Functional Analysis of Proteins Involved in Translational Regulation

Thesis advisor: Laura E. HakeCytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo.Thesis (BS) — Boston College, 2003.Submitted to: Boston College. College of Arts and Sciences.Discipline: Biology.Discipline: College Honors Program

Adiponectin Decreases Pulmonary Arterial Remodeling in Murine Models of Pulmonary Hypertension

Remodeling of the pulmonary arteries is a common feature among the heterogeneous disorders that cause pulmonary hypertension. In these disorders, the remodeled pulmonary arteries often demonstrate inflammation and an accumulation of pulmonary artery smooth muscle cells (PASMCs) within the vessels. Adipose tissue secretes multiple bioactive mediators (adipokines) that can influence both inflammation and remodeling, suggesting that adipokines may contribute to the development of pulmonary hypertension. We recently reported on a model of pulmonary hypertension induced by vascular inflammation, in which a deficiency of the adipokine adiponectin (APN) was associated with the extensive proliferation of PASMCs and increased pulmonary artery pressures. Based on these data, we hypothesize that APN can suppress pulmonary hypertension by directly inhibiting the proliferation of PASMCs. Here, we tested the effects of APN overexpression on pulmonary arterial remodeling by using APN-overexpressing mice in a model of pulmonary hypertension induced by inflammation. Consistent with our hypothesis, mice that overexpressed APN manfiested reduced pulmonary hypertension and remodeling compared with wild-type mice, despite developing similar levels of pulmonary vascular inflammation in the model. The overexpression of APN was also protective in a hypoxic model of pulmonary hypertension. Furthermore, APN suppressed the proliferation of PASMCs, and reduced the activity of the serum response factor–serum response element pathway, which is a critical signaling pathway for smooth muscle cell proliferation. Overall, these data suggest that APN can regulate pulmonary hypertension and pulmonary arterial remodeling through its direct effects on PASMCs. Hence, the activation of APN-like activity in the pulmonary vasculature may be beneficial in pulmonary hypertension

Validation of Noninvasive Measurements of Cardiac Output in Mice Using Echocardiography

Although multiple echocardiographic methods exist to calculate cardiac output (CO), they have not been validated in mice using a reference method. Echocardiographic and flow probe measurements of CO were obtained in mice before and after albumin infusion and inferior vena cava occlusions. Echocardiography was also performed before and after endotoxin injection. Cardiac output was calculated using LV volumes obtained from a M Mode or a 2D view, LV stroke volume calculated using the pulmonary flow, or estimated using pulmonary VTI. Close correlations were demonstrated between flow probe-measured CO and all echocardiographic measurements of CO. All echocardiographic-derived CO overestimated the flow-probe measured CO. 2D images-derived CO was associated with the smallest overestimation of CO. Interobserver variability was lowest for pulmonary VTI derived CO. In mice, CO calculated from 2D parasternal long axis images is most accurate when compared to flow probe measurements, however, pulmonary VTI-derived CO is subject to less variability

Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice

Objective: Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection. Methods and Results: We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation. Conclusions and Significance: Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.American Heart Association (Predoctoral Fellowship)National Institutes of Health (U.S.) (R01 NS33335)National Institutes of Health (U.S.) (R01 HL57818

Genetic modifiers of hypertension in soluble guanylate cyclase α1–deficient mice

Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling

Adiponectin Decreases Pulmonary Arterial Remodeling in Murine Models of Pulmonary Hypertension

Remodeling of the pulmonary arteries is a common feature among the heterogeneous disorders that cause pulmonary hypertension. In these disorders, the remodeled pulmonary arteries often demonstrate inflammation and an accumulation of pulmonary artery smooth muscle cells (PASMCs) within the vessels. Adipose tissue secretes multiple bioactive mediators (adipokines) that can influence both inflammation and remodeling, suggesting that adipokines may contribute to the development of pulmonary hypertension. We recently reported on a model of pulmonary hypertension induced by vascular inflammation, in which a deficiency of the adipokine adiponectin (APN) was associated with the extensive proliferation of PASMCs and increased pulmonary artery pressures. Based on these data, we hypothesize that APN can suppress pulmonary hypertension by directly inhibiting the proliferation of PASMCs. Here, we tested the effects of APN overexpression on pulmonary arterial remodeling by using APN-overexpressing mice in a model of pulmonary hypertension induced by inflammation. Consistent with our hypothesis, mice that overexpressed APN manfiested reduced pulmonary hypertension and remodeling compared with wild-type mice, despite developing similar levels of pulmonary vascular inflammation in the model. The overexpression of APN was also protective in a hypoxic model of pulmonary hypertension. Furthermore, APN suppressed the proliferation of PASMCs, and reduced the activity of the serum response factor–serum response element pathway, which is a critical signaling pathway for smooth muscle cell proliferation. Overall, these data suggest that APN can regulate pulmonary hypertension and pulmonary arterial remodeling through its direct effects on PASMCs. Hence, the activation of APN-like activity in the pulmonary vasculature may be beneficial in pulmonary hypertension