Hamdy H. Hassanain

Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene.

Interferon (IFN)-γ induces the expression of the indoleamine 2,3-dioxygenase (INDO) gene in human cells, which plays a role in the inhibitory effect of IFN-γ on intracellular pathogens and on cell proliferation. Earlier studies established that the IFN-γ-inducible expression of the INDO gene was dependent on two upstream elements: (i) a 14-base pair sequence homologous to an interferon-stimulated response element (ISRE) sequence found in IFN-α-inducible genes and (ii) a 9-base pair palindromic sequence (palindromic element (PE) II) homologous to an interferon-γ-activated site (GAS) element found in IFN-γ-inducible genes. A second GAS element (PE I), between ISRE and PE II, was ineffective in supporting a response to IFN-γ. Studies were carried out to determine the distinction between the two GAS elements and the relative role of the two elements (ISRE and PE II) required for a response to IFN-γ. The PE I element was able to form a complex with IFN-γ-activated p91 (STAT1) factor but with lower efficiency than the complex formed with PE II sequence. However, switching the positions of PE I and II sequences in reporter plasmid constructs (containing chloramphenicol acetyltransferase gene) showed that both PE I and PE II were able to support a response to IFN-γ if located at the position of PE II but not at the position of PE I. Increasing the distance between the ISRE and PE II also affected the level of response, suggesting that the relative position of the two elements is important for optimal stimulus. To explore whether an interaction between the IFN-γ-regulated factors (IRF-1 and p91) binding to the ISRE and PE II might be important, we tested whether the ISRE sequence could be replaced by another response element, NF-κB. The plasmid construct with NF-κB element in place of the ISRE was responsive to IFN-γ, indicating that an interaction between the IRF-1 and p91 factors was not required. The results indicate that the response of INDO gene to IFN-γ depends on a cooperative role of IFN-γ-responsive factors binding to the ISRE and GAS elements.

Methylation of the estrogen receptor-α gene promoter is selectively increased in proliferating human aortic smooth muscle cells

OBJECTIVE Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-alpha gene (ERalpha) promoter blocks the expression of ERalpha, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERalpha is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. METHODS We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERalpha in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). RESULTS We provide evidence that methylation of the ERalpha in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERalpha promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERalpha and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. CONCLUSIONS Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERalpha, could contribute to the switch in phenotype observed in these cells.

ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis

&NA; Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)‐(3‐iodobenzyl)‐adenosine‐5‐N‐methyluronamide (IB‐MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine‐based drugs in experimental inflammatory bowel disease. High‐density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS‐treated (7 days), and oral IB‐MECA‐treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin‐trap monitoring of free radicals. Remarkable colitis‐induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis‐related dysregulation. Downregulation occurs in membrane transporter, mitogen‐activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox‐sensitive genes. Oral IB‐MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB‐MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB‐MECA blocked the colitis‐induced upregulation (≤20‐fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real‐time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB‐MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high‐density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine‐based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Thrombospondin 2 Regulates Cell Proliferation Induced by Rac1 Redox-Dependent Signaling

ABSTRACT Thrombospondin 2 (TSP2) is a matricellular protein controlling the apoptosis-proliferation balance in endothelial cells. Little is known about its transcriptional regulation compared with that of TSP1. We found that overexpression of a constitutively active mutant of Rac (RacV12) specifically increases TSP2 mRNA levels without affecting TSP1 in human aortic endothelial cells (HAEC). Moreover, TSP2 induction by RacV12 is dependent upon reactive oxygen species (ROS) production, as gp91ds-tat peptide, an inhibitor of NADPH oxidase, and the flavoprotein inhibitor diphenylene iodinium (DPI) block TSP2 synthesis. Furthermore, we found that increasing RacV12 expression results in a biphasic proliferative curve, with proliferation initially increasing as RacV12 expression increases and then returning to levels less than that of control cells at higher expression. This growth inhibition is mediated by TSP2, as either DPI treatment, which blocks TSP2 synthesis, or pan-TSP blocking antibodies restore the proliferative ability of HAEC with high expression. Mechanistically, we show that the effect of TSP2 on cell proliferation is independent of the antiangiogenic TSP2 Hep1 sequence, which is capable of altering actin cytoskeletal reorganization but not proliferation in our experimental conditions. Finally, we show in vivo that Rac-induced TSP2 expression is observed in the aorta of transgenic mice selectively expressing RacV12 in smooth muscle cells. These results identify Rac-induced ROS as a new pathway involved in the regulation of TSP2 expression.

