A Correlative, Three‐dimensional Approach to Studying Coronary Collateral Growth Using Lineage Tracing, Micro‐computed Tomography and Multiphoton Imaging in a Mouse Model of Repetitive Ischemia

Abstract

Coronary heart diseases (CHD) are the leading cause of mortality and morbidity in the United States. Inherent coronary collateral network in the heart represents a native circulation that is capable of undergoing tremendous abluminal expansion to provide flow to an ischemic area of the myocardium. A well‐developed coronary collateral circulation ameliorates the consequences of CHD, reducing the incidence of sudden death and infarct size following coronary occlusion. Stimulation of coronary collateral growth (CCG) is an alternative therapeutic approach for patients with intractable angina pectoris who are not indicated for percutaneous coronary intervention and/or coronary artery bypass grafting. Even though the importance of CCG is undisputable, the mechanism underlying the CCG is not clear, such as its time course and sequence of process, critical cell type(s) and genes/factors. We developed a mouse model of CCG by repetitive ischemia (RI) and validated the CCG with contrast echo to measure the coronary blood flow and 3D imaging using micro‐CT (μCT) to quantitate the CCG. Figure 1B shows the micro‐CT images of grown coronary collaterals after RI while Figure 1A shows no native coronary collaterals in mouse heart. Moreover, we adopted lineage tracing to dissect the process of CCG. We crossed lineage tracing (ROSA mT/mG floxed) mice with tissue specific cre expressing lines (endothelial cells, smooth muscle cells, inflammatory cells, etc.) and these mice were subjected to RI surgery and protocol. Mice were echoed for blood flow measurement and sacrificed at various time‐points. Mouse hearts were harvested for micro‐CT or multi‐photon fluorescent imaging. Tiled and stitched z‐stacks of heart slices containing the growing collaterals were acquired for fluorescent target cells as well as vasculature. Figure 2 shows vasculature of a ROSA‐tie2‐GFP mouse heart by multiphoton without RI. Also, datasets from μCT and multiphoton modalities could be merged in Amira™ so that the final 3D reconstruction included precise locations of lineage cells within the growing collaterals. Post‐imaging, the heart slices could also be embedded and sectioned for fluorescent IHC for proliferation and other proteins critical for arteriogenesis. In conclusion, we have developed a method for exploring the process of CCG in greater detail at the molecular and cellular level in a mouse CCG model. Understanding this process at such level could provide a therapeutic strategy to induce CCG in ischemic heart diseases.Support or Funding InformationThe research is funded by National Institutes of Health grant 2R01HL103227‐05 (YZ, LY), 1R01HL135110‐01 (WMC, LY), 1R01 HL137008‐01A1 (LY), 1R15HL115540‐01 (LY) and 14BGIA18770028 from American Heart Association (LY).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.