Pulmonary arterial hypertension (PAH) is a devastating disorder. An increase in pulmonary artery (PA) stiffness is a strong predictor of mortality in patients with PH. During blood flow, shear stress-induced endothelial cell (EC) signaling related to nitric oxide (NO), calcium (Ca2+) and ATP appears to play important roles in vascular smooth muscle cell stiffness that contributes to arterial stiffness. Little is known how local shear stiffness of the endothelial layer influences ATP production, ATP release, NO and Ca signaling because it has not been possible to measure the local stiffness of ECs during simulated blood flow. Since cell stiffness is related to subcortical actin tension, our hypothesis s that in pulmonary hypertension, individual pulmonary arterial ECs with increased stiffness act as seeds for local vasoactive mediator signaling which reduces ATP production and release through direct mechanotransduction to the mitochondria via the subcortical actin and microtubules. We propose to test this hypothesis with the following aims using ECs from normal and GATA-6 null mice with PAH:
Aim 1 : Develop an in vitro method to measure the shear stiffness of ECs in culture using specific quantum dots during simulated blood flow.
Aim 2 : Correlate the topographical distributions of shear stiffness with intracellular Ca2+ and NO, mitochondrial ATP production and ATP level, to identify biological links between EC stiffness, Ca2+ and vasoactive mediators.
Aim 3 : Determine the roles of cellular mechanical components in shear stress-induced NO, ATP and Ca2+ signaling through selective inhibition of subcortical actin and microtubules. We have built a microfluidic chamber to expose cells in culture to controlled shear stress while imaging intracellular structures. We have also developed a novel concept that allows us compute the shear stiffness of individual cells by computing shear stress along cells and imaging of displacements of fluorescent beads bound to cell surface. We will use quantum dots to measure nm-scale bead displacements to estimate the stiffness of individual cells. We have successfully labeled cells under flow for various intracellular structures. We have also visualized mitochondrial inner membrane voltage as a surrogate of ATP production, during biaxial stretch of cells in culture and showed that mitochondrial mechanotransduction is critically dependent on subcortical actin and microtubule organization. The significance of these studies is that our new device will open new potentials to test ideas related to cell stiffness and signaling that have not been possible. Additionally, if our hypothesi is confirmed, we will have much better mechanistic underpinnings of the early events in shear stress related EC signaling in PAH. If this 2-year project is successful, we will use the results a preliminary data for a full R01 application. We will extend the methodology to visualize intracellular structures and measure cell stiffness of PA tissue strips from normal and hypertensive rats under physiological pulsatile blood flow waveforms and test the interaction of shear stress-induced EC signaling, vascular smooth muscle contraction and longer-term vascular wall remodeling.
Pulmonary hypertension is a devastating disorder with high morbidity and early mortality. The proposed studies will introduce a novel technique to elucidate the relation between arterial wall stiffness, a strong predictor of disease outcome, and several signaling molecules known to play a role in hypertension under simulated blood flow conditions.
