J. Bembry

Endothelial cell cultures derived from isolated cerenrbal microvessels

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to the capillaries of other organs. The intercellular diffusion is restricted by the tight junction separating the continuous layer of endothelial cells, while the vesicular transport is limited in the normal endothelium. Many harmful conditions such as anoxia, ischemia, metabolic and/or degenerative disturbances may alter the endothelium and thus the barrier function. Therefore the elucidation of mechanisms involved in various cerebrovascular disorders such as cerebral ischemia could be facilitated by the study of cellular components of the cerebral vessels in a living state. The present study was designed to establish a cultured cell line originating from cerebral microvessels. In this communication we will describe the establishment of a cell line culture showing the characteristic cytochemical features of cerebral endothelial cells, and which originated from dissociated cells of isolated cerebral microvessels. The brains of 2-day-old Osborne-Mendel rats (36-40) were used under sterile conditions for the separation of the microvessels from the non-vascular tissue. The cerebral hemispheres were freed of leptomeninges, minced with scissors and homogenized in buffered (10 mm Hepes) 0.1 ~ albumin-Ringer solution at pH 7.4. The fraction of microvessels was obtained by centrifugation and discontinuous sucrose gradient (1.0-1.5 m), a simple technique described previouslylk Two samples of microvessels (pellets) were pooled and washed twice with 10 ml of modified Simms Balanced Salt Solution (BSS) containing 100 U penicillin, 100/~g streptomycin and 0.25 #g Fungizone (Gibco, Long Island, N.Y.). They were centrifuged immediately after the first wash and then washed again for 30 min in the refrigerator prior to centrifugation at

SUMOylation participates in induction of ischemic tolerance

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non‐preconditioned neurons. Preconditioned neurons maintained elevated SUMO‐1 conjugation levels (and, to a lesser extent those of SUMO‐2/3) on western blots in contrast to non‐preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO‐1 or SUMO‐2 were over‐expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO‐1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO‐1 (and maybe SUMO‐2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.

Human Brain Capillary Endothelium: 2-Arachidonoglycerol (Endocannabinoid) Interacts With Endothelin-1

In brain, the regulatory mechanism of the endothelial reactivity to nitric oxide and endothelin-1 may involve Ca2+, cytoskeleton, and vasodilator-stimulated phosphoprotein changes mediated by the cGMP/cGMP kinase system. 1 Endothelium of human brain capillaries or microvessels is used to examine the interplay of endothelin-1 with the putative vasorelaxant 2-arachidonoyl glycerol, an endogenous cannabimimetic derivative of arachidonic acid. This study demonstrates that 2-arachidonoyl glycerol counteracts Ca2+ mobilization and cytoskeleton rearrangement induced by endothelin-1. This event is independent of nitric oxide, cyclooxygenase, and lipoxygenase and is mediated in part by cannabimimetic CB1 receptor, G protein, phosphoinositol signal transduction pathway, and Ca2+-activated K+ channels. The induced rearrangements of cellular cytoskeleton (actin or vimentin) are partly prevented by inhibition of protein kinase C or high levels of potassium chloride. The 2-arachidonoyl glycerol–induced phosphorylation of vasodilator-stimulated phosphoprotein is mediated by cAMP. These findings suggest that 2-arachidonoyl glycerol may contribute to the regulation of cerebral capillary and microvascular function.

Effect of arachidonic acid on cultured cerebromicrovascular endothelium: permeability, lipid peroxidation and membrane "fluidity".

SummaryThe relationship of free arachidonic acid (AA) to cellular permeability, lipid peroxidation and physical state “fluidity” of the membrane was investigated in cultured endothelial cells (EC) dissociated from cerebral microvessels of rats. The results demonstrate that AA can induce a reversible alteration of endothelial permeability to trypan blue albumin (TBA). Exposure of EC to AA increases membrane “fluidity” as measured by fluorescence anisotropy using 1,6-diphenyl-1,3,5 hexatriene as a fluorescent probe. The AA modification of EC membrane “fluidity” is not associated with changes in EC permeability. Addition of AA and H2O2 to the incubation medium of EC leads to persistant alteration of EC permeability which can be prevented by catalase treatment. Both AA and H2O2 induce a greater formation of malondialdehyde, the product of lipid peroxidation, than AA alone. These findings strongly suggest that a release of AA either from the capillary or cellular membrane of the brain under a pathological condition may alone or through a peroxidative process alter the function of blood-brain barrier.

Nitric Oxide Modulates Endothelin 1-Induced Ca2+ Mobilization and Cytoskeletal F-Actin Filaments in Human Cerebromicrovascular Endothelial Cells

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1–stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1–mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO–ET-1 interactions may contribute to regulation of microvascular function.

Disruption of downstream MyD88 or TRIF Toll-like receptor Signaling does not protect against cerebral ischemia

Toll-like receptor (TLR) signaling plays an important role in cerebral ischemia, but downstream signaling events, which can be organ-specific, are incompletely understood. We thereby investigated involvement of the MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling pathways in 2 in vitro and 2 in vivo models of cerebral ischemia. For in vitro studies, we used a model of oxygen-glucose deprivation (OGD) followed by flow cytometric analysis to determine:1) viability of PC12 cells following knock-down with MyD88 siRNA compared to negative control siRNA and 2) viability, apoptosis and necrosis of cortical neurons from MyD88 null (-/-) , TRIF mutant, and wild type (WT) mice. In addition, in vivo, 1) We examined CA1 neuronal survival 7 days after global forebrain ischemia and 2) infarct volumes 24h after Middle Cerebral Artery Occlusion (MCAO) in all 3 types of mice. OGD: 1) There were no differences in either percent viability of PC12 cells transfected with MyD88 compared to negative control siRNA or 2) in percent viability, apoptosis and necrosis of cortical neurons from MyD88-/-,TRIF mutant and WT mice. Global ischemia: neuronal survival was similar in all 3 groups of mice. Finally, MCAO: infarct volumes were not statistically different among all 3 groups of mice: MyD88-/-, 23.9±9.9 mm(3), TRIF mutant, 26.7±5.8 mm(3) and WT, 17.9±8.4mm(3). These findings show that disruption of MyD88 or TRIF signaling does not confer protection in brain ischemia and suggests the possibility of additional or alternate downstream adaptors during TLR signaling in cerebral ischemia.

