Beyond Lipid-Lowering: Effects of Statins on Cardiovascular and Cerebrovascular Diseases and Cancer.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are administered as first-line therapy for hypercholesterolemia, both as primary and secondary prevention. Besides the lipid-lowering effect, statins have been suggested to inhibit the development of cardiovascular disease through anti-inflammatory, antioxidant, vascular endothelial function-improving, plaque-stabilizing, and platelet aggregation-inhibiting effects. The preventive effect of statins on atherothrombotic stroke has been well established, but statins can influence other cerebrovascular diseases. This suggests that statins have many neuroprotective effects in addition to lowering cholesterol. Furthermore, research suggests that statins cause pro-apoptotic, growth-inhibitory, and pro-differentiation effects in various malignancies. Preclinical and clinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. The pleiotropic effects of statins on cardiovascular and cerebrovascular diseases have been well established; however, the effects of statins on cancer patients have not been fully elucidated and are still controversial. This review discusses the recent evidence on the effects of statins on cardiovascular and cerebrovascular diseases and cancer. Additionally, this study describes the pharmacological action of statins, focusing on the aspect of 'beyond lipid-lowering'.

Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood-Brain Barrier In Vitro Model.

Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood-brain barrier (BBB) implication is supposed to play a major role in brain metastasis. However, the role of pericytes remains to be elucidated in the brain metastasis. This pilot study described the expression profile of interactions between pericytes, endothelial cells, and cancer cells. We applied an in vitro BBB model with rat primary cultured BBB-related cells (endothelial cells and pericytes), and performed the gene expression analyses of pericytes under the lung cancer cells coculture conditions. Pericytes demonstrated inhibition of the cancer cell proliferation significantly (p < 0.05). RNA was extracted from the pericytes, complementary DNA library was prepared, and RNA-seq was performed. The sequence read data were analyzed on the Management and Analysis System for Enormous Reads and Tag Count Comparison-Graphical User Interface platforms. No statistically or biologically significant differentially expressed genes (DEGs) were detected in the explanatory analyses. Lot-specific DEG detection demonstrated significant decreases in the expression of two genes (Wwtr1 and Acin1), and enrichment analyses using Metascape software revealed the inhibition of apoptotic processes in fibroblasts. Our results suggest that the expression profiles of brain pericytes are partially implicated in the prevention of lung cancer metastasis to the brain. Pericytes exerted an anti-metastatic effect in the BBB model, and their neurohumoral factors remain to be elucidated.

Pericytes Suppress Brain Metastasis from Lung Cancer In Vitro.

Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

BACKGROUND: Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. METHOD: To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. RESULTS: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. CONCLUSION: These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

Pentosan polysulfate treatment ameliorates motor function with increased serum soluble vascular cell adhesion molecule-1 in HTLV-1-associated neurologic disease.

The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-α, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP.

Hepatocyte growth factor enhances the barrier function in primary cultures of rat brain microvascular endothelial cells.

The effects of hepatocyte growth factor (HGF) on barrier functions were investigated by a blood-brain barrier (BBB) in vitro model comprising a primary culture of rat brain capillary endothelial cells (RBEC). In order to examine the response of the peripheral endothelial cells to HGF, human umbilical vascular endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) were also treated with HGF. HGF decreased the permeability of RBEC to sodium fluorescein and Evans blue albumin, and dose-dependently increased transendothelial electrical resistance (TEER) in RBEC. HGF altered the immunochemical staining pattern of F-actin bands and made ZO-1 staining more distinct on the linear cell borders in RBEC. In contrast, HGF increased sodium fluorescein and Evans blue albumin permeability in HMVEC and HUVEC, and decreased TEER in HMVEC. In HMVEC, HGF reduced cortical actin bands and increased stress fiber density, and increased the zipper-like appearance of ZO-1 staining. Western blot analysis showed that HGF significantly increased the amount of ZO-1 and VE-cadherin. HGF seems to act on the BBB to strengthen BBB integrity. These findings indicated that cytoskeletal rearrangement and cell-cell adhesion, such as through VE-cadherin and ZO-1, are candidate mechanisms for the influence of HGF on the BBB. The possibility that HGF has therapeutic significance in protecting the BBB from damage needs to be considered.

Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 µm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

Cilostazol strengthens barrier integrity in brain endothelial cells.

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 µM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 µM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered.

Pravastatin reduces steroid-induced osteonecrosis of the femoral head in SHRSP rats.

