Intracerebral hemorrhage and thrombin-induced alterations in cerebral microvessel matrix.

Four phase III clinical trials of oral direct factor Xa or thrombin inhibitors demonstrated significantly lower intracranial hemorrhage compared to warfarin in patients with nonvalvular-atrial fibrillation. This is counter-intuitive to the principle that inhibiting thrombosis should increase hemorrhagic risk. We tested the novel hypothesis that anti-thrombin activity decreases the risk of intracerebral hemorrhage by directly inhibiting thrombin-mediated degradation of cerebral microvessel basal lamina matrix, responsible for preventing hemorrhage. Collagen IV, laminin, and perlecan each contain one or more copies of the unique α-thrombin cleavage site consensus sequence. In blinded controlled experiments, α-thrombin significantly degraded each matrix protein in vitro and in vivo in a concentration-dependent fashion. In vivo stereotaxic injection of α-thrombin significantly increased permeability, local IgG extravasation, and hemoglobin (Hgb) deposition together with microvessel matrix degradation in a mouse model. In all formats the direct anti-thrombin dabigatran completely inhibited matrix degradation by α-thrombin. Fourteen-day oral exposure to dabigatran etexilate-containing chow completely inhibited matrix degradation, the permeability to large molecules, and cerebral hemorrhage associated with α-thrombin. These experiments demonstrate that thrombin can degrade microvessel matrix, leading to hemorrhage, and that inhibition of microvessel matrix degradation by α-thrombin decreases cerebral hemorrhage. Implications for focal ischemia and other conditions are discussed.

ß1-integrin-matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability.

Acutely following focal cerebral ischemia disruption of the microvessel blood-brain barrier allows transit of plasma proteins into the neuropil as edema formation that coincides with loss of microvessel endothelial ß1-integrins. We extend previous findings to show that interference with endothelial ß1-integrin-matrix adhesion by the monoclonal IgM Ha2/5 increases the permeability of primary cerebral microvascular endothelial cell monolayers through reorganization of claudin-5, occludin, and zonula occludens-1 (ZO-1) from inter-endothelial borders. Interference with ß1-integrin-matrix adhesion initiates F-actin conformational changes that coincide with claudin-5 redistribution. ß1-integrin-matrix interference simultaneously increases phosphorylation of myosin light chain (MLC), while inhibition of MLC kinase (MLCK) and Rho kinase (ROCK) abolishes the Ha2/5-dependent increased endothelial permeability by 6 h after ß1-integrin-matrix interference. These observations are supported by concordant observations in the cortex of a high-quality murine conditional ß1-integrin deletion construct. Together they support the hypothesis that detachment of ß1-integrins from abluminal matrix ligands increases vascular endothelial permeability through reorganization of tight junction (TJ) proteins via altered F-actin conformation, and indicate that the ß1-integrin-MLC signaling pathway is engaged when ß1-integrin detachment occurs. These findings provide a novel approach to the research and treatment of cerebral disorders where the breakdown of the blood-brain barrier accounts for their progression and complication.

Cathepsin L acutely alters microvessel integrity within the neurovascular unit during focal cerebral ischemia.

During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.

Dabigatran abrogates brain endothelial cell permeability in response to thrombin.

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell ß1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

Disruption of dystroglycan-laminin interactions modulates water uptake by astrocytes.

Cerebral edema is a serious complication of ischemic brain injury. Cerebral edema includes accumulation of extracellular fluid due to leakage of the brain's microvessel permeability barrier, and swelling of astrocytes as they absorb water from the extracellular space. Expression of matrix adhesion receptors in brain microvessels decreases in ischemic stroke; this contributes to increased microvessel permeability and detachment of astrocytes from the extracellular matrix (ECM). Since loss of the astrocyte adhesion receptor dystroglycan has been associated with disrupted polarization of ion and water channels, we hypothesized that adhesion of astrocytes to the ECM contributes to regulation of water uptake, and that disruption of matrix adhesion impairs the ability of astrocytes to direct water transport. To test this hypothesis, the capacity of astrocytes to take up water was measured using a fluorescence self-quenching assay under both oxygen/glucose deprivation (OGD) and direct antibody-mediated blockade of α-dystroglycan. Both conditions decreased the rate of water uptake. Moreover, inhibiting proteolytic cleavage of dystroglycan that occurs in OGD abrogated the effect of OGD, but not direct blockade of α-dystroglycan, indicating that interfering with dystroglycan-matrix binding itself affects water uptake. Activation of extracellular signal-related kinase (ERK) by OGD was dependent on α-dystroglycan binding, and inhibition of ERK activity with U0126 abrogated the loss of water uptake following OGD. These studies demonstrate for the first time that water uptake in astrocytes is regulated by dystroglycan-dependent signaling associated with matrix adhesion. This presents a novel potential approach to the treatment of cerebral edema.

