Cardiovascular diseases (CVD), associated with dysfunction of endothelial cells (ECs), are the leading cause of mortality worldwide, and abdominal aortic aneurysm (AAA) is one of the deadliest. More than many other cells, ECs are critically reliant on heme availability to maintain mitochondrial respiration and metabolic homeostasis.
The impairment of heme synthesis has recently been recognized as one of the EC defects' primary drivers. On the other hand, free heme can be toxic, and it is degraded by heme oxygenases (HO1 and HO2). Our RNAseq data suggest that primary ECs lacking HO1 have altered cellular metabolism and deregulated cell cycle. Intriguingly, we observed a lower incidence of Ang II-induced AAA in HO1-deficient mice. Therefore, we hypothesize that increased availability of heme in endothelial cells reshapes cellular metabolism and thus makes blood vessels less prone to injury.

 

The main aim of the project is to verify this hypothesis and to answer the following questions:

1. Which cells are the most important for heme-dependent protection against AAA formation?

2. Does the decreased heme degradation rate take the strain off vessel wall cells?

3. Can we prevent AAA formation with supplementation with heme precursors?

4. Does the supplementation with heme precursors change levels of free heme in the blood vessel wall?

 

We will answer those questions with a set of complementary experiments in vitro and in vivo. We observed lower AAA incidence in mice with total HO1 knockout in comparison to wild type animals. To answer the first question, we will analyze AAA formation in mice with endothelial cell-specific or smooth muscle cell-specific knockout of HO1 by using Hmox1fl/fl mice. To evaluate the influence of decreased heme degradation in hematopoietic cells, we will transplant Hmox1-/- bone marrow to wild-type recipients. We found that human primary endothelial cells with decreased heme degradation had unchanged total heme levels but downregulated heme synthesis enzymes. To answer the second question, first, we will check the levels of free heme in cells with a low heme degradation rate. To do so, we will use a genetically encoded FRET-based heme sensor system in cytoplasm and mitochondria. Furthermore, we hypothesize that EC and smooth muscle cells with decreased heme degradation have reshaped cellular metabolism. To test that, we will use metabolomics and transcriptomics. We will also apply routine functional assays to test mitochondrial function in primary cells with low heme degradation. Using 13C-glucose and glycine, we will evaluate changes in metabolic flux and the contribution of both metabolites to cellular metabolism and heme synthesis in cells with lower heme degradation. We will also attempt to analyze endothelial metabolism in situ in mouse and human AAA samples using metabolomic imaging. We suspect that heme can affect cellular metabolism via the eIF2ak1-eIF2α-ATF4 axis. In our in vitro setting, we will test that hypothesis in primary human endothelial cells with specific inhibitors and silencing of eIF2α phosphorylation repressors. Aortic dissection patients have lower serum levels of both glycine and serine. Therefore, to answer the third question, we will test if heme precursors, e.g., δ-aminolevulinate or metabolites selected in earlier tasks, can decrease AAA formation in animals with normal levels of heme degradation. Finally, there is no available animal model that would allow us to analyze levels of free heme in tissues. Therefore, it is impossible to precisely assess if supplementation with heme precursors would increase free heme in vivo. To that end, we will generate the mouse model with the FRET-based free heme sensor's expression.

 

Understanding of the molecular mechanisms behind cardiovascular diseases is quintessential for the development of new therapies. We believe that free heme alters cellular metabolism, regulates endothelial and smooth muscle cells' function, and renders them more resistant to injury. In this project, we will fill the gap in the knowledge on the role of heme in endothelium and evaluate if the maintenance of a constant heme level in vascular cells is more important for a healthy vessel than a rapid heme removal.