(566e) Transcriptional Regulation Underlying the Central Nervous System Response to Acute Hypertension

Essential hypertension is one of the most common disorders affecting human health. The central nervous system (CNS) has a critical role in the development and maintenance of elevated arterial pressure. Blood pressure is encoded by baroreceptors in the carotid sinus and the aortic arch, which relay signals to the nucleus tractus solitarius (NTS). The NTS uses this input to modulate sympathetic and parasympathetic activity, which adjusts heart rate, peripheral resistance, and cardiac output in a response called the baroreflex. However, the molecular mechanisms of blood pressure regulation are largely unknown. It is postulated that long-term central resetting of the baroreflex set point is mediated by long-term changes in the molecular state of baroreceptive cells in the NTS. System-wide transcriptional changes would be necessary to effect such a change in the molecular state of the cells.

The primary objective of the present study is to characterize the genomic response of the NTS during acute hypertension and relate the changes in neuronal state to the central resetting of the blood pressure set point. The focus is on the acute rather than chronic hypertension in order to observe the dynamics of resetting of the adapting system rather than the steady state reflecting the adapted system. To this end, we take a genomics approach to obtain a time series of the system-wide transcriptional changes in the NTS during acute hypertension. The time series gene expression data was analyzed in two domains: (i) the ?output' domain as to which signaling pathways, ion channels and other functionally relevant systems constitute the response, and (ii) the ?input' domain as to which gene regulatory mechanisms are involved. The first analysis includes mapping the annotation of the differentially expressed genes at various time points to the known function (via Gene Ontology, http://www.geneontology.org) and pathways (via Panther Database, http://www.pantherdb.org), thus building a system-wide map of the temporal dynamics of various neuronal functions and pathways in the acute hypertension response. The second analysis focuses ?upstream' to predict the transcription factors involved based on the enriched transcriptional regulatory elements in various genes grouped by temporal co-expression.

Adult Sprague-Dawley rats were subjected to elevated blood pressure for 60 minutes by infusing phenylephrine (200ug/ml) through the femoral vein. The corresponding control animals were treated with saline infusions for 60 minutes. Animals were sacrificed at 0, 15, 30, 45 and 60 minutes following the saline or phenylephrine infusion. NTS was isolated from each animal by a micropunch (75 um diameter) of 250 um slices of brainstem. A total of 40 samples were collected from four animals per condition (acute hypertensive or control) at each time point. RNA was isolated and processed using microarrays in a reference design to measure the expression of 8832 brain-expressed genes.

Analysis of the gene expression data using ANOVA indicates that a total of 2890 genes were responsive to acute hypertension at any time point at a 10% false discovery rate threshold. An important characteristic of this system-wide response is that almost all of the statistically significant expression changes were in the range of 20-50%. The data was clustered using an iterative approach. First, the 2890 responsive genes were grouped into 40 clusters by hierarchical clustering. In each of the 40 clusters, the genes with negative silhouette coefficients (indicating they are incorrectly clustered) were moved to the closest cluster until all genes had positive silhouette coefficients. These clusters were further pruned such that the Euclidian distance from the cluster mediod was less than 1. This resulted in a set of clusters with very similarly co-expressed genes. Analysis of the enriched Gene Ontology terms in these clusters indicates RNA splicing, ribosomal complex assembly, translation-related processes, and various metabolic functions, as being affected in the NTS in response to acute hypertension. This result are consistent with the early stages of a molecular program to modulate the neuronal state through altered protein expression. Analysis of the intracellular signaling pathways via Panther Database implicates several well known receptors including G-protein coupled receptor Gq and Gi pathways, serotonin receptor pathways (especially 5HT-3), ionotropic and metabotropic glutamate receptor pathways, and alpha adrenergic receptor pathways. Enrichment Analysis of the transcriptional regulatory elements upstream of these genes resulted in hypotheses on 17 transcription factors acting at different time points. Together, these results provide us a system-wide view of the early gene regulatory events and downstream effects in the modulation of the molecular state of the baroreceptive NTS neurons during acute hypertension.