Rabbit chronic ileitis leads to up-regulation of adenosine A1/A3 gene products, oxidative stress, and immune modulation.

A rabbit model of chronic ileitis has helped decipher the mechanism of alteration of multiple electrolyte and nutrient malabsorptions in inflammatory bowel disease (IBD). This study examined alterations in the adenosine A1/A3 receptor, oxidant, antioxidant, and immune-inflammatory pathways in chronic ileitis. Chronic ileal inflammation was induced 13-15 days after infection with 10,000 Eimeria magna oocytes. Quantitative analysis in 16 rabbits was done for oxidants, antioxidants, A1 and A3 transcripts, transport, injury, and inflammatory mediators. Inflamed gut had villus blunting, crypt hyperplasia and fusion, and immune cell infiltration. Alkaline phosphatase and Na-glucose co-transport were reduced by 78% (P=0.001) and 89% (P=0.001), respectively. Real-time fluorescence monitoring (TaqMan)-polymerase chain reaction revealed a transcriptional up-regulation of 1.34-fold for A1 and 5.40-fold for A3 receptors in inflamed gut. Lipid peroxidation increased in the mucosa (78%, P=0.012), longitudinal muscle-myenteric plexus (118%, P=0.042), and plasma (104%, P=0.001). Mucosal antioxidants were altered by inflammation: reductions occurred in superoxide dismutase (32%, P=0.001) and catalase (43%, P=0.001), whereas increases occurred in glutathione (75%, P=0.0271) and glutathione reductase (86%, P=0.0007). Oxidant enzyme activities were elevated by 21% for xanthine oxidase (P=0.004), 172% for chloramine (P=0.022), 47% for gelatinase (P=0.041), and 190% for myeloperoxidase (P=0.002). Mast cell tryptase increased by 79% (P=0.006). Increases occurred in the plasma concentration of leukotriene B(4) (13-fold, P=0.003), thromboxane B(2) (61-fold, P=0.018), and tumor necrosis factor-alpha (9-fold, P=0.002). In conclusion, chronic ileitis and tissue injury are associated with discrete alterations in complex multi-level oxidant, antioxidant, and immune inflammatory components. The rabbit ileitis model is a suitable model to gain further insight into chronic inflammation and IBD. We hypothesize that adenosine A3 and A1 receptors may provide a novel target for therapy in chronic ileitis and perhaps IBD.

Vascular Hypertrophy and Hypertension Caused by Transgenic Overexpression of Profilin 1