Microglial cell activation is a source of metalloproteinase generation during hemorrhagic transformation.

Hemorrhage and edema accompany evolving brain tissue injury after ischemic stroke. In patients, these events have been associated with metalloproteinase (MMP)-9 in plasma. Both the causes and cellular sources of MMP-9 generation in this setting have not been defined. MMP-2 and MMP-9 in nonhuman primate tissue in regions of plasma leakage, and primary murine microglia and astrocytes, were assayed by immunocytochemistry, zymography, and real-time RT-PCR. Ischemia-related hemorrhage was associated with microglial activation in vivo, and with the leakage of plasma fibronectin and vitronectin into the surrounding tissue. In strict serum-depleted primary cultures, by zymography, pro-MMP-9 was generated by primary murine microglia when exposed to vitronectin and fibronectin. Protease secretion was enhanced by experimental ischemia (oxygen-glucose deprivation, OGD). Primary astrocytes, on each matrix, generated only pro-MMP-2, which decreased during OGD. Microglia-astrocyte contact enhanced pro-MMP-9 generation in a cell density-dependent manner under normoxia and OGD. Compatible with observations in a high quality model of focal cerebral ischemia, microglia, but not astrocytes, respond to vitronectin and fibronectin, found when plasma extravasates into the injured region. Astrocytes alone do not generate pro-MMP-9. These events explain the appearance of MMP-9 antigen in association with ischemia-induced cerebral hemorrhage and edema.

Use of gel zymography to examine matrix metalloproteinase (gelatinase) expression in brain tissue or in primary glial cultures.

Glia synthesize, package, and secrete several species of matrix proteases, including the gelatinases (pro-)MMP-2 and (pro-)MMP-9. In appropriate settings (e.g., experimental ischemia), these MMPs can be assayed from cerebral tissues or from astrocytes and microglia in culture by enzymatic substrate-dependent assays and by gelatin-based zymography. We describe the methodologies for the sensitive quantitative development of the inactive and active forms of both MMP-2 and MMP-9 from tissues and cells, by means of lysis of the collagen substrate in collagen-impregnated gel electropheresis by the zymogen and active gelatinases. These methodologies are a refinement of those used commonly, with instructions to increase sensitivity. Serious and often overlooked issues regarding sources of sample contamination and elements confounding the MMP band development and their interpretation are discussed.

Interendothelial claudin-5 expression depends on cerebral endothelial cell-matrix adhesion by ß(1)-integrins.

The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by ß(1)-integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with ß(1)-integrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 kDa dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of ß(1)-integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell-matrix interactions via ß(1)-integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability.

The vitamin C:vitamin K3 system - enhancers and inhibitors of the anticancer effect.

The oxidizing anticancer system of vitamin C and vitamin K3 (VC:VK3, producing hydrogen peroxide via superoxide) was combined individually with melatonin, curcumin, quercetin, or cholecalciferol (VD3) to determine interactions. Substrates were LNCaP and PC-3 prostate cancer cell lines. Three of the tested antioxidants displayed differences in cell line cytotoxicity. Melatonin combined with VC:VK3 quenched the oxidizing effect, while VC:VK3 applied 24 hours after melatonin showed no quenching. With increasing curcumin concentrations, an apparent combined effect of VC:VK3 and curcumin occurred in LNCaP cells, but not PC-3 cells. Quercetin alone was cytotoxic on both cell lines, but demonstrated an additional 50-percent cytotoxicity on PC-3 cells when combined with VC:VK3. VD3 was effective against both cell lines, with more effect on PC-3. This effect was negated on LNCaP cells with the addition of VC:VK3. In conclusion, a natural antioxidant can enhance or decrease the cytotoxicity of an oxidizing anticancer system in vitro, but generalizations about antioxidants cannot be made.

Cytotoxic effect of oyster mushroom Pleurotus ostreatus on human androgen-independent prostate cancer PC-3 cells.

