Treatment of ß-thujaplicin counteracts di(2-ethylhexyl)phthalate (DEHP)-exposed vascular smooth muscle activation, inflammation and atherosclerosis progression.

The initiation of atherosclerosis involves up-regulation of molecules such as E-selectin, VCAM-1, and ICAM-1. The progression of atherosclerosis is linked to proliferation and migration of vascular smooth muscle cell via MMP-2 and MMP-9 activities. However, the etiology of atherosclerosis concerning plasticizers is unknown. We evaluated ß-thujaplicin in preventing the development of atherosclerosis in a model induced by pro-inflammatory cytokines. Moreover, we established a new atherosclerosis model in vascular smooth muscle cells (VSMC) exposed to a common contact plasticizer, di(2-ethylhexyl)phthalate (DEHP). SEVC4-10 endothelial cells were treated with 50% RAW conditioned medium and A7r5 VSMC was treated with the plasticizer, with/without ß-thujaplicin (4 or 12 µM). Production of E-selectin, ICAM-1, and VCAM-1 in SEVC4-10 cells as well as MMP-2/MMP-9 (both expression and activity) in VSMC were monitored. Results showed that the conditioned medium induced E-selectin and ICAM were significantly prevented by ß-thujaplicin. However, inhibition on the production of VCAM by ß-thujaplicin was only seen in a concentration of 12 µM. Both concentrations of ß-thujaplicin also significantly prevented DEHP-induced MMP-2 and MMP-9 expression and activities. Evidence uncovers that ß-thujaplicin has additional factors in amelioration of atherosclerosis and corroborates that ß-thujaplicin is a strong candidate in preventing the initiation and progression of atherosclerosis.

Possible Mechanisms of Di(2-ethylhexyl) Phthalate-Induced MMP-2 and MMP-9 Expression in A7r5 Rat Vascular Smooth Muscle Cells.

Proliferation and migration of vascular smooth muscle cells (VSMC) are important in the development and/or progression of many cardiovascular diseases, including atherosclerosis. Evidence shows that matrix metalloproteinase (MMP)-2 and MMP-9 are related to the pathogenesis of atherosclerosis. The expressions of MMP-2 and MMP-9 in atherosclerosis are regulated via various pathways, such as p38 mitogen activated protein kinase (MAPK), extracellular signal regulated kinase 1 and 2 (ERK1/2), Akt, and nuclear factor kappa (NF-κB). Di(2-ethylhexyl) phthalate (DEHP) has been shown to induce atherosclerosis by increasing tumor necrosis factor (TNF)-α, interleukin (IL)-6, and intercellular adhesion molecule (ICAM) productions. However, whether DEHP poses any effects on MMP-2 or MMP-9 expression in VSMC has not yet been answered. In our studies, rat aorta VSMC was treated with DEHP (between 2 and 17.5 ppm) and p38 MAPK, ERK1/2, Akt, NF-κB, and MMP-2 and MMP-9 proteins and activities were measured. Results showed that the presence of DEHP can induce higher MMP-2 and MMP-9 expression than the controls. Similar results on MMP-regulating proteins, i.e., p38 MAPK, ERK1/2, Akt, and NF-κB, were also observed. In summary, our current results have showed that DEHP can be a potent inducer of atherosclerosis by increasing MMP-2 and MMP-9 expression at least through the regulations of p38 MAPK, ERK1/2, Akt, and NF-κB.

Alternative activation in systemic juvenile idiopathic arthritis monocytes.

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1ß after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

Correlation analyses of clinical and molecular findings identify candidate biological pathways in systemic juvenile idiopathic arthritis.

BACKGROUND: Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC). METHODS: PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways. RESULTS: Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup. CONCLUSIONS: The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.

Stereotactic targeting and dose verification for age-related macular degeneration.

PURPOSE: Validation of the targeting and dose delivery of the IRay low voltage age-related macular degeneration treatment system. METHODS: Ten human cadaver eyes were obtained for this study and mounted in the IRay system. Using gel and vacuum, an I-Guide immobilization device was coupled to the eyes and radiochromic film was affixed to the posterior aspect of the globes. Three narrow x-ray beams were delivered through the pars plana to overlap on the predicted nominal fovea. A needle was placed through the center of the film's beam spot and into the eye to register the film and the inner retina. The process was performed three times for each of the ten eyes (30 simulated treatments; 90 individual beams). The globes were dissected to assess the targeting accuracy by measuring the distances from the needles to the fovea. The dose to the fovea was calculated from the radiochromic film. RESULTS: X-ray targeting on the retina averaged 0.6 +/- 0.4 mm from the fovea. Repeated treatments on the same eye showed a reproducibility of 0.4 +/- 0.4 mm. The optic nerve was safely avoided, with the 90% isodose edge of the beam spot between 0.4 and 2.6 mm from the edge of the optic disk. Measured dose matched that prescribed. CONCLUSIONS: This study provides confidence that the IRay, with an average accuracy of 0.6 mm and a precision of 0.4 mm, can reliably treat most AMD lesions centered on the fovea. With the exception of motion, all sources of error are included.

