A precision medicine approach to stress testing using metabolomics and microribonucleic acids.

Both transcriptomics and metabolomics hold promise for identifying acute coronary syndrome (ACS) but they have not been used in combination, nor have dynamic changes in levels been assessed as a diagnostic tool. We assessed integrated analysis of peripheral blood miRNA and metabolite analytes to distinguish patients with myocardial ischemia on cardiac stress testing. We isolated and quantified miRNA and metabolites before and after stress testing from seven patients with myocardial ischemia and 1:1 matched controls. The combined miRNA and metabolomic data were analyzed jointly in a supervised, dimension-reducing discriminant analysis. We implemented a baseline model (T0) and a stress-delta model. This novel integrative analysis of the baseline levels of metabolites and miRNA expression showed modest performance for distinguishing cases from controls. The stress-delta model showed worse performance. This pilot study shows potential for an integrated precision medicine approach to cardiac stress testing.

The study of small sequences of ribonucleic acids (miRNAs) and byproducts of cellular metabolism (metabolites) could help us to identify important cardiac conditions such as not enough blood and oxygen supply to the heart (acute coronary syndrome). We obtained blood samples from patients getting cardiac stress tests (a noninvasive test to see if the patient has enough blood flow to their heart) before and after their test, then compared the levels of miRNAs and metabolites in them. We compared the levels in patients who had abnormal stress tests with those that had normal tests. We believe this could be a model for a new type of cardiac stress test if validated in more patients.

Dexamethasone increases pigment epithelium-derived factor in perfused human eyes.

PURPOSE: To investigate the effects of dexamethasone (DEX) on pigment epithelium-derived factor (PEDF) cDNA and secreted protein in human trabecular meshwork (TM). METHODS: Anterior segment organ cultures were perfused with 0.1 microM DEX (OD) and vehicle (OS). Primary human TM cells (HTM) were treated with DEX under similar conditions. PEDF mRNA and secreted PEDF protein were quantitated by RT-PCR and Western blot. RESULTS: PEDF mRNA and secreted PEDF protein levels were significantly higher in DEX over vehicle-treated cultures. In contrast, DEX decreased the activity of a 92-kDa gelatinolytic zymogen in organ culture effluents. CONCLUSION: DEX action in the human TM might include a PEDF-mediating pathway.

Assessing incomplete deprotection of microarray oligonucleotides in situ.

En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

Tissue differential microarray analysis of dexamethasone induction reveals potential mechanisms of steroid glaucoma.

PURPOSE: To identify myocilin (TIGR/MYOC) properties that are specific to the human trabecular meshwork (HTM). To search for genes highly expressed in dexamethasone (DEX)-induced HTM cells that are barely expressed or absent in DEX-induced cells from other tissues. METHODS: TIGR/MYOC induction by DEX (10(-7) M for 8-10 days) was analyzed by Northern and Western blot analyses in HTM, human umbilical vein endothelial cells, HeLa cells, and human embryonic skeletal muscle cells and optic nerve head (ONH) astrocytes at confluence. Processing and secretion were analyzed after the cells were infected with adenoviruses overexpressing wild-type and mutant forms of TIGR/MYOC. Affymetrix U95Av2 GeneChips (n = 6) and software were used to compare paired expression profiles of HTM, HTM-DEX, ONH astrocytes, and ONH astrocytes-DEX. Identification of HTM-DEX-specific genes (compared with ONH astrocytes-DEX) was performed by selecting genes with the highest fold change values (>/=20). Genes with fold change values of four or more were matched with loci linked to glaucoma, by using gene databases. RESULTS: TIGR/MYOC induction by DEX occurred only in HTM cells. Secretory and glycosylation characteristics remained the same across cell types. Expression profile analysis revealed multiple genes differentially upregulated in HTM-DEX including, in addition to TIGR/MYOC, a serine protease inhibitor (alpha1-antichymotrypsin), a neuroprotective factor (pigment epithelium-derived factor), an antiangiogenesis factor (cornea-derived transcript 6), and a prostaglandin synthase (prostaglandin D(2) synthase). Fifteen of the 249 genes with fold change values of four or more mapped to glaucoma-linked loci. CONCLUSIONS: The induction of TIGR/MYOC by DEX is HTM-specific, whereas its secretory and glycosylation characteristics are ubiquitous. The known functions of HTM-DEX-specific genes reveal the presence of protective and damaging mechanisms for regulation of IOP during DEX treatment. Besides TIGR/MYOC, other HTM-DEX-specific genes may be good candidates for linkage to glaucoma.

Gene transfer of dominant-negative RhoA increases outflow facility in perfused human anterior segment cultures.

