Calcium calmodulin dependent protein kinase II in the development of atherosclerosis
|dc.contributor.author||Worthington, Luke Philip Ian|
|dc.identifier.citation||Worthington, L. P. I. (2021). Calcium calmodulin dependent protein kinase II in the development of atherosclerosis (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/11921||en|
|dc.description.abstract||Atherosclerosis is the leading cause of death in the developed world. The accumulation of low-density lipoprotein cholesterol (LDL-C) in the arterial wall leads to the formation of foam cell lesions. The growth of lesions is influenced by a number of genetic and lifestyle factors. Over-time, lesions encroach the vessel lumen, impeding blood flow and increasing risk of cardiovascular events including myocardial infarction and stroke. Statins have been effective at reducing risk for cardiovascular events, however, even when target LDL-C levels are met there remains a significant residual risk. Clearly, a detailed investigation into the mechanism of foam cell lesion growth is required to explore new drug targets to ease the burden on the health care system. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a nodal signalling protein in endothelial cell (EC) and vascular smooth muscle cell (VSMC) physiology. In vascular disease, CaMKII has been shown to promote endothelial dysfunction and vascular smooth muscle cell proliferation. Both of these pathologies are involved in the early stages of atherosclerosis so it is intriguing to speculate that CaMKII also contributes to atherosclerosis. We have previously shown systemic inhibition of CaMKII in an atherosclerotic model (ApoE-/- mice) reduces foam cell lesion development. However, there are multiple isoforms of CaMKII and associated splice variants. These sub-types are differentially expressed in the tissues of the body and have shown contrasting roles. A global inhibition of CaMKII is not a feasible target due to the important physiological functions of these multiple isoforms. Therefore, it is crucial we identify the isoform(s) contributing to foam cell lesion growth so a more specific therapeutic target can be explored. Firstly, the predominant isoforms of CaMKII were identified in the major cell types of the vasculature. The aortic tree was dissected from ApoE-/- mice at 13- (early atherosclerosis) and 20-weeks (mid-atherosclerosis). In addition, human umbilical vein endothelial cells (HUVECs), human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) were cultured. PCR and Western blotting was run for CaMKII delta and gamma isoforms. Results showed CaMKII delta as the predominant isoform in human and mouse vascular cells. In addition, at the mRNA level, delta splice variants 2, 3 and 6 were detected. CaMKII delta expression was high at 13-weeks in the ApoE-/- mouse and levels persisted at 20-weeks, suggesting the delta isoform is the most likely isoform contributing to atherosclerosis. To investigate if CaMKII signalling contributed to foam cell lesion development, we next employed a genetic approach. ApoE-/-‑mice were crossed with CaMKIIdelta-/- to generate a novel ApoE-/-CaMKIIdelta-/- (dKO) mouse model. At 20-weeks there was extensive atherosclerosis in the aortic sinus of female, but not male groups. Histological analysis showed there was a strikingly significant reduction in foam cell lesion content in female ApoE-/-CaMKIIdelta-/-, compared to ApoE-/-CaMKIIdelta+/+ litter mate controls. This indicates the CaMKII isoform is a critical player in the early development of atherosclerosis. To further test the mechanistic role of CaMKII delta in foam cell lesion development, an opposite strategy was performed whereby an AAV-mediated overexpression approach was utilised. ApoE-/-CaMKIIdelta-/- mice were put on a high-fat diet at 12-weeks to accelerate atherogenesis. At 16-weeks of age, the left carotid arteries of ApoE-/-CaMKII-/- mice were ligated to increase shear stress and further promote foam cell lesion growth in a localised area. AAV-particles harbouring the CaMKIIdelta2 or control (scrambled) gene sequence were then introduced to the ligated left carotid artery. Ligation of the carotid artery led to a range of variation in foam cell lesion progression. Histological analysis of the carotid artery revealed the foam cell lesion area and stenosis from CaMKIIdelta2-mCherry mice showed a trend towards an increase, despite low n numbers due to adverse complications in 20% of the ligated mice. In summary, a number of genetic and molecular biological techniques have been used to provide evidence to show CaMKII contributes to the early stages of atherosclerosis. Importantly, there is still work that needs to be done to further validate this mechanism and the potential contribution of other delta variants and/or other isoforms. This novel study has provided foundation evidence of CaMKII as a specific target for treatment. The results provide impetus to continue the investigation of CaMKII that ultimately, could lead to a pharmacological intervention to treat the early progression of atherosclerosis.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Calcium calmodulin dependent protein kinase II in the development of atherosclerosis|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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