Twenty species of edible mushrooms and three purified mushroom polysaccharides were screened for their antitumor potential on human androgen-independent cancer PC-3 cells. A water-soluble extract (POE) prepared from the fresh oyster mushroom Pleurotus ostreatus produced the most significant cytotoxicity on PC-3 cells among the mushroom species tested. At the same time, POE induced a rapid apoptosis on PC-3 cells detected with annexin V-fluorescein isothiocyanate flow cytometry when the cells were exposed to POE (150 microg/mL) for 2 hours. Induced apoptosis was also confirmed by DNA fragment terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling staining while POE (200 microg/mL) was added to PC-3 cells for 6 hours. Both cytotoxicity and induced apoptosis mediated by POE in PC-3 cells are dose-dependent. Interestingly, PC-3 cells appeared to be more sensitive to POE in anchorage-independent growth condition. Tumor colony-forming efficiency was dramatically reduced to 4.5% or 0.5% in POE (60 or 120 microg/mL)-supplemented soft agar medium compared with that of POE-free medium (defined as 100%). Temperature in POE processing plays a decisive role for the cytotoxic activity. Bioactivity of POE was eliminated by exposure to high temperature (80 degrees C) for 2 hours; however, it remained stable at a series temperatures of below 40 degrees C. The active fraction POE-F2 was analyzed and identified by size exclusion of high performance liquid chromatography and the CellTiter 96 AQueous Cell Proliferation Assay (Promega, Madison, WI). Since POE-F2 is also sensitive to heat and has strong 280 nm absorption, the results imply that active compounds recovered from P. ostreatus are water-soluble proteins or polypeptides.

In vitro effects on proliferation, apoptosis and colony inhibition in ER-dependent and ER-independent human breast cancer cells by selected mushroom species.

Breast cancer is the most commonly diagnosed cancer among women in Western countries. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. We have screened 38 species of edible mushroom on human estrogen-receptor positive (MCF-7) and estrogen-receptor negative (MDA-MB-231, BT-20) breast cancer cells to select potential agents with broad-spectrum antitumor activity against breast cancer cells. Water-based extracts of three mushroom species, Coprinellus sp., Coprinus comatus, Flammulina velutipes (CME, CCE and FVE, respectively), were identified as novel anti-breast cancer agents. The anti-tumor activities include: 1) marked growth inhibition of both ER+ and ER- breast cancer cells; 2) induction of rapid apoptosis on both ER+ and ER- cells; 3) significant inhibition of MCF-7 tumor colony formation in vitro. The antiproliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ER- breast cancer cells was very high, while the IC50 of mushroom extract CME was found to be as low as 40 microg/ml on MDA-MB-231 cells and the IC50 of mushroom extract FVE was only 30 microg/ml on BT-20 cells. More interestingly, mushroom extracts CME and FVE induced an exceptionally rapid apoptosis on MCF-7 and MDA-MB-231 detected by Annexin V-FITC within 2 h of treatment and DNA fragment end-labeling assay (TUNEL) in 5 h of treatment. Anchorage-independent growth assays indicated that the MCF-7 tumor colony formation rate was reduced by 60% in CCE- and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that mushroom species Coprinus comatus, Coprinellus sp. and Flammulina velutipes contain potent antitumor compounds for breast cancer. Our finding is important due to the lack of chemotherapeutic and chemopreventive agents for ER- human breast cancer.

Evidence for mitochondrial gene control of mating types in Phytophthora.

When protoplasts carrying metalaxyl-resistant (Mr) nuclei from the A1 isolate of Phytophthora parasitica were fused with protoplasts carrying chloroneb-resistant (Cnr) nuclei from the A2 isolate of the same species, fusion products carrying Mr nuclei were either the A2 or A1A2 type, while those carrying Cnr nuclei were the A1, A2, or A1A2 type. Fusion products carrying Mr and Cnr nuclei also behaved as the A1, A2, or A1A2 type. The result refutes the hypothesis that mating types in Phytophthora are controlled by nuclear genes. When nuclei from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species and vice versa, all of the nuclear hybrids expressed the mating type characteristics of the protoplast parent. The same was true when the nuclei from the A1 isolate of P. parasitica were fused with the protoplasts from the A0 isolate of Phytophthora capsici and vice versa. These results confirm the observation that mating type genes are not located in the nuclei and suggest the presence of mating type genes in the cytoplasms of the recipient protoplasts. When mitochondria from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species, the mating type of three out of five regenerated protoplasts was changed to the A1 type. The result demonstrated the decisive effect of mitochondrial donor sexuality on mating type characteristics of mitochondrial hybrids and suggested the presence of mating type genes in mitochondria. All of the mitochondrial hybrids resulting from the transfer of mitochondria from the A0 isolate of P. capsici into protoplasts from the A1 isolate of P. parasitica were all of the A0 type. The result supports the hypothesis of the presence of mating type genes in mitochondria in Phytophthora.

