Occlusive arterial disease remains the leading cause of mortality and morbidity in Americans and constitutes a tremendous financial burden to society. Arterial occlusion results in arteriogenesis, a process by which small collateral arterioles remodel into larger conduit arteries that reroute blood and improve flow to the ischemic tissue. The capacity of pre-existing collaterals and their growth following occlusion is strongly modulated by genetic background in both humans and mice, resulting in a wide range of outcomes. Increased hemodynamic shear stress in these collateral vessels promotes arteriogenesis but the molecular pathways and mechano-responses mediating this growth are not well understood. Notch receptor and ligand loss-of-function studies demonstrate that the Notch signaling pathway is necessary for arteriogenesis of pre- existing vessels. The goal of our study is to elucidate the mechanism by which endothelial Notch signaling is regulated after arterial occlusion and to establish a role for Notch signaling in enhancing arteriogenesis of cerebral collaterals after occlusive injury. We observe decreased cerebral arteriogenesis after arterial occlusion in mice with genetic ablation of Notch signaling in endothelial cells (ECs). Conversely, we observe impressive enlargement of cerebral collaterals following arterial occlusion in mice with expression of constitutively active Notch4 (Notch4*) in ECs. Our preliminary results suggest that arteriogenesis of cerebral collaterals occurs specifically in vessel segments that deliver increased flow to the region of injury. We hypothesize that increased hemodynamic shear stress activates endothelial Notch signaling, and that Notch is a necessary and potent enhancer of shear-induced arteriogenesis.
In aim 1, we develop methods to dynamically study cerebral collateral enlargement and blood flow in the same animals over time by combining a surgical model for middle cerebral artery (MCA) ligation and intravital two-photon excited fluorescence microscopy. We generate new analytical methods to quantify hemodynamics and WSS with high accuracy and improvement over current approximations.
In aim 2, we will determine if Notch signaling in ECs is activated by shear stress. We will use an in vivo reporter of canonical Notch signaling and immunofluorescence to determine when Notch is activated in endothelium after MCA ligation. We will determine whether Notch activation is limited to vessels with increased WSS, which we have observed is correlated with arteriogenesis.
In aim 3, we will determine whether Notch signaling in ECs controls cerebral arteriogenesis through a shear-responsive program. Specifically, we will determine whether Notch signaling in ECs is critical for arteriogenesis after MCA ligation. We will also determine whether Notch4* in ECs is sufficient to enhance arteriogenesis after MCA ligation and if elevated WSS is also required. Upon completion of this project, we will have advanced the mechanistic understanding of Notch-mediated arteriogenesis. Knowledge gained from this study will help therapeutic development for disease associated with cerebral arterial occlusion.
Occlusive arterial disease, including stroke and heart attack, is the leading cause of mortality and morbidity in Americans. There is significant interest in developing molecular therapy to stimulate blood vessel growth and return blood flow to dying tissue. Our research may elucidate an important biological mechanism that controls artery growth, helping in the development of molecular treatments for occlusive arterial disease.
|Murphy, Patrick A; Kim, Tyson N; Huang, Lawrence et al. (2014) Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels. Proc Natl Acad Sci U S A 111:18007-12|
|Kim, Tyson N; Goodwill, Patrick W; Chen, Yeni et al. (2012) Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One 7:e38590|
|Murphy, Patrick A; Kim, Tyson N; Lu, Gloria et al. (2012) Notch4 normalization reduces blood vessel size in arteriovenous malformations. Sci Transl Med 4:117ra8|