Tsuyoshi Nishioku

Brain pericytes among cells constituting the blood-brain barrier are highly sensitive to tumor necrosis factor-α, releasing matrix metalloproteinase-9 and migrating in vitro

BackgroundIncreased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs), pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains.MethodsThe culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs) and phosphoinositide-3-kinase (PI3K)/Akt in the mediation of tumor necrosis factor (TNF)-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay.ResultsZymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL)-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody.ConclusionThese findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte-derived MMP-9 initiated cellular migration of pericytes, which might be involved in pericyte loss in the damaged BBB.

AT1 Receptor Blockade Regulates the Local Angiotensin II System in Cerebral Microvessels From Spontaneously Hypertensive Rats

Background and Purpose— Blockade of angiotensin II AT1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. Methods— Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. Results— We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT1 receptors were localized to the endothelium. There was no evidence of AT2 receptor localization in the microvascular endothelium. In SHR, (pro)renin receptor mRNA and AT1 receptor mRNA and protein expression were higher, whereas Aogen, ACE mRNA and AT2 receptor mRNA and protein expression were lower than in WKY rats. Candesartan treatment increased Aogen, ACE and AT2 receptor in SHR, and increased ACE and decreased Aogen in WKY rats, without affecting the (pro)renin and AT1 receptors. Conclusions— Increased (pro)renin and AT1 receptor expression in SHR substantiates the importance of the local RAS overdrive in the cerebrovascular pathophysiology in hypertension. AT1 receptor blockade and increased AT2 receptor stimulation after administration of candesartan may contribute to the protection against brain ischemia and inflammation.

Cathepsin E Deficiency Induces a Novel Form of Lysosomal Storage Disorder Showing the Accumulation of Lysosomal Membrane Sialoglycoproteins and the Elevation of Lysosomal pH in Macrophages

Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE-/-) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE-/- macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H+-ATPase activity in both cell types, the elevated lysosomal pH in CatE-/- macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder.

Involvement of caspase 3-like protease in methylmercury-induced apoptosis of primary cultured rat cerebral microglia.

Methylmercury (MeHg) has been implicated to induce massive neurodegeneration by disruption of neuron-glia interactions besides a direct potent neurotoxicity. In the present study, we examined potential cytotoxic effects of MeHg on primary cultured rat microglia. Following treatment with a relatively low concentration (0.5 microM) of MeHg, microglia had induced cell death accompanied by DNA fragmentation and an activation of caspase-3-like protease. MeHg-induced microglial death was significantly suppressed by the caspase-3-like protease inhibitor benzyloxycarbonyl-Try-Val-Ala-Asp-fluoromethyl-ketone indicating the occurrence of caspase-3-like protease-executed apoptosis. The aspartic protease inhibitor pepstatin A had a partial but significant inhibitory effect on MeHg-induced microglial apoptosis. These results indicate that a relatively low concentration of MeHg predominantly induces caspase-3-like protease-executed apoptosis of microglia, while the endosomal/lysosomal system is also partially involved in the cell death pathway.

Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen

ANG II AT(1) receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT(1) receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 microg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-alpha, IL-1beta, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-alpha, IL-1beta, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT(1) receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT(1) receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-alpha and IL-6 mRNA and protein; expression of IL-1beta and IkappaB-alpha mRNA; COX-2 mRNA and protein expression and PGE(2) concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT(1) receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

Angiotensin II AT1 Receptor Blockade Decreases Lipopolysaccharide-Induced Inflammation in the Rat Adrenal Gland

Peripheral administration of bacterial endotoxin [lipopolysaccharide (LPS)] to rodents produces an innate immune response and hypothalamic-pituitary-adrenal axis stimulation. Renin-angiotensin-aldosterone system inhibition by angiotensin II AT1 receptor blockade has antiinflammatory effects in the vasculature. We studied whether angiotensin II receptor blockers (ARBs) prevent the LPS response. We focused on the adrenal gland, one organ responsive to LPS and expressing a local renin-angiotensin-aldosterone system. LPS (50 microg/kg, ip) produced a generalized inflammatory response with increased release of TNF-alpha and IL-6 to the circulation, enhanced adrenal aldosterone synthesis and release, and enhanced adrenal cyclooxygenase-2, IL-6, and TNF-alpha gene expression. ACTH and corticosterone release were also increased by LPS. Pretreatment with the ARB candesartan (1 mg/kg.d, sc for 3 d before the LPS administration) decreased LPS-induced cytokine release to the circulation, adrenal aldosterone synthesis and release, and cyclooxygenase-2 and IL-6 gene expression. Candesartan did not prevent the LPS-induced ACTH and corticosterone release. Our results suggest that AT1 receptors are essential for the development of the full innate immune and stress responses to bacterial endotoxin. The ARB decreased the general peripheral inflammatory response to LPS, partially decreased the inflammatory response in the adrenal gland, prevented the release of the pro-inflammatory hormone aldosterone, and protected the antiinflammatory effects of glucocorticoid release. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that the ARBs may have therapeutic effects on inflammatory conditions.