We have overexpressed either the cDNA of human profilin 1 or expressed the mutant (88R/L) in the blood vessels of transgenic FVB/N mice. Reverse transcription-PCR indicated selective overexpression of profilin 1 and 88R/L in vascular smooth muscle cells. Polyproline binding showed increased profilin 1 and 88R/L proteins in transgenic mice compared with control (∼30%, p < 0.05). Rhodamine-phalloidin staining revealed increase stress fiber formation in vascular smooth muscle cells of profilin 1 compared with 88R/L and control. Hematoxylin and eosin staining showed clear signs of vascular hypertrophy in the aorta of profilin 1 mice versus 88R/L and control. However, there were no differences between 88R/L and control mice. Western blotting confirmed the activation of the hypertrophic signaling cascades in aortas of profilin 1 mice. Phospho-ERK1/2 was significantly higher in profilin 1 than 88R/L and control (512.3 and 361.7%, respectively, p < 0.05). Profilin 1 mice had significant increases in phospho-JNK as compared with 88R/L and control (371.4 and 346%, respectively, p < 0.05). However, there were no differences between 88R/L and control mice in both kinases. There was a significant increase in ROCK II kinase in the aorta of profilin 1 mice compared with controls (>400%, p < 0.05). Tail cuff and circadian monitoring of blood pressure showed significant increases in systolic and mean arterial blood pressures of profilin 1 mice starting at age 6 months compared with controls (∼25 mm Hg, p < 0.05). These results suggest that increased actin polymerization in blood vessels triggers activation of the hypertrophic signaling cascades and results in elevation of blood pressure at advanced age.

Impact of disrupting adenosine A3 receptors (A3−/−AR) on colonic motility or progression of colitis in the mouse

Background: Pharmacological studies suggest that adenosine A3AR influences motility and colitis. Functional A3−/−AR knockout mice were used to prove whether A3AR activation is involved in modulating either motility or colitis. Methods: A3AR was probed by polymerase chain reaction (PCR) genotyping, Western blot, and immunochemistry. Motility was assessed in vivo by artificial bead‐expulsion, stool‐frequency, and FITC‐dextran transit. Colitis was induced with dextran sodium sulfate (DSS) in A3−/−AR or wildtype (WT) age‐ and sex‐matched controls. Progression of colitis was evaluated by histopathology, changes in myeloperoxidase (MPO), colon length, CD4+‐cells, weight‐loss, diarrhea, and the guaiac test. Results: Goat anti‐hu‐A3 antiserum identified a 66 kDa immunogenic band in colon. A3AR‐immunoreactivity is expressed in SYN+‐nerve varicosities, s‐100+‐glia, and crypt cells, but not 5‐HT+ (EC), CD4+ (T), tryptase+ (MC), or muscle cells. A3AR immunoreactivity in myenteric ganglia of distal colon ⟩ proximal colon by a ratio of 2:1. Intestinal transit and bead expulsion were accelerated in A3−/−AR mice compared to WT; stool retention was lower by 40%–60% and stool frequency by 67%. DSS downregulated A3AR in epithelia. DSS histopathology scores indicated less mucosal damage in A3−/−AR mice than WT. A3−/−AR phenotype protected against DSS‐induced weight loss, neutrophil (MPO), or CD4+‐T cell infiltration, colon shortening, change in splenic weight, diarrhea, or occult‐fecal blood. Conclusions: Functional disruption of A3AR in A3−/−AR mice alters intestinal motility. We postulate that ongoing release of adenosine and activation of presynaptic‐inhibitory A3AR can slow down transit and inhibit the defecation reflex. A3AR may be involved in gliotransmission. In separate studies, A3−/−AR protects against DSS colitis, consistent with a novel hypothesis that A3AR activation contributes to development of colitis. (Inflamm Bowel Dis 2010)

Vascular hypertrophy-associated hypertension of profilin1 transgenic mouse model leads to functional remodeling of peripheral arteries

Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. Vascular hypertrophy of resistance vessels leads to reduced compliance and elevation of blood pressure. We showed before that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our hypothesis is that profilin1 induced vascular hypertrophy in resistance vessels, which led to elevation of blood pressure, both of which contributed to the modulation of vascular function. Our results showed significant increases in the expression of alpha(1)- and beta(1)-integrins (280 + or - 6.3 and 325 + or - 7.4%, respectively) and the activation of the Rho/Rho-associated kinase (ROCK) II pathway (260 and 350%, respectively, P < 0.05) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of endothelial nitric oxide synthase expression (39 + or - 5.4%; P < 0.05) and phosphorylation (35 + or - 4.5%; P < 0.05) but also an increase in myosin light chain 20 phosphorylation (372%, P < 0.05). There were also increases in hypertrophic signaling pathways in the mesenteric arteries of profilin1 mice such as phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase (312.15 and 232.5%, respectively, P < 0.05). Functional analyses of mesenteric arteries toward the vasoactive drugs were assessed using wire-myograph and showed significant increases in the vascular responses of profilin1 mesenteric arteries toward phenylephrine, but significant decreases in response toward ROCK inhibitor Y-27632, ACh, sodium nitrite, and cytochalasin D. The changes in vascular responses in the mesenteric arteries of profilin1 mice are due to vascular hypertrophy and the elevation of blood pressure in the profilin1 transgenic mice.