BACKGROUND AND PURPOSE: Although the definite cause of steroid-induced osteonecrosis of the femoral head (ONFH) is unknown, peripheral circulatory failure, lipid metabolism disturbance, and increased oxidative stress are considered to be possible causes. We investigated whether pravastatin as a statin treatment reduces (1) the incidence of ONFH, (2) the adipocyte area, and (3) bone marrow changes in the femoral head. METHODS: We divided up 81 thirteen-week-old spontaneously hypertensive stroke-prone (SHRSP)/Izm male rats into 4 groups: a control group (group C), a group given pravastatin (group P), a group given steroid (group S), and a group given both pravastatin and steroid (Group PS). The steroid was administered at 15 weeks of age. Pravastatin, as a statin, was administered in the drinking water for 4 weeks. The rats were killed when 17 weeks old. Osteonecrosis was diagnosed based on histopathological examination. Oxidative stress was assessed from immunostaining. RESULTS: The incidence of histological osteonecrosis was lower in the groups given pravastatin. The percentage of adipocyte area in the bone marrow was lower in the PS group than in the S group. Immunohistochemical staining for oxidative stress showed that staining was less in the PS group than in the S group. Pravastatin had no effect on the blood-derived biochemical findings on lipid metabolism. However, it reduced the incidence of steroid-induced ONFH in these SHRSP rats. We presume that this occurred by reducing oxidative stress and by reducing the percentage of adipocyte area in the femoral heads. INTERPRETATION: Our data suggest that pravastatin may be effective in reducing steroid-induced ONFH.

Patented in vitro blood-brain barrier models in CNS drug discovery.

The blood-brain barrier (BBB) is a regulatory interface between the circulation and the central nervous system (CNS). Therapy of neurological diseases is limited due to restricted penetration of pharmacons across the BBB. Models for screening the brain penetration of drug candidates are needed early in drug discovery. Culture-based models are useful tools for both basic research on BBB, and testing the permeability of new therapeutical molecules. This review focuses on patented in vitro BBB models and their potential application in CNS drug discovery. Cell culture models using primary and immortalized brain endothelial cells of non-human and human origin, in co-culture or mono-culture setting, in static or dynamic conditions are discussed, as well as methods to induce BBB properties in such in vitro models. The aim of these models is to reproduce as many aspects as possible of the in vivo BBB. All models should show some elements of general endothelial and specific BBB properties, like physiologically realistic cell architecture, restrictive paracellular pathway, and functional expression of transport mechanisms. Though no "ideal in vitro BBB model" has been constructed yet, the currently available models provide valuable information on BBB permeability and are useful tools in CNS drug discovery.

Pentosan reduces osteonecrosis of femoral head in SHRSP.

Increased oxidative stress is considered one of the main causes of steroid-induced osteonecrosis of the femoral head (ONFH). The aim of this study was to evaluate the effects of a steroid hormone and pentosan polysulfate sodium (pentosan), a heparin analog, in stroke-prone spontaneously hypertensive rats (SHRSP) as a model of ONFH. One hundred twenty-three 13-week-old male SHRSP/Izm rats were divided into four groups: a control group (group C), pentosan-administered group (group P), steroid-administered group (group S), and group administered pentosan plus steroid (group PS). Methylprednisolone acetate, as the steroid hormone, at a dose of 4 mg (15 mg/kg) was administered at 15 weeks of age. Pentosan at a dose of 3 mg/day/kg was continuously administered intraperitoneally from 13 weeks of age for 4 weeks. Rats were sacrificed at 17 weeks of age, and heart blood and both femora were collected. Triglyceride levels were significantly lower in group PS than in group S, indicating that pentosan improves lipid metabolism. The incidence of histologic ONFH was significantly lower in group P, at 14.8% (10/71 femoral heads), than in group C, at 30.4% (17/56 femoral heads), and significantly lower in group PS, at 40.8% (29/71 femoral heads), than in group S, at 91.3% (42/46 femoral heads), indicating that pentosan markedly inhibits ONFH. Immunohistochemical staining for oxidative stress showed that the stainability was significantly lower in group PS than in group S. Pentosan seems to reduce the incidence of ONFH in SHRSP by improving lipid metabolism and decreasing oxidative stress.

Protection of the blood-brain barrier by pentosan against amyloid-ß-induced toxicity.