Expression of long-term potentiation of the striatum in methamphetamine- sensitized rats

We examined the change of corticostriatal glutamatergic neuronal transmission in striatal slices of methamphetamine (MAP)-sensitized rats in vitro. Tetanic stimulation induced long-term depression (LTD) of the field potential in the striatum of saline-treated rats. However, it induced long-term potentiation (LTP) in the striatum of MAP-sensitized rats. This LTP was significantly suppressed by a N-methyl-D-aspartate (NMDA) receptor antagonist, aminophosphonovaleric acid (APV). These results suggest that LTP is expressed in the striatum of MAP-sensitized rats, and that NMDA receptors are indispensable for the LTP formation.

A Predominant Apoptotic Death Pathway of Neuronal PC12 Cells Induced by Activated Microglia Is Displaced by A Non-apoptotic Death Pathway Following Blockage of Caspase-3-dependent Cascade*

Activated microglia have been implicated in the regulation of neuronal cell death. However, the biochemical mechanism for neuronal death triggered by activated microglia is still unclear. When treated with activated microglia, neuronal PC12 cells undergo apoptosis accompanied by caspase-3-like protease activation and DNA fragmentation. Apoptotic bodies formed were subsequently phagocytosed by neighboring activated microglia. Pretreatment of the cells with the caspase-3-like protease inhibitorN-acetyl-Asp-Glu-Val-Asp-aldehyde did not reverse this cell death. Although Bcl-2 overexpression in the cells caused the inhibition of caspase-3-like protease activity and DNA fragmentation and the effective interference of apoptosis induced by deprivation of trophic factors, it could not suppress the activated microglia-induced neuronal death. At the electron microscopic level, degenerating cells with high levels of Bcl-2 were characterized by slightly condensed chromatins forming irregular-shaped masses, severely disintegrated perikarya, and marked vacuolation. Various protease inhibitors tested did not inhibit this cell death, whereas the radical oxygen species scavengerN-acetyl-l-cysteine significantly suppressed this death. Altogether, our study provides an alternative death pathway for the activated microglia-induced neuronal death by blockage of the caspase-3 protease cascade.

Differential Regulation of the Nature and Functions of Dendritic Cells and Macrophages by Cathepsin E

The aspartic proteinase cathepsin E is localized mainly in the endosomal structures of APCs and has been implicated in a variety of immune responses, however, the precise roles of cathepsin E in these cells remain speculative. In this study, we report the effect of disrupting the gene encoding cathepsin E on the nature and functions of dendritic cells (DCs) and macrophages derived from mouse bone marrow precursors, as well as mouse peritoneal macrophages. Whereas cathepsin E deficiency induced the accumulation of the lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and elevated the lysosomal pH in macrophages, it did not have these effects on DCs. Although cathepsin E deficiency also caused a marked decrease in degradation of phagocytosed OVA and chemotactic responses to MCP-1 and fMLP by macrophages, these abilities were little affected in DCs by the absence of cathepsin E. Interestingly, cathepsin E deficiency markedly decreased the ability of macrophages to present intact OVA, as well as an OVA-derived antigenic peptide (266–281), to cognate T cells, while that of DCs was inversely enhanced by the absence of this protein. This paradox was resolved, in part, by the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which amplify the response of T cells, in cathepsin E-deficient DCs compared with the wild-type cells. These results indicate that cathepsin E differentially regulates the nature and function of DCs and macrophages.

Expression and transport of Angiotensin II AT1 receptors in spinal cord, dorsal root ganglia and sciatic nerve of the rat

To clarify the role of Angiotensin II in the regulation of peripheral sensory and motor systems, we initiated a study of the expression, localization and transport of Angiotensin II receptor types in the rat sciatic nerve pathway, including L(4)-L(5) spinal cord segments, the corresponding dorsal root ganglia (DRGs) and the sciatic nerve. We used quantitative autoradiography for AT(1) and AT(2) receptors, and in situ hybridization to detect AT(1A), AT(1B) and AT(2) mRNAs. We found substantial expression and discrete localization of Angiotensin II AT(1) receptors, with much higher numbers in the grey than in the white matter. A very high AT(1) receptor expression was detected in the superficial dorsal horns and in neuronal clusters of the DRGs. Expression of AT(1A) mRNA was significantly higher than that of AT(1B). AT(1) receptor binding and AT(1A) and AT(1B) mRNAs were especially prominent in ventral horn motor neurons, and in the DRG neuronal cells. Unilateral dorsal rhizotomy significantly reduced AT(1) receptor binding in the ipsilateral side of the superficial dorsal horn, indicating that a substantial number of dorsal horn AT(1) receptors have their origin in the DRGs. After ligation of the sciatic nerve, there was a high accumulation of AT(1) receptors proximal to the ligature, a demonstration of anterograde receptor transport. We found inconsistent levels of AT(2) receptor binding and mRNA. Our results suggest multiple roles of Angiotensin II AT(1) receptors in the regulation of sensory and motor functions.