Homocysteine causes cerebrovascular leakage in mice

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 microM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9-/- + Hcy); and 4) MMP-9-/- with topical application of histamine (10(-4) M) (MMP-9-/- + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9-/- + Hcy mice. Increased cerebrovascular leakage in the MMP-9-/- + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 microM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 microM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.     

Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Deltapsi(m)) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide (JC-1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl(2)/Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)-mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death.     

Innate immunity in multiple sclerosis: myeloid dendritic cells in secondary progressive multiple sclerosis are activated and drive a proinflammatory immune response

Multiple sclerosis (MS) is postulated to be a T cell-mediated autoimmune disease characterized clinically by a relapsing-remitting (RR) stage followed by a secondary progressive (SP) phase. The progressive phase is felt to be secondary to neuronal degenerative changes triggered by inflammation. The status of the innate immune system and its relationship to the stages of MS is not well understood. Dendritic cells (DCs) are professional APCs that are central cells of the innate immune system and have the unique capacity to induce primary immune responses. We investigated circulating myeloid DCs isolated directly from the blood to determine whether there were abnormalities in myeloid DCs in MS and whether they were related to disease stage. We found that SP-MS subjects had an increased percentage of DCs expressing CD80, a decreased percentage expressing PD-L1, and an increased percentage producing IL-12 and TNF-alpha compared with RR-MS or controls. A higher percentage of DCs from both RR and SP-MS patients expressed CD40 compared with controls. We then investigated the polarization effect of DCs from MS patients on naive T cells taken from cord blood using a MLR assay. Whereas DCs from RR-MS induced higher levels of Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4, IL-13) cytokines compared with controls, DCs from SP-MS only induced a polarized Th1 response. These results demonstrate abnormalities of DCs in MS and may explain the immunologic basis for the different stages and clinical patterns of MS.     

GABA receptors ameliorate Hcy-mediated integrin shedding and constrictive collagen remodeling in microvascular endothelial cells

Mammalian endothelial cells are deficient in cystathionine β synthetase (CBS) activity, which is responsible for homocysteine (Hcy) clearance. This deficiency makes the endothelium theprime target for Hcy toxicity. Hcy induces integrin shedding in microvascular endothelial cells (MVEC) by increasing matrix metalloproteinase (MMP). Hcy competes with inhibitory neurotransmitter γ aminobutyric acid (GABA)-A receptor. We hypothesized that Hcy transduces MVEC remodeling by increasing metalloproteinase activity and shedding β-1 integrin by inactivating the GABA-A/B receptors, thus behaving as an excitatory neurotransmitter. MVEC were isolated from mouse brain. The presence of GABA-A receptor was determined by immunolabeling. It was induced by muscimol, an agonist of GABA-A receptors as measured by Western blot analysis. Hcy induced MMP-2 activity in a dose- and time-dependent maner, measured by zymography. GABA-A/B receptors ameliorated the Hcy-mediated MMP-2 activation. Hcy selectively increased the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 but decreased the levels of TIMP-4. Treatment with muscimol decreased the levels of TIMP-1 and TIMP-3 and increased the levels of TIMP-4 to control. Hcy caused a robust increae in the levels of a disintegrin and metalloproteinase (ADAM)-12. In the medium of MVEC reated with Hcy, the levels of β-1 integrin were significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels significantly increased. Treatment with muscimol or baclofen (GABA-B receptor agonist) ameliorated the levels of β-1 integrin in the medium. These results suggested that Hcy induced DAM-12. Significantly, Hcy facilitated the β-1 integrin shedding. Treatment of MVEC with muscimole or baclofen during Hcy administration ameliorated the expression of metalloproteinase, integrin-shedding, and constrictive collagen remodeling, suggesting a role of Hcy in GABA receptor-mediated cerebrovascular remodeling.     

Comparison of Disease Activity in SPMS and PPMS in the Context of Multicenter Clinical Trials

Background

Retrospective single center natural history studies have shown that times to reach disability milestones and ages at which they are reached are similar in primary (PPMS) and secondary (SPMS) progressive multiple sclerosis suggesting that they may be phenotypic variations of the same disease.

Objective

Here we compared longitudinal disease activity in SPMS and PPMS in the context of international multicenter clinical trials.

Methods

We analyzed all objective outcome measures that were systematically collected over 2 years for all subjects randomized to placebo arms in one SPMS and one PPMS clinical trial over the last decade. Conventional and exploratory definitions of clinical disease activity were used. Disease activity was analyzed in 3 different categories intermittent activity, progression, and improvement. Conventional MRI measures and one patient reported outcome measure of quality of life were included when available for comparison. Heat maps were drawn for all results followed by hierarchical clustering.

Results

There were 101 outcome variables from 206 SPMS subjects and 79 outcome variables from 135 PPMS subjects. The comparison revealed that SPMS and PPMS subjects exhibited similar disease activity over 2 years in all but two of the variables in common worsening in the EDSS sensory system was more common in PPMS while worsening on the 9 hole PEG was more common in SPMS. Intermittent activity was the most common pattern of disease activity in SPMS and PPMS. Clinical worsening and improvement occurred at similar frequency in both.

Conclusion

Longitudinal disease activity was nearly identical in SPMS and PPMS subjects in the context of the two multicenter international clinical trials we examined.     

CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea

Arrays of clustered, regularly interspaced short palindromic repeats (CRISPRs) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats that are separated by similarly sized non-repetitive spacers. CRISPR arrays, together with a group of associated proteins, confer resistance to phages, possibly by an RNA-interference-like mechanism. This Progress discusses the structure and function of this newly recognized antiviral mechanism.