Abstract-A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)-regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase-polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II-mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

Toll-like receptor 4 (TLR4) is present on monocytes and other cell types, and mediates inflammatory events such as the release of TNF after exposure to LPS. C3H/HeJ mice are resistant to LPS-induced mortality, due to a naturally occurring mutation in TLR4. We therefore hypothesized that LPS-induced acute renal failure (ARF) requires systemic TNF release triggered by LPS acting on extrarenal TLR4. We injected C3H/HeJ mice and C3H/HeOuJ controls with 0.25 mg of LPS, and sacrificed them 6 h later for analysis of blood urea nitrogen (BUN) and kidney tissue (n = 8 per group). In contrast to C3H/HeOuJ controls, C3H/HeJ mice were completely resistant to LPS-induced ARF (6-h BUN of 32.3 ± 1.1 vs 61.7 ± 5.6 mg/dl). C3H/HeJ mice released no TNF into the circulation at 2 h (0.00 vs 1.24 ± 0.16 ng/ml), had less renal neutrophil infiltration (6.4 ± 1.0 vs 11.4 ± 1.3 neutrophils per high power field), and less renal apoptosis, as assessed by DNA laddering. Transplant studies showed that C3H/HeJ recipients of wild-type kidneys (n = 9) were protected from LPS-induced ARF, while wild-type recipients of C3H/HeJ kidneys (n = 11) developed severe LPS-induced ARF (24-h BUN 44.0 ± 4.1 vs 112.1 ± 20.0 mg/dl). These experiments support our hypothesis that LPS acts on extrarenal TLR4, thereby leading to systemic TNF release and subsequent ARF. Renal neutrophil infiltration and renal cell apoptosis are potential mechanisms by which endotoxemia leads to functional ARF.

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs.Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

Acute graft-versus-host disease (aGVHD) remains a major complication of allogeneic hematopoietic stem cell transplant (alloHSCT), underscoring the need to further elucidate its mechanisms and develop novel treatments. Based on recent observations that microRNA-155 (miR-155) is up-regulated during T-cell activation, we hypothesized that miR-155 is involved in the modulation of aGVHD.Here we show that miR-155 expression was up-regulated in T cells from mice developing aGVHD after alloHSCT. Mice receiving miR-155-deficient donor lymphocytes had markedly reduced lethal aGVHD, whereas lethal aGVHD developed rapidly in mice recipients of miR-155 overexpressing T cells. Blocking miR-155 expression using a synthetic antimiR-155 after alloHSCT decreased aGVHD severity and prolonged survival in mice. Finally, miR-155 up-regulation was shown in specimens from patients with pathologic evidence of intestinal aGVHD. Altogether, our data indicate a role for miR-155 in the regulation of GVHD and point to miR-155 as a novel target for therapeutic intervention in this disease.

PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G0/1 into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Rationale Although Nox5, (Nox2 homologue), has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives To test if vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+ -sensitive Nox5, associated with Rac-1, generates superoxide (•O2−) and activates growth and inflammatory responses via MAP kinases in human endothelial cells (ECs). Methods and results Cultured ECs, exposed to AngII and ET-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor) and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with siRNA. AngII and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated •O2− production and inhibited phosphorylation of ERK1/2, but not p38MAPK or SAPK/JNK. Nox5 siRNA blunted AngII-induced, but not ET-1-induced, upregulation of PCNA and VCAM-1, important in growth and inflammation. Conclusions Human ECs possess functionally active Nox5, regulated by AngII and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by AngII and ET-1 induces ROS generation and ERK 1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by AngII but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by AngII and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by AngII and ET-1.

Objective-Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results-VSMCs from WKY rats were studied. At low concentrations (10 Ϫ10 mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (PϽ0.01), an effect blocked by eplerenone (Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 (inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (PϽ0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions-Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent.

Abstract-This study assesses the receptor subtype (AT 1 and AT 2 ) through which angiotensin II (Ang II) mediates contraction in small arteries of young and adult spontaneously hypertensive rats (SHR). Segments of third-order mesenteric arteries (Ϸ200 m in lumen diameter) were mounted in a pressurized system. Systolic blood pressure and media:lumen ratio of small arteries were significantly greater (PϽ0.001) in young SHR and adult SHR than in age-matched Wistar-Kyoto rats (WKY). Ang II-induced contractile effects were significantly increased (PϽ0.05) in young SHR compared with age-matched WKY. AT 1 blockade with losartan, and combined AT 1 and AT 2 blockade with losartan and PD123319, abolished Ang II-stimulated contraction in young and adult rats. AT 2 blockade (PD123319) significantly reduced (PϽ0.01) Ang II-elicited contraction in young SHR but had no effect in WKY or adult SHR, indicating that AT 2 receptors may contribute to Ang II-induced contraction in young SHR. To determine the Ang receptor status in rat mesenteric vessels, AT 1 and AT 2 receptor mRNA expression was determined by reverse transcription-polymerase chain reaction. AT 1 and AT 2 receptor protein expression were detected by Western blot analysis. AT 1 receptor mRNA was equally expressed in age-matched rats, but expression was significantly lower in young rats compared with adult rats. AT 2 receptor mRNA was weakly expressed in WKY and adult SHR. In vessels from young SHR, AT 2 receptor mRNA expression was significantly increased compared with the other groups. AT 1 receptor protein was equally expressed in adult rats of both strains but was undetectable in young rats. AT 2 receptor protein was only detectable in young rats, with the magnitude of expression greater in SHR than WKY. Key Words: resistance Ⅲ arteries Ⅲ hypertension Ⅲ receptors, angiotensin Ⅲ vasoconstriction Ⅲ PD123319 Ⅲ losartan A ngiotensin II (Ang II), the final mediator of the reninangiotensin system, plays a pivotal physiological role in cardiovascular homeostasis. It is a potent vasoconstrictor of the peripheral vasculature and induces hypertrophy, hyperplasia, or both in small resistance arteries, in vascular smooth muscle cells, in endothelial cells, and in cardiomyocytes. [1][2][3][4] Because of these actions, Ang II may play an important pathophysiological role in the development and maintenance of hypertension.Cellular responses to Ang II are mediated by specific cell membrane receptors. Two main subtypes of Ang receptors have been pharmacologically defined: AT 1 and AT 2 , which are blocked specifically by losartan and PD123319, respectively. 5-7 AT 1 has 2 subtypes in rodents, AT 1A and AT 1B , with greater than 95% amino acid sequence homology. 6 Most of the known physiological effects of Ang II are mediated by AT 1 receptors and, until recently, it was believed that vascular Ang II receptors were exclusively of the AT 1 subtype. However, it has recently been demonstrated that the adult rat aorta expresses a small but significant amount of AT 2 receptors as well. 8...