PURPOSE: To investigate the regulation of expression and the role of the RhoA gene in the human trabecular meshwork (TM). To attempt to modulate outflow facility by gene transfer of the RhoA gene's dominant-negative mutant protein. METHODS: Total RNA extracted from cultured human trabecular meshwork (HTM) cells treated with outflow facility drugs were analyzed by northern blot hybridization using an amplified human RhoA cDNA from plasmid pZip-RhoA wild type (wt) [1]. A dominant-negative form of RhoA (single amino acid substitution of Thr19 to Asn) was placed under the control of the CMV promoter and inserted into a replication-deficient adenoviral vector by overlapping recombination (AdhRhoA2). AdhRhoA2 was infected into perfused anterior segment cultures from post-mortem human donors and HTM and Schlemm's canal cells in culture. Changes in outflow facility (flow/pressure) were calculated as percent changes from baseline values (C0), pooled into treated and control groups and expressed as the mean plus minus standard error. HTM and Schlemm's Canal (SC) cells were fluorescently double-labeled for the RhoA protein and actin, paxillin, or ZO-1. RESULTS: Transcription of RhoA in HTM cells was not considerably affected by treatment of the cells with cytoskeletal/outflow facility drugs. At 66 h post-injection, anterior segments treated with AdhRhoA2 (n=9) exhibited an increase in outflow facility of 32.5 +/- 7.7% while that of the vehicle-injected controls (n=6) was 5.1 +/- 4.0% (p=0.02). HTM cells treated with AdhRhoA2 showed a marked change in morphology with a reduction in actin stress fibers and of the focal adhesion-containing protein, paxillin. Confluent monolayers of SC cells infected with AdhRhoA2 were devoid of peripheral ZO-1 staining indicating a loss of intercellular junctions. CONCLUSIONS: In the HTM cells, cytoskeletal/outflow facility drugs do not seem to affect the levels of RhoA mRNA, possibly suggesting the importance of mRNA availability to allow rapid turnover of its function. Gene transfer of inactive RhoA to the intact human TM results in an increase in outflow facility. This increase appears to be correlated with a loosening of the cell-substrate and cell-cell attachments in the cells of the outflow pathway. Adenoviral vectors carrying the dominant negative form of RhoA could potentially be utilized as a gene therapy to modulate outflow facility.

Effects of elevated intraocular pressure on outflow facility and TIGR/MYOC expression in perfused human anterior segments.

PURPOSE: To investigate the effects of high intraocular pressure (h-IOP) on TIGR/MYOC expression, extracellular matrix (ECM) deposition, and outflow facility (C) in perfused human anterior segment cultures. METHODS: Anterior segments of 31 pairs of normal human eyes from postmortem donors were perfused at constant flow (3 microl/min). After reaching stable baseline, the flow of one eye from each of 31 pairs was raised to obtain a continuous pressure of 60 to 70 mm Hg for a period of 1 hour (3 pairs), 6 hours (10 pairs), 24 hours (2 pairs), 48 hours (3 pairs), and 7 days (13 pairs). Sixteen of these pairs were used to study trabecular meshwork expression of TIGR/MYOC and stromelysin by Northern blot analysis hybridization. Nine pairs (1 pair each at h-IOP for 1, 6, and 48 hours and 6 pairs at 7 days) were fixed at pressure for analysis by electron microscopy. Eyes selected for C measurements fulfilled the inclusion criteria of C0 values between 0.06 and 0.4, intact RNA recovery and normal light microscopy morphology. Percent change of facility from the baseline (C/C0) was computed at 6 and 24 hours and 2, 4, and 7 days from the long-term perfusion experiments (n = 9 h-IOP, n = 8 controls). RESULTS: No induction of TIGR/MYOC expression was observed after h-IOP for 1 and 6 h. A slight induction was seen after 24 and 48 hours. At 7 days, the treated eye from 4 of 5 pairs showed a clear induction, which was very pronounced in one of the pairs. In contrast, stromelysin expression was induced at 6 hours and not at 7 days. Morphometric electron microscopy after 7 days showed no significant difference in the amounts of fine fibrillar material or plaque material in the juxtacanalicular (JCT) region. The percent increase of C of the treated eye at 6 hours was 11.0% +/- 4.6% compared with 3.7% +/- 3.8% in the control eyes (P = 0.26). However, after longer time periods, the facility of the h-IOP eyes increased, whereas that of the contralateral eyes remained unchanged. This difference reached peak, significant values at 4 days (32.9% +/- 8.4% versus 7.4% +/- 7.6%, respectively; P = 0.04) and decreased to 8.9% +/- 7.9% versus 1.1% +/- 12.7% (P = 0.6) at 7 days. CONCLUSIONS: Elevated IOP appears to cause a decrease in outflow pathway resistance at 1 to 4 days, and this effect seems to disappear with further time. In contrast, induction of TIGR/MYOC appears to be strongest at 7 days. We speculate that this induction pattern might indicate a stress-related, rather than a possible homeostatic, role for the TIGR/MYOC protein.