UVM Theses and Dissertations
Format:
Online
Author:
Rose, Patricia Camela
Title:
Dept./Program:
Pharmacology
Year:
2007
Degree:
PhD
Abstract:
Vascular diseases such as hypertension are marked by changes in calcium (Ca²⁺) and extracellular signal regulated kinase (ERK) signaling in the arterial wall. The overall goal of this project was to better understand pathways leading to altered gene regulation in cerebral arteries. Two models were tested to determine if, 1) Ca²⁺/cAMP response element binding protein (CREB) is regulated in intact cerebral arteries by multiple sources of Ca²⁺, and 2) hypertensive disease causes changes in genes regulated by ERK and CREB. Ca²⁺-mediated phosphorylation of CREB (P-CREB) was measured by immunofluorescence in both cultured vascular smooth muscle cells (VSMCs) and in intact cerebral arteries. The level of P-CREB was increased by both Ca²⁺ influx through voltage-dependent calcium channels (VDCCs) and store-operated Ca²⁺ entry (SOCE) in VSMCs. A similar increase in P-CREB was observed following stimulation of VDCCs and SOCE in intact cerebral arteries. However, unlike the results obtained from VSMCs phosphorylation of CREB following Ca²⁺ store depletion using thapsigargin, was partially dependent on Ca²⁺ entry through VDCCs, suggesting that communication between Ca²⁺ entry pathways in intact arteries may be lost during cell culture.
The second model was tested using immunocytochemistry and RNA analysis to measure differences in cerebral artery signal transduction and gene expression caused by chronic hypertension in the Dahl salt sensitive genetic hypertensive rat model. Arteries from hypertensive animals exhibited increased phosphorylation of ERK and expression of Ki-67, a marker of proliferation, when compared to controls. In addition, microarray analysis of arterial RNA revealed overexpression of the matricellular ERK-regulated genes osteopontin (OPN), and plasminogen activator inhibitor 1 (PAI-1), and the activator protein transcription factor (AP-1) member junB in cerebral arteries, with validation using RT qPCR. To elucidate a role for CREB, ERK and JunB in the transcriptional regulation of OPN and PAI-1, VSMCs were treated with angiotensin II (Ang II), a vasoconstrictor linked to hypertension, and confirmed activator of OPN and PAI-1 transcription. Ang II induced an ERK-dependent transient increase in junB mRNA and protein prior to OPN, and PAI-1 induction. Gene silencing experiments indicated that OPN and PAI-1 are reciprocally regulated by junB and CREB, respectively, and that CREB is a negative regulator of OPN. Data from cell culture confirms that the Ang II response in VSMCs is transient, in contrast to the hypertensive in vivo model, suggesting that the CREB and ERK response induces long term changes. Together, these data have revealed mechanisms for regulation of gene expression that are linked to proliferation and remodeling in the arterial wall. Future experiments will explore an in vivo role for Ang II and SOCE in the mediation of ERK- and CREB-regulated gene expression. This research has the potential to help in defining therapeutic strategies to prevent arterial remodeling caused by arterial pathologies such as hypertension.
The second model was tested using immunocytochemistry and RNA analysis to measure differences in cerebral artery signal transduction and gene expression caused by chronic hypertension in the Dahl salt sensitive genetic hypertensive rat model. Arteries from hypertensive animals exhibited increased phosphorylation of ERK and expression of Ki-67, a marker of proliferation, when compared to controls. In addition, microarray analysis of arterial RNA revealed overexpression of the matricellular ERK-regulated genes osteopontin (OPN), and plasminogen activator inhibitor 1 (PAI-1), and the activator protein transcription factor (AP-1) member junB in cerebral arteries, with validation using RT qPCR. To elucidate a role for CREB, ERK and JunB in the transcriptional regulation of OPN and PAI-1, VSMCs were treated with angiotensin II (Ang II), a vasoconstrictor linked to hypertension, and confirmed activator of OPN and PAI-1 transcription. Ang II induced an ERK-dependent transient increase in junB mRNA and protein prior to OPN, and PAI-1 induction. Gene silencing experiments indicated that OPN and PAI-1 are reciprocally regulated by junB and CREB, respectively, and that CREB is a negative regulator of OPN. Data from cell culture confirms that the Ang II response in VSMCs is transient, in contrast to the hypertensive in vivo model, suggesting that the CREB and ERK response induces long term changes. Together, these data have revealed mechanisms for regulation of gene expression that are linked to proliferation and remodeling in the arterial wall. Future experiments will explore an in vivo role for Ang II and SOCE in the mediation of ERK- and CREB-regulated gene expression. This research has the potential to help in defining therapeutic strategies to prevent arterial remodeling caused by arterial pathologies such as hypertension.