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Advanced Imaging and Bioengineering Approaches to Cardiovascular Disease

  • Author(s): Packard, Rene Rupen Sevag
  • Advisor(s): Hsiai, Tzung K
  • et al.
Abstract

Light Sheet Fluorescence Microscopy enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin chemotherapy treatment. We incorporate light-