3 hr.

Pericytes were found to express the Macrophage markers ED-2 and the Integrin subunit CD11b (alpha M) in culture as well as on isolated MicroVessels.

Pericytes did not express the Macrophage marker ED-1. We confirm the heterogeneity of cultured CNS Pericytes with regard to expression of alpha-smooth muscle Actin.

In conclusion, Pericytes express Macrophage surface Antigens and have the ability to perform at least some Immune function.

CNS Pericytes may therefore have a role in NeuroImmune Networks at the BBB.