Endothelin-1 and Nitric Oxide Affect Human Cerebromicrovascular Endothelial Responses and Signal Transduction

Endothelium plays a central role in regulating the vascular tone, blood flow and blood brain barrier (BBB) permeability. The experiments presented here examine the mechanisms by which nitric oxide (NO) and endothelin-1 (ET-1) may be involved in these processes. The findings indicate that ET-1-stimulated [Ca2+]i accumulation occurs through activation of ETA receptor. The capacity of NO to affect this response was indicated by results showing: 1) a two-fold increase in ET-1-stimulated [Ca2+]i by L-NAME, the inhibitor of nitric oxide synthase, and 2) a dose-dependent decrease in [Ca2+]i accumulation by pretreatment with Nor-1 (NO donor). Abrogation of this Nor-1 effect by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-pCPT-cGMPS (an inhibitor of protein kinase G) and inhibition of ET-1 stimulated intracellular Ca2+ accumulation by 8-bromo-cGMP (a permeable, analog of cGMP) substantiate the involvement of interplay between ET-1 and NO in [Ca2+]i accumulation in HBMEC. ET-1 treatment also increased thickness of F-actin cytoskeletal filaments in HBMEC. This effect was attenuated by pretreatment with NO; NO also rarefied F-actin filaments in control cultures. The findings support a linkage between NO and ET-1 in regulating microvascular tone, microcirculation and BBB permeability and indicate a role for cGMP/cGMP protein kinase system and cytoskeletal changes in responses of HBMEC.

Interaction between histamine and adenosine in human cerebromicrovascular endothelial cells: Modulation of second messengers

This study demonstrates the presence of histamine H1 and H2 receptors and purinoreceptors A1 and A2 on endothelial cells derived from human brain microvessels (HBEC). Histamine induced formation of both inositol triphosphate (IP3) (EC50=10.2±0.9 μM) and cyclic adenosine monophosphate (cAMP) (EC50=5.2±0.9 μM) in HBEC in a concentration-dependent fashion. IP3 formation was inhibited by H1 receptor antagonists mepyramine maleate and chlorphenyramine, but not by H2 receptor antagonist cimetidine. Production of cAMP was efficiently inhibited by cimetidine. Selective A1 receptor agonists decreased, whereas A2 receptor agonists increased cAMP production in HBEC. When added together with histamine to HBEC cultures, both A1 and A2 receptor agonists diminished histamine-induced IP3 stimulation. This effect was reversed in the presence of specific A1 and A2 receptor antagonists, respectively. Marked augmentation of histamine-induced cAMP production by HBEC was observed in the presence of A2 agonist. This response was dependent on H1 receptors, since it was reduced in the presence of H1-receptor antagonist. It is suggested that interaction between histamine and adenosine modulating induction of second messengers in HBEC may influence endothelium-dependent responses of brain microvascular compartments.

Cerebromicrovascular endothelial permeability

SummaryPermeability of cerebromicrovascular endothelium has been investigated in a new model of cultured cells. The endothelial cells are grown on dextran microcarriers and constitute a barrier for trypan blue (TB) binding to the dextran beads. Changes in the permeability of microcarrier-cultured endothelium have been investigated during the exposure of cells to arachidonic acid or substances involved either in arachidonate metabolism or stimulation of cAMP. The results demonstrate enhanced TB passage through the endothelial barrier during exposure to high concentrations of arachidonic acid and indomethacin, but not to ibuprofen. The effect of indomethacin could be prevented by pretreatment with dexamethasone. Dexamethasone alone did not influence the barrier. Forskolin, a drug which stimulates the catalytic unit of adenylate cyclase, did not affect the endothelial permeability to TB. These findings support the contention that substances derived from a disturbed cellular membrane contribute to the altered blood-brain barrier function found under pathological conditions.

Effect of vasoactive peptides on prostacyclin formation in cerebromicrovascular cellular elements and glia: a comparative study.

The aim of the present study was to determine basal and stimulated release of prostacyclin from the separately cultured endothelial and smooth muscle cells derived from rat brain microvessels and from glial cells. The basal release of PGI(2) (measured as a 6-keto-PGF(1?) formation by radioimmunoassay method) was significantly greater in cultured endothelial cells than in cultured smooth muscle or glial cells (254 +/- 32, 140.7 +/- 17 and 76.8 +/- 5.8 pg/mg protein, respectively). Prostacyclin formation stimulated by angiotensin I, angiotensin II and bradykinin was significantly increased in the smooth muscle cells. A significant enhancement of PGI(2) formation was also observed in the glial cells exposed to angiotensin II or bradykinin. Vasoactive peptides did not affect prostacyclin production in the endothelial cells. Presented results indicate that the smooth muscle cells represent the most sensitive site of prostacyclinpeptide interaction. These data also suggest that the endothelial and the glial cells may protect the cerebromicrovascular smooth muscle by inactivating vasoactive peptides derived from either the blood or the brain.