An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT-PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.

Disparate effects of telomere attrition on gene expression during replicative senescence of human mammary epithelial cells cultured under different conditions.

Telomere shortening in populations of human mammary epithelial cells (HMECs) that survive early replicative arrest (M0) by the inactivation of p16(INK4A) during cell culture on plastic dishes leads to a state of permanent replicative arrest termed senescence. While culture of HMECs on feeder layers abrogates M0 and p16(INK4A) inactivation, progressive telomere attrition in these cells also eventually results in permanent replicative arrest. Expression of telomerase prevents both senescence on plastic (S-P) and senescence on feeder layers (S-FL) in HMECs, as it does also in cultured primary human fibroblasts. We report here that the gene expression profiles of senescence in HMECs of the same lineage maintained under different culture conditions showed surprisingly little commonality. Moreover, neither of these senescence-associated profiles in HMECs resembles the profile for senescence in human fibroblasts. These results indicate that senescence-associated alterations in gene expression resulting from telomere attrition are affected by culture conditions as well as by cell origins, and argue that replicative senescence at the molecular level is a diverse rather than unique cellular process.

Effects of threshold choice on biological conclusions reached during analysis of gene expression by DNA microarrays.

Global analysis of gene expression by using DNA microarrays is employed increasingly to search for differences in biological properties between normal and diseased tissue. In such studies, expression that deviates from defined thresholds commonly is used for creating genetic signatures that characterize disease vs. normality. Although it is axiomatic that the threshold parameters applied to microarray analysis will alter the contents of such genetic signatures, the extent to which threshold choice can affect the fundamental conclusions made from microarray-based studies has not been elucidated. We used GABRIEL (Genetic Analysis By Rules Incorporating Expert Logic), a platform of knowledge-based algorithms for the global analysis of gene expression, together with conventional statistical approaches, to examine the sensitivity of conclusions to threshold choice in recently published microarray-based studies. An analysis of the effects of threshold decisions in one of these studies [Ramaswamy, S., Ross, K. N., Lander, E. S. & Golub, T. R. (2003) Nat. Genet. 33, 49-54], which arrived at the important conclusion that the metastatic potential of primary tumors is encoded by the bulk of cells in the tumor, is the focus of this article. We discovered that support for this conclusion highly depends on the threshold used to create gene expression signatures. We also found that threshold choice dramatically affected the gene function categories represented nonrandomly in signatures. Our results suggest that the robustness of biological conclusions made by using microarray analysis should be routinely assessed by examining the validity of the conclusions by using a range of threshold parameters.

Senescence-specific gene expression fingerprints reveal cell-type-dependent physical clustering of up-regulated chromosomal loci.

Replicative senescence is the state of irreversible proliferative arrest that occurs as a concomitant of progressive telomere shortening. By using cDNA microarrays and the gabriel system of computer programs to apply domain-specific and procedural knowledge for data analysis, we investigated global changes in gene transcription occurring during replicative senescence in human fibroblasts and mammary epithelial cells (HMECs). Here we report the identification of transcriptional "fingerprints" unique to senescence, the finding that gene expression perturbations during senescence differ greatly in fibroblasts and HMECs, and the discovery that despite the disparate nature of the chromosomal loci affected by senescence in fibroblasts and HMECs, the up-regulated loci in both types of cells show physical clustering. This clustering, which contrasts with the random distribution of genes down-regulated during senescence or up-regulated during reversible proliferative arrest (i.e., quiescence), supports the view that replicative senescence is associated with alteration of chromatin structure.

Analysis of DNA microarrays using algorithms that employ rule-based expert knowledge.

The ability to investigate the transcription of thousands of genes concurrently by using DNA microarrays offers both major scientific opportunities and significant analytical challenges. Here we describe GABRIEL, a rule-based system of computer programs designed to apply domain-specific and procedural knowledge systematically and uniformly for the analysis and interpretation of data from DNA microarrays. GABRIEL'S problem-solving rules direct stereotypical tasks, whereas domain-specific knowledge pertains to gene functions and relationships or to experimental conditions. Additionally, GABRIEL can learn novel rules through genetic algorithms, which define patterns that best match the data being analyzed and can identify groupings in gene expression profiles preordered by chromosomal position or by a nonsupervised algorithm such as hierarchical clustering. GABRIEL subsystems explain the logic that underlies conclusions and provide a graphical interface and interactive platform for the acquisition of new knowledge. The present report compares GABRIEL'S output with published findings in which expert knowledge has been applied post hoc to microarray groupings generated by hierarchical clustering.