Selective induction of apoptosis in murine skin carcinoma cells (CH72) by an ethanol extract of Lentinula edodes.

The effects of ethanol extracts from four species of mushroom fruiting bodies, mushroom spores and mushroom cultured broth, were assessed for modulation of cell proliferation and apoptosis in murine skin carcinoma cells (CH72) and non-tumorigenic epidermal cells (C50). While extracts from mycelia of Grifola frondosa, Ganoderma lucidum, Hericium erinaceus, or from spores of G. lucidum exerted little, if any, effect on proliferation, the ethanol-soluble extract of Lentinula edodes (L. edodes) significantly decreased cell proliferation of CH72 cells. There were no changes in the proliferative response of the non-tumorigenic keratinocyte cell line, C50, to any of the mushroom extracts tested. To analyze cell proliferation and apoptosis, fluorescent DNA-microscopy with ethidium bromide and acridine orange staining of cells revealed L. edodes reduced cell proliferation and induced apoptosis in time- and dose-dependent manners in carcinoma cells but had no effect in non-tumorigenic cells (C50). Cell cycle analysis demonstrated that L. edodes extract induced a transient G(1) arrest, with no changes observed in the non-tumorigenic cells (C50).

Wheat Genotype-Specific Induction of Soil Microbial Communities Suppressive to Disease Incited by Rhizoctonia solani Anastomosis Group (AG)-5 and AG-8.

ABSTRACT The induction of disease-suppressive soils in response to specific cropping sequences has been demonstrated for numerous plant-pathogen systems. The role of host genotype in elicitation of the essential transformations in soil microbial community structure that lead to disease suppression has not been fully recognized. Apple orchard soils were planted with three successive 28-day cycles of specific wheat cultivars in the greenhouse prior to infestation with Rhizoctonia solani anastomosis group (AG)-5 or AG-8. Suppressiveness to Rhizoctonia root rot of apple caused by the introduced isolate of R. solani AG-5 was induced in a wheat cultivar-specific manner. Pasteurization of soils after wheat cultivation and prior to pathogen introduction eliminated the disease suppressive potential of the soil. Wheat cultivars that induced disease suppression enhanced populations of specific fluorescent pseudomonad genotypes with antagonistic activity toward R. solani AG-5 and AG-8, but cultivars that did not elicit a disease suppressive soil did not modify the antagonistic capacity of this bacterial community. When soils were infested prior to the initial wheat planting, all cultivars were uniformly susceptible to R. solani AG-8. However, when pathogen inoculum was added after three growth-cycles, wheat root infection during the fourth growth-cycle varied in a cultivar specific manner. The same wheat cultivar-specific response in terms of transformation of the fluorescent pseudomonad community and subsequent suppression of Rhizoctonia root rot of apple was observed in three different orchard soils. These results demonstrate the importance of host genotype in modification of indigenous saprophytic microbial communities and suggest an important role for host genotype in the success of biological control.

Cultural management of microbial community structure to enhance growth of apple in replant soils.

ABSTRACT Apple replant disease typically is managed through pre-plant application of broad-spectrum soil fumigants including methyl bromide. The impending loss or restricted use of soil fumigants and the needs of an expanding organic tree fruit industry necessitate the development of alternative control measures. The microbial community resident in a wheat field soil was shown to suppress components of the microbial complex that incites apple replant disease. Pseudomonas putida was the primary fluorescent pseudomonad recovered from suppressive soil, whereas Pseudomonas fluorescens bv. III was dominant in a conducive soil; the latter developed within 3 years of orchard establishment at the same site. In greenhouse studies, cultivation of wheat in replant orchard soils prior to planting apple suppressed disease development. Disease suppression was induced in a wheat cultivar-specific manner. Wheat cultivars that enhanced apple seedling growth altered the dominant fluorescent pseudo-monad from Pseudomonas fluorescens bv. III to Pseudomonas putida. The microbial community resident in replant orchard soils after growing wheat also was suppressive to an introduced isolate of Rhizoctonia solani anastomosis group 5, which causes root rot of apple. Incorporation of high glucosinolate containing rapeseed ('Dwarf Essex') meal also enhanced growth of apple in replant soils through suppression of Rhizoc-tonia spp., Cylindrocarpon spp., and Pratylenchus penetrans. Integration of these methods will require knowledge of the impact of the biofumigant component on the wheat-induced disease-suppressive microbial community. Implementation of these control strategies for management of apple replant disease awaits confirmation from ongoing field validation trials.