Anti-growth properties of BAY 41-2272 in vascular smooth muscle cells

Vascular smooth muscle (VSM) growth is integral in the pathophysiology of blood vessel diseases, and identifying approaches that have capacity to regulate VSM growth is critically essential. Cyclic nucleotide signaling has been generally considered protective in cardiac and vascular tissues and has been the target of numerous basic science and clinical studies. In this project, the influence of BAY 41-2272 (BAY), a recently described soluble guanylate cyclase (sGC) stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis, on VSM cell growth was analyzed. In rat A7R5 VSM cells BAY significantly reduced proliferation in dose- and time-dependent fashion. BAY activated cGMP and cyclic adenosine monophosphate (cAMP) signaling evidenced through elevated cGMP and cAMP content, increased expression of cyclic nucleotide-dependent protein kinases, and differential vasodilator-stimulated phosphoprotein (VASP) phosphorylation. BAY significantly elevated cyclin E expression, decreased expression of the regulatory cyclin-dependent kinases (Cdk)-2 and -6, increased expression of cell cycle inhibitory p21WAF1/Cip1 and p27Kip1, and reduced expression of phosphorylated focal adhesion kinase (FAK). These comprehensive findings provide first evidence for the anti-growth, cell cycle-regulatory properties of the neoteric agent BAY 41-2272 in VSM and lend support for its continued study in the clinical and basic cardiovascular sciences. Originally published Journal of Cardiovascular Pharmacology, Vol. 53, No. 2, Feb 200

Coenzyme Q10 Ameliorates Ultraviolet B Irradiation Induced Cell Death Through Inhibition of Mitochondrial Intrinsic Cell Death Pathway

Ultraviolet B (UVB) induces cell death by increasing free radical production, activating apoptotic cell death pathways and depolarizing mitochondrial membrane potential. Coenzyme Q10 (CoQ10), an essential cofactor in the mitochondrial electron transport chain, serves as a potent antioxidant in the mitochondria. The aim of the present study is to establish whether CoQ10 is capable of protecting neuronal cells against UVB-induced damage. Murine hippocampal HT22 cells were treated with 0.01, 0.1 or 1 μM of CoQ10 3 or 24 h prior to the cells being exposed to UVB irradiation. The CoQ10 concentrations were maintained during irradiation and 24 h post-UVB. Cell viability was assessed by counting viable cells and MTT conversion assay. Superoxide production and mitochondrial membrane potential were measured using fluorescent probes. Levels of cleaved caspase-9, caspase-3, and apoptosis-inducing factor (AIF) were detected using immunocytochemistry and Western blotting. The results showed that UVB irradiation decreased cell viability and such damaging effect was associated with increased superoxide production, mitochondrial depolarization, and activation of caspase-9 and caspase-3. Treatment with CoQ10 at three different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide production, normalization of mitochondrial membrane potential and inhibition of caspase-9 and caspase-3 activation. It is concluded that the neuroprotective effect of CoQ10 results from inhibiting oxidative stress and blocking caspase-3 dependent cell death pathway

YC-1 Stimulates the Expression of Gaseous Monoxide-Generating Enzymes in Vascular Smooth Muscle Cells

The benzylindazole derivative 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-α]quinozalin-1-one (ODQ) or by the protein kinase G inhibitors (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823) and YGRKKRRQRRRPPLRKKKKKH-amide (DT-2) and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator 5-cyclopropyl-2[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b] pyridine-3-yl] pyrimidin-4-ylamine (BAY 41-2272). However, the YC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor (9S,10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1′-kl)pyrrolo(3,4-i)(1,6)-benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and was mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides and simultaneously stimulate their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified. Originally published Molecular Pharmacology, Vol. 75, No. 1, Jan 200

Contribution of Age, Brain Region, Mood Disorder Pathology, and Interindividual Factors on the Methylome of Human Microglia