Endothelial cells of brain capillaries forming the blood-brain barrier play an important role in the pathogenesis and therapy of Alzheimer's disease. Amyloid-ß (Aß) peptides are key pathological elements in the development of the disease. A blood-brain barrier model, based on primary rat brain endothelial cells was used in which the barrier properties were induced by glial cells. The effects of amyloid peptides have been tested on cell viability and barrier functions. Aß showed toxic effects on primary rat brain endothelial cells measured by MTT dye conversion and the lactate dehydrogenase release. Morphologically cytoplasmic vacuolization, disruption of the structure of cytoplasmic organelles and tight junctions could be observed in brain endothelial cells. Treatment with Aß1-42 decreased the electrical resistance, and increased the permeability of brain endothelial cell monolayers for both fluorescein and albumin. Serum amyloid P component which stabilizes Aß fibrils in cortical amyloid plaques and cerebrovascular amyloid deposits significantly potentiated the barrier-weakening effect of Aß1-42. Sulfated polysaccharide pentosan could decrease the toxic effects of Aß peptides in brain endothelial cells. It could also significantly protect the barrier integrity of monolayers from damaging actions of peptides. Pentosan modified the size, and significantly decreased the number of amyloid aggregates demonstrated by atomic force microscopy. The present data further support the toxic effects of amyloid peptides on brain endothelial cells, and can contribute to the development of molecules protecting the blood-brain barrier in Alzheimer's disease.

Sodium pentosan polysulfate resulted in cartilage improvement in knee osteoarthritis--an open clinical trial.

BACKGROUND: Pentosan polysulfate sodium (pentosan) is a semi-synthetic drug manufactured from beech-wood hemicellulose by sulfate esterification of the xylopyranose hydroxyl groups. From in vitro and animal model studies, pentosan has been proposed as a disease modifying osteoarthritis drug (DMOAD). The objective of this study was to assess the efficacy, safety, and patient satisfaction in patients with mild radiographic knee osteoarthritis (OA) findings and OA-associated symptoms and signs. METHODS: Twenty patients were assessed clinically at Nagasaki University Hospital. The radiographic indications of OA were grade 1 to 3 using the Kellgren-Lawrence Grading System (K/L grade). Pentosan used in this study was manufactured and supplied in sterile injectable vials (100 mg/ml) by bene GmbH, Munich, Germany. The study was a single-center, open-label trial. Treatment consisted of 6 weekly subcutaneous injections (sc) of pentosan (2 mg/kg). Patients were clinically assessed at entry and 1 to 8, 11, 15, 24 & 52 weeks post treatment. The results were analyzed using one way ANOVA and Dunnett's method. RESULTS: Hydrarthroses were reduced quickly in all cases. The clinical assessments, i.e., knee flexion, pain while walking, pain after climbing up and down stairs, etc, were improved significantly and these clinical improvements continued for almost one year. The dose used in this study affected the blood coagulation test, but was within safe levels. Slightly abnormal findings were noted in serum triglycerides. CONCLUSIONS: Pentosan treatment in twenty patients with mild knee OA seemed to provide improvements in clinical assessments and C2C level of cartilage metabolism.

Development of a novel cell-based assay to evaluate the malignant potential of cancer in vitro.

BACKGROUND: Metastasis is a leading cause of cancer death. To evaluate the complex metastatic process in vitro, an attempt was made to develop a cell-based assay (Can kit) that could evaluate the late stages of metastasis. MATERIALS AND METHODS: Two membrane chambers were set up of which the upper membrane chamber with 8 mum pores was covered with normal cell layers. Cancer cells were introduced to the upper chamber and after passing through the normal cell layers dropped through onto the lower chamber membrane where cancer colonies formed and were evaluated based on the reduction of transepithelial electrical resistance (TEER) with a Madin-Darby canine kidney (MDCK) cell monolayer. RESULTS: When two pairs of cancer cell lines, with different metastatic potentials in vivo, were applied to the Can kit assay, differences in potentials between the two cell lines in vitro were demonstrated. The reduction in the TEER was correlated with the total area of the cancer colonies and the production of matrix metalloproteinases (MMPs). CONCLUSION: A cell-based assay able to evaluate the malignant potential of cancer in vitro was developed and is considered to be useful for research and the clinical examination of cancer metastatic potential.

DJ-1-binding compounds prevent oxidative stress-induced cell death and movement defect in Parkinson's disease model rats.

