Date of Award
Doctoral Dissertation - Restricted (NYMC/Touro only) Access
Doctor of Philosophy
Michael S. Wolin, Ph.D.
Carl I. Thompson, Ph.D.
Dong Sun, M.D., Ph.D.
Pulmonary Hypertension (PH) is a complex disorder with a prominent feature of increased vascular resistance and elevated pulmonary arterial blood pressure (PAP), leading to right ventricular hypertrophy and failure. It can result from various underlying conditions that make controlling the disease difficult and reflect poorly on the patient’s prognosis. Altered redox state is associated with all forms of PH and can trigger pathological pathways influencing vascular tone and remodeling. Understanding the mechanical approach at which increased ROS act to facilitate the progression of the disease can serve as a target for future treatments. The effect of changes in extracellular matrix structure (ECM) on regulating the vascular intracellular signaling is poorly understood. Hence, we hypothesized that PH mediators potentiate the release of superoxide from pulmonary arterial smooth muscle cells and induce a pathological loop inducing MMP9 and degrading matrix proteins that have an essential role in controlling vascular hemostasis. Matrix metalloproteinase 9 (MMP9) is an inducible enzyme belongs to end proteinases family and has been shown to increase in many pathological conditions. MMP9 mediates its actions by proteolytic remodeling of the basement membrane and by releasing extracellular matrix (ECM) bound proteins leading to modulation of intracellular signaling. To test our hypothesis, we used Sugen in combination with chronic hypoxia mice served as our animal model of PH in wild type and MMP9 knockout mice in addition to bovine pulmonary arteries, and human pulmonary arterial smooth muscle cells (PASMC) to perform our experiments. The hemodynamic measures, as shown by an increased right ventricular systolic pressure and a reduced pulmonary artery acceleration time to ejection time ratio confirmed the establishment of PH in our animal model. These measures were associated with an increased right ventricular hypertrophy and significant increases in MMP9 expression in the lung tissues. In comparison to Sugen/Hypoxia (SuHx) wild type xxiii mice, MMP9 KO mice showed an attenuated effect with less elevation of right ventricular systolic pressure and partial restoration of PAP values. Vascular reactivity studies showed that SuHx mice were presented with impaired endothelium-dependent and endothelium-independent vasodilatory responses. To examine if superoxide is contributing to the defective vasodilatory response, we investigated the effect of superoxide dismutase (SOD) and TEMPO on Acetylcholine and NONOate relaxation responses, respectively. Our findings showed only TEMPO seem to partially improve NONOate vasodilation. Furthermore, the loss of MMP9 partially enhanced Acetylcholine relaxation responses at larger doses with more prominent effects on the NONOate relaxation responses. MMP9 can degrade essential proteins that function to protect the integrity of vascular cells. In this thesis, we focused on examining the relationship between MMP9 and cartilage oligomeric matrix protein (COMP) considering its role in maintaining the contractility of pulmonary arteries. COMP was found to be depleted in SuHx animal model, while SuHx MMP9 KO mice showed preserved COMP expression suggesting that MMP9 participated in COMP depletion in this animal model. To further understand the consequences of COMP depletion, we incubated endothelium-denuded bovine pulmonary arteries (BPA) with the PH mediator Endothelin-1(ET1). ET-1 induced reductions in heme levels and impairment of NONOate vasodilatory responses in BPA. Incubation with COMP protected against ET-1-induced decreases in Guanylate Cyclase and Ferrochelatase expression. Impaired function of bone morphogenetic protein type 2 receptor (BMPR2) has been linked to PH, hence, we examined the interaction between MMP9 and BMPR2. Lungs from SuHx mice showed a significant decrease in BMPR2 expression that was mitigated in MMP9 KO mice. MMP9 is hypothesized to deplete BMPR2 via the removal of a previously reported COMP-dependent stabilization of BMPR2. Treatment with ET-1 significantly increased mitochondrial superoxide levels concomitant with reduced expression of the mitochondrial antioxidant enzymes and increased PASMC proliferation. These detrimental effects seem to be mediated by a depletion of BMPR2 induced by ET-1, considering that treatment with FK506, a BMPR2 stabilizer, reversed xxiv these effects. While superoxide is known to be an essential factor in PH, specific aspects of its mechanisms remain to be defined. Our results showed that extracellular superoxide levels were elevated in lungs and pulmonary arteries from SuHx mice that could function to potentiate the induction of MMP9 levels without changes in the endogenous MMP9 inhibitor, TIMP1, or in extracellular SOD expression. In BPA treated PH mediators, superoxide levels were significantly increased concomitant with increased MMP9 levels which was decreased when BPA were incubated in combination with superoxide scavenger or NADPH oxidase-2 (NOX2) inhibitor. Collectively, these findings expand our understanding of roles for superoxide and matrix reconstitution in PH, and suggest a superoxide-induction of a pathological loop of increased MMP9 and reduced COMP and BMPR2 levels in pulmonary arteries. Targeting this loop can serve as a potential therapeutic approach to protect against PH development.
Alruwaili, Norah, "Superoxide as a Driver of Pulmonary Hypertension in the Regulation of Matrix Proteins and Vascular Function" (2020). NYMC Student Theses and Dissertations. 45.