Abstract
Accurate control of vasodilatory signals is critical to the maintenance of blood pressure in mammals. One mechanism whereby vascular endothelium can control the diameter of the vessel is through nitric oxide (NO) signaling, a small gaseous molecule that is endogenously produced in endothelium and diffuses to smooth muscle. NO synthesis is tightly regulated through endothelial NO synthase (eNOS), the main enzyme contributing to NO production. One recently-discovered regulator of NO signaling is endothelial expression of a hemoglobin, specifically alpha globin (without its beta chain partner). Alpha globin binds directly to eNOS and, through its prosthetic heme group, can scavenge NO at the source of production. Disrupting the interaction of alpha globin and eNOS is a druggable goal with implications in anti-hypertensive therapy. The work presented in this thesis is focused on understanding the signaling and molecular interactions involved in, and the physiological impacts of, endothelial alpha globin regulating of vascular NO signaling.
The research presented in chapter 2 focuses on understanding the signals that induce endothelial alpha globin expression. Forcing endothelial contact with smooth muscle in an artery is sufficient to cause alpha globin production. The production of alpha globin is needed to change the vasodilatory mechanism by decreasing the proportion of dilation signals that come from NO. In chapter 3, I show that displacing alpha globin from eNOS increases NO signaling in the resistance vasculature. Using a peptide that mimics the region of alpha globin that binds with eNOS (named HbαX), we can disrupt alpha globin/eNOS complex formation to increase perfusion and decrease systemic blood pressure. This HbαX peptide binds directly to eNOS and has therapeutic relevance for hypertensive disease states.
The research presented in chapter 4 was completed during an internship at Heinrich Heine Universität and focuses on the role of NO in protecting the intrinsic deformability of red blood cells and, thus, blood pressure regulation. Reactive oxygen species can modify the red blood cell cytoskeleton and cause an increase in rigidity, which was correlated with hypertension in humans. I show that NO acts to protect these cells from increased reactive oxygen species through increasing the red blood cell antioxidant capacity and, therefore, protects the cells from increased rigidity.
Another disease context with dysfunctional NO signaling is pulmonary hypertension, which is the focus of chapter 5. Using a model of hypoxia-induced hypertension and our HbαX peptide to increase NO availability, we hypothesized that pulmonary hypertension could be alleviated by increasing dilatory signals using the HbαX peptide. Interestingly, the chronic administration of HbαX seemed detrimental to the pulmonary tissue. We observed increased nitrosative stress that damaged lung tissue downstream of increasing NO, rather than a vasodilatory response and alleviating pulmonary hypertension. Although the predicted increase in NO was observed with HbαX, the physiological consequence was not the one predicted, thus reminding us that the total physiological context of the disease state is more complex than one regulatory interaction.
In order to further our knowledge of how alpha globin can regulate eNOS and NO signaling, an understanding of the molecular interactions determining the alpha globin/eNOS complex using recombinant proteins was pursued and described in chapter 6. After attempting docking of the two protein structures in silico, the experimental approach of crosslinking mass spectrometry was established to define the residues on eNOS that interact with alpha globin. Work in this area is still ongoing, but will provide valuable context for how NO is controlled in the endothelium by this protein-protein interaction.
Finally, in chapter 7, research focused on an engineered a mouse model harboring a deletion within the sequence of alpha globin that binds to eNOS is described. This deletion decreases alpha globin and eNOS association in the endothelium and increases vasodilatory NO. The increase in NO impacts blood pressure homeostasis, because although the total blood pressure was normal in the mice harboring the heterozygous deletion, the dilatory capacity of individual arteries was decreased due to decreased NO-response proteins.
Overall, this work furthers the understanding of NO signaling, the role of alpha globin in controlling NO flux, and therapeutic potential of targeted disruption of the alpha globin and eNOS interaction in the vasculature.
