Abstract
Endothelial cells (ECs), traditionally known for their pivotal role in blood pressure regulation, are increasingly understood as key players in metabolic regulation. This thesis explores a multitude of ways in which ECs can affect systemic metabolism with the hopes to uncover novel mechanism for therapeutically targeting the vasculature in cardiovascular disease and metabolic syndrome. Specifically, I focus on adipose ECs as they are the main site of lipid uptake due to their proximity to adipocytes. The work presented here can be broken up into EC lipid uptake (Chapter 2), handling of fatty acids by endothelium (Chapter 3), and EC-adipocyte communication (Chapter 4). Additionally, data from a brief study on the role of a key metabolic gene, FTO, in vascular smooth muscle cells (VSMCs) is shown in Chapter 5. The guiding theme throughout the data presented here is the vascular endothelium impacts systemic metabolism.
The work presented in Chapter 2 shows a novel mechanism involving the modification of EC CD36 by nitric oxide (NO) and the regulation of lipid uptake. Nitrosation of CD36 on cysteines 3 and 466 inhibits palmitoylation of those same cysteines. Palmitoylation of cysteines 3 and 466 is necessary for proper membrane trafficking of CD36. Therefore, nitrosation inhibits CD36 localization to the plasma membrane and decreases lipid uptake into endothelium. This mechanism is regulated by Caveolin1 (Cav1) as it is a negative regulator of endothelial nitric oxide synthase (eNOS) the main producer of NO in ECs. With HFD Cav1 expression decreases, increasing NO production and increasing the nitrosation of CD36. We propose this is an inherent cellular mechanism aimed at protecting ECs from lipotoxicity induce by excess fatty acid accumulation.
Chapter 3 outlines the heterogenous transcriptomic response in ECs from the mesenteric and adipose vasculature with an obesogenic diet. We discovered the response of ECs to the insult of HFD is based on their vascular type (i.e. artery, capillary, vein, lymphatic) and tissue of origin. EC heterogeneity is an emerging topic in the vascular field and this data, with a single cell resolution, adds to the idea that not all ECs have the same transcriptomic profile. We show under physiological conditions adipose arterial and capillary ECs have a higher expression of genes involved in mitochondrial respiration and a higher mitochondrial content compared to mesenteric ECs. However, Chapter 3 contains data showing upon lipid loading during HFD arterial and capillary ECs from adipose tissue lose these key mitochondrial gene transcription signatures. Contrary to adipose, mesenteric ECs from arteries and capillaries remain unaltered by HFD. Cumulatively this data shows HFD decreases tissue specific EC heterogeneity. It also suggests adipose ECs are faced with the main lipid uptake burden as they are the most affected by HFD.
The data shown in Chapter 4 presents a novel signaling axis between capillary adipose ECs (CaECs) and adipocytes. We show that ECs and adipocytes make heterocellular contact in vitro and in vivo. This contact is facilitated by Cx43 gap junctions, which in HFD are phosphorylated and closed decreasing communication between ECs and adipocytes ultimately dysregulating lipid handling. Using a combination of in vitro method development and novel Cx43 mutant mice (Cx43S368A developed by Dr. Scott Johnstone at Virginia Tech) we were able to test the effect of EC Cx43 on systemic metabolic parameters. We show that loss of EC Cx43 increases whole body and epididymal fat pad mass as well as serum triglycerides and cholesterol. This chapter serves as further evidence for the crucial role in which endothelium can regulate metabolism.
Chapter 5 turns away from ECs and focuses on understanding the role of FTO in SMCs. Previous work by the lab had established loss of EC FTO to antagonize obesity induced metabolic and vascular dysfunction. We hypothesized that loss of SMC FTO would have a similar phenotype to loss of EC FTO. The phenotypes between loss of EC and loss of SMC FTO could not be more opposite. SMC FTO null mice were spontaneously hypotensive and lacked appropriate myogenic tone. We discovered this loss of myogenic tone to be due to a loss of key SMC contractile genes (Srf, Myocd, Tagln, Acta2). Due to the m6A RNA demethylase activity of FTO we hypothesized FTO to be an upstream master regulator of SRF, the key transcription factor influencing SMC function. By stabilizing or destabilizing SRF RNA via m6A methylation, FTO can control the amount of SRF RNA present and subsequently its transcriptional activity. This chapter shows how a gene, FTO, which was previously thought to have solely metabolic functions can actually regulate arterial function and systemic blood pressure.
Lastly, chapter 6 is a culmination of how type 2 diabetes medications and insulin-based therapies act on the endothelium. The data included in this chapter was collected in collaboration with Uta Erdburgger and Steve Malin at UVA. The key finding from this chapter is that when healthy 3rd order mesenteric arteries are perfused with extracellular vesicles (EVs) from patients with metabolic syndrome, the vessels have a diminished dilatory response to insulin compared to healthy patient EV controls. First, this data shows how ECs are insulin responsive and implicates insulin resistance as a potential mechanism for hypertension associated with metabolic disease. Secondly, this data points to EVs as primary orchestrators of insulin signaling. When considering the contribution ECs have to systemic metabolism understanding the endothelium’s response to insulin is crucial as insulin sensitivity is a primary determinate of metabolic function.
