Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE.</p> <p>Methods</p> <p>SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits.</p> <p>Results</p> <p>Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen). Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R) infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC.</p> <p>Conclusion</p> <p>These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss.</p

Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

BACKGROUND: Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs(Shh+)) that are able to regulate in vitro angiogenesis.METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated the ability of MPs(Shh+) to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs(Shh+), MPs(Shh+) plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs(Shh+)-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs(Shh+)-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs(Shh+) increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs(Shh+) enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs(Shh+) on eNOS pathway. Quantitative RT-PCR revealed that MPs(Shh+) treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. CONCLUSIONS/SIGNIFICANCE: These findings suggest that MPs(Shh+) may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis

Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

<p>Abstract</p> <p>Background</p> <p>VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein.</p> <p>Methods</p> <p>Tissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's <it>F</it>-test for multiple comparisons.</p> <p>Results</p> <p>Of the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM.</p> <p>Conclusion</p> <p>These data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling.</p

Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions

3′,4′-Dihydroxyflavonol Antioxidant Attenuates Diastolic Dysfunction and Cardiac Remodeling in Streptozotocin-Induced Diabetic m(Ren2)27 Rats

Diabetic cardiomyopathy (DCM) is an increasingly recognized cause of chronic heart failure amongst diabetic patients. Both increased reactive oxygen species (ROS) generation and impaired ROS scavenging have been implicated in the pathogenesis of hyperglycemia-induced left ventricular dysfunction, cardiac fibrosis, apoptosis and hypertrophy. We hypothesized that 3',4'-dihydroxyflavonol (DiOHF), a small highly lipid soluble synthetic flavonol, may prevent DCM by scavenging ROS, thus preventing ROS-induced cardiac damage.Six week old homozygous Ren-2 rats were randomized to receive either streptozotocin or citrate buffer, then further randomized to receive either DiOHF (1 mg/kg/day) by oral gavage or vehicle for six weeks. Cardiac function was assessed via echocardiography and left ventricular cardiac catheterization before the animals were sacrificed and hearts removed for histological and molecular analyses. Diabetic Ren-2 rats showed evidence of diastolic dysfunction with prolonged deceleration time, reduced E/A ratio, and increased slope of end-diastolic pressure volume relationship (EDPVR) in association with marked interstitial fibrosis and oxidative stress (all P<0.05 vs control Ren-2). Treatment with DiOHF prevented the development of diastolic dysfunction and was associated with reduced oxidative stress and interstitial fibrosis (all P<0.05 vs untreated diabetic Ren-2 rats). In contrast, few changes were seen in non-diabetic treated animals compared to untreated counterparts.Inhibition of ROS production and action by DiOHF improved diastolic function and reduced myocyte hypertrophy as well as collagen deposition. These findings suggest the potential clinical utility of antioxidative compounds such as flavonols in the prevention of diabetes-associated cardiac dysfunction

The Regulation of MS-KIF18A Expression and Cross Talk with Estrogen Receptor

This study provides a novel view on the interactions between the MS-KIF18A, a kinesin protein, and estrogen receptor alpha (ERα) which were studied in vivo and in vitro. Additionally, the regulation of MS-KIF18A expression by estrogen was investigated at the gene and protein levels. An association between recombinant proteins; ERα and MS-KIF18A was demonstrated in vitro in a pull down assay. Such interactions were proven also for endogenous proteins in MBA-15 cells were detected prominently in the cytoplasm and are up-regulated by estrogen. Additionally, an association between these proteins and the transcription factor NF-κB was identified. MS-KIF18A mRNA expression was measured in vivo in relation to age and estrogen level in mice and rats models. A decrease in MS-KIF18A mRNA level was measured in old and in OVX-estrogen depleted rats as compared to young animals. The low MS-KIF18A mRNA expression in OVX rats was restored by estrogen treatment. We studied the regulation of MS-KIF18A transcription by estrogen using the luciferase reporter gene and chromatin immuno-percipitation (ChIP) assays. The luciferase reporter gene assay demonstrated an increase in MS-KIF18A promoter activity in response to 10−8 M estrogen and 10−7M ICI-182,780. Complimentary, the ChIP assay quantified the binding of ERα and pcJun to the MS-KIF18A promoter that was enhanced in cells treated by estrogen and ICI-182,780. In addition, cells treated by estrogen expressed higher levels of MS-KIF18A mRNA and protein and the protein turnover in MBA-15 cells was accelerated. Presented data demonstrated that ERα is a defined cargo of MS-KIF18A and added novel insight on the role of estrogen in regulation of MS-KIF18A expression both in vivo and in vitro