Parkinson's disease (PD) is caused by neuronal cell death. Although a precursor of dopamine and inhibitors of dopamine degradation have been used for PD therapy, cell death progresses during treatment. DJ-1, a causative gene product of a familial form of PD, PARK7, plays roles in transcriptional regulation and anti-oxidative stress, and loss of its function is thought to result in the onset of PD. Superfluous oxidation of cysteine at amino acid 106 (C106) of DJ-1 renders DJ-1 inactive, and such oxidized DJ-1 has been observed in patients with the sporadic form of PD. In this study, we isolated compounds that bind to the region at C106 by a virtual screening. These compounds prevented oxidative stress-induced death of SH-SY5Y cells, embryonic stem cell-derived dopaminergic cells and primary neuronal cells of the ventral mesencephalon, but not that of DJ-1-knockdown cells of SH-SY5Y and NIH3T3 cells, indicating that the effect of the compounds is specific to DJ-1. These compounds inhibited production of reactive oxygen species and restored activities of mitochondrial complex I and tyrosine hydroxylase that had been compromised by oxidative stress. These compounds prevented dopaminergic cell death in the substantia nigra and restored movement abnormality in 6-hydroxyldopamine-injected PD model rats. One mechanism of action of these compounds is prevention of superfluous oxidation of DJ-1, and the compounds passed through the blood-brain barrier in vitro. Taken together, the results indicate that these compounds should become fundamental drugs for PD therapy.

New method for separation of subpopulations from a heterogeneous colon cancer cell line.

BACKGROUND: To understand the heterogeneity of human colon cancers, a new method to separate cancer subpopulations was developed. MATERIALS AND METHODS: Cells from a human colon cancer cell line, DLD-1, were seeded on an 8 microm pore membrane. After six hours, the cells which remained beneath the membrane as well as the cells which dropped onto the 24-well plate were collected. To clarify the differences between the two subpopulations, transepithelial electrical resistance (TEER) and immunocytochemistry were evaluated. RESULTS: Two subpopulations, clones D and A, were separated from DLD-1 with the newly developed method. Both subpopulations showed quite different TEER values and different arrangements of cell-cell contact. In addition, the distinct subcellular localizations of claudin family proteins and zonula occludens-1 (ZO-1) were identified. CONCLUSION: A new separation method to isolate colon cancer subpopulations was established in which the intercellular junctions differed. This method can be considered as a helpful tool in the investigation of colon cancer heterogeneity.

Possible protection of sinusoidal endothelial cells by endothelin B receptor during hepatic warm ischemia-reperfusion.

PURPOSE: Endothelins (ETs) are important regulators of the hepatic microcirculation. We investigated the pure biological roles of endothelin B receptors (ETB-Rs) on hepatic warm ischemia-reperfusion (I/R) injury using ETB-R deficient spotting lethal (sl) rats. METHODS: Homozygous (sl/sl) and wild-type (+/+) rats were exposed to 60 min of 92% partial hepatic ischemia and then were killed at 2, 6, and 24 h, and 3 and 7 days after reperfusion. We measured the serum alanine aminotransferase (ALT) levels to assess hepatocyte injury, and the serum hyaluronic acid (HA) levels and factor VIII-related antigen (FVIIIRAg) staining to assess sinusoidal endothelial cell (SEC) injury. We also measured the concentrations of ET-1 and nitrite (NO2-) and nitrate (NO3-) of liver tissue samples. RESULTS: Although no significant difference was observed in the ALT levels, the HA levels were significantly elevated at an early stage after reperfusion in the sl/sl rats. Regarding FVIIIRAg staining, positive SECs were enhanced in the sl/sl rats. The ET-1 levels were also significantly elevated at an early stage after reperfusion in the sl/sl rats. Regarding the NO2- and NO3- levels, no significant difference was observed. CONCLUSION: Endothelin B receptor was shown to have a protective effect on SECs through the inhibition of ET-1 during hepatic warm I/R injury.

Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

Neuroprotective effect of pentosan polysulphate on ischemia-related neuronal death of the hippocampus.

Pentosan polysulphate (PPS) negatively charged sulphated glycosaminoglycan was studied in ischemia-related hippocampal neuronal death and compared with a low molecular weight of heparin, named dalteparin in rats. Transient global ischemia was produced by four vessel-occlusion, the occlusion of the bilateral common carotid arteries following the electrocautherization of the vertebral arteries. 3mg/kg of PPS or 300IU/kg of dalteparin was administered i.v. immediately after 7min-occlusion/reperfusion. Seven days after the operation, the animals were perfused with 4% paraformaldehyde, and paraffinized coronal brain sections measuring 6microm in thickness were stained with hematoxylin and eosin. Neuronal damage was then estimated as a ratio of the number of degenerated neurons to that of both the surviving and degenerated neurons in three distinct area of the CA1 subfield. The ratio of neuronal death increased with the length of the occlusion-time, at 5, 7 and 10min. Both PPS and dalteparin significantly inhibited the neuronal damage induced by 7min-occlusion. These results demonstrated that both PPS and dalteparin could thus protect brain neurons against ischemia/reperfusion-induced damage thus suggesting that they may be potentially useful therapeutic agents for acute ischemic stroke.

Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production.

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.