Dietary sodium regulates angiotensin AT1a and AT1b mRNA expression in mouse brain.

Previous results showed that angiotensin (Ang) AT1a and AT1b receptor mRNA are expressed in mouse hypothalamus (HYP), brainstem (BS) and anterior pituitary (PIT). To extend these findings, we developed a real-time polymerase chain reaction (PCR) method to differentiate and quantify Ang AT1a and AT1b mRNA in mouse brain. An experiment was conducted in male C57Bl/6J mice to determine the effects of low and high dietary salt (0.04 or 8% NaCl for 2 weeks) on mRNA expression. Physiological measurements showed that high salt increased water intake (15.1 +/- 0.6 ml/day), whereas low salt decreased water intake (3.2 +/- 0.1 ml/day). There were no significant changes in body weight, hematocrit or plasma osmolality. Real-time PCR was effective in distinguishing AT1a and AT1b receptor mRNA. The PCR efficiencies for AT1a, AT1b and 18S ribosome were tested to be identical, making it possible to quantify mRNA levels. There were differences in angiotensin receptor expression, related to diet and brain region. In hypothalamus, both the high salt and low salt diet decreased AT1a expression (to 63 +/- 4% and 62 +/- 1%), although there were no changes in AT1b. In brainstem, there was a marked increase in AT1a (to 365 +/- 60%) and AT1b (to 372 +/- 23%) after high salt, although there was only a marked decrease for AT1b (to 23 +/- 5%) after low salt. In anterior pituitary, both high salt and low salt diet increased AT1a expression (to 152 +/- 8% and 123 +/- 9%), although there were no changes in AT1b. Results document that both AT1 receptor subtypes are present in mouse hypothalamus, brainstem and anterior pituitary, and that there is differential regulation of expression in response to changes in dietary salt.

Vascular remodeling-associated hypertension leads to left ventricular hypertrophy and contractile dysfunction in profilin-1 transgenic mice.

Abstract: Hypertension is a major health problem and a main risk factor for cardiovascular diseases. We have shown that overexpression of profilin-1 in blood vessels of transgenic mice generates mechanical tone and led to vascular remodeling/hypertension. However, little is known whether cardiac contractile performance in these mice is compromised. We investigated the in vivo contractile function and in vitro contractile performance using isolated papillary muscles from both right ventricle and left ventricle of profilin-1 mice at older age. Our results showed mild left ventricular hypertrophy and moderate systolic dysfunction in profilin-1 mice as evident by increased heart/body weight ratio and echocardiography analysis. Under near physiological conditions, right ventricle papillary muscles of profilin-1 mice maintained their peak isometric active developed tension, and the rate of force development over the entire frequency range of 4–14 Hz. Positive inotropic responses to increasing Ca2+ and &bgr;-adrenergic stimulation were also maintained. Conversely, left ventricular papillary muscles of profilin-1 mice exhibited depressed peak isometric, peak isometric active developed tension and rate of force development, and depressed positive inotropic responses to increasing Ca2+ and &bgr;-adrenergic stimulation. We here provide functional evidence that a significant contractile dysfunction in profilin-1 mice exists. Targeting vascular profilin-1 signaling could represent a promising therapeutic approach in hypertensive patients.