Background: Transcriptome studies have revealed age-, disease-, and region-associated microglial phenotypes reflecting changes in microglial function during development, aging, central nervous system homeostasis, and pathology. The molecular mechanisms that contribute to these transcriptomic changes are largely unknown. The aim of this study was to characterize the DNA methylation landscape of human microglia and the factors that contribute to variations in the microglia methylome. We hypothesized that both age and brain region would have a large impact on DNA methylation in microglia. Methods: Microglia from postmortem brain tissue of four different brain regions of 22 donors, encompassing 1 patient with schizophrenia, 13 patients with mood disorder pathology, and 8 control subjects, were isolated and assayed using a genome-wide methylation array. Results: We found that human microglial cells have a methylation profile distinct from bulk brain tissue and neurons, and age explained a considerable part of the variation. Additionally, we showed that interindividual factors had a much larger effect on the methylation landscape of microglia than brain region, which was also seen at the transcriptome level. In our exploratory analysis, we found various differentially methylated regions that were related to disease status (mood disorder vs. control). This included differentially methylated regions that are linked to gene expression in microglia, as well as to myeloid cell function or neuropsychiatric disorders. Conclusions: Although based on relatively small samples, these findings suggest that the methylation profile of microglia is responsive to interindividual variations and thereby plays an important role in the heterogeneity of microglia observed at the transcriptome level

Glycated collagen I (GC) impairs angiogenesis in vitro: a study using an innovative chamber for cell research

Studies of cell-matrix, cell-cell interaction, or angiogenesis on different matrices require simultaneous comparison of read-out parameters from differently treated companion cells. The culture conditions (cell number, temperature, volume of culture medium) in different chambers are not completely equalized using conventional methods. It has been reported that cells growing in different environmental conditions may exhibit different proliferation patterns (2). Herein we describe an innovative chamber which could resolve this problem by significantly improving the standardization of experimental conditions. The chamber was manufactured from a standard cell culture well by its division with a septum into two sections. We utilized the chamber and recently developed topological analysis to examine the effects of glycated matrices on the capillary network by endothelial cells. Glycated Collagen I resulted in dose-dependent changes to all measured topological characteristics of the capillary-like network, such as the number of branching points, number of meshes and total capillary length. These differences were observed only in neighbored compartments coated with different matrices, but not in the compartments coated with the same matrix. The novel chamber brings an opportunity for better standardization of experimental conditions and simultaneous observation of different experimental groups, reducing the possible effect of any systematic error

Selenite stimulates mitochondrial biogenesis signaling and enhances mitochondrial functional performance in murine hippocampal neuronal cells.

Supplementation of selenium has been shown to protect cells against free radical mediated cell damage. The objectives of this study are to examine whether supplementation of selenium stimulates mitochondrial biogenesis signaling pathways and whether selenium enhances mitochondrial functional performance. Murine hippocampal neuronal HT22 cells were treated with sodium selenite for 24 hours. Mitochondrial biogenesis markers, mitochondrial respiratory rate and activities of mitochondrial electron transport chain complexes were measured and compared to non-treated cells. The results revealed that treatment of selenium to the HT22 cells elevated the levels of nuclear mitochondrial biogenesis regulators PGC-1α and NRF1, as well as mitochondrial proteins cytochrome c and cytochrome c oxidase IV (COX IV). These effects are associated with phosphorylation of Akt and cAMP response element-binding (CREB). Supplementation of selenium significantly increased mitochondrial respiration and improved the activities of mitochondrial respiratory complexes. We conclude that selenium activates mitochondrial biogenesis signaling pathway and improves mitochondrial function. These effects may be associated with modulation of AKT-CREB pathway

Anti-growth properties of BAY 41-2272 in vascular smooth muscle cells

Vascular smooth muscle (VSM) growth is integral in the pathophysiology of blood vessel diseases and identifying approaches that have capacity to regulate VSM growth is critically essential. Cyclic nucleotide signaling has been generally considered protective in cardiac and vascular tissues and has been the target of numerous basic science and clinical studies. In this project the influence of BAY 41-2272 (BAY) a recently described soluble guanylate cyclase (sGC) stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis on VSM cell growth was analyzed. In rat A7R5 VSM cells BAY significantly reduced proliferation in dose- and time-dependent fashion. BAY activated cGMP and cyclic adenosine monophosphate (cAMP) signaling evidenced through elevated cGMP and cAMP content increased expression of cyclic nucleotide-dependent protein kinases and differential vasodilator-stimulated phosphoprotein (VASP) phosphorylation. BAY significantly elevated cyclin E expression decreased expression of the regulatory cyclin-dependent kinases (Cdk)-2 and -6 increased expression of cell cycle inhibitory p21WAF1/Cip1 and p27Kip1 and reduced expression of phosphorylated focal adhesion kinase (FAK). These comprehensive findings provide first evidence for the anti-growth cell cycle-regulatory properties of the neoteric agent BAY 41-2272 in VSM and lend support for its continued study in the clinical and basic cardiovascular sciences. Originally published Journal of Cardiovascular Pharmacology Vol. 53 No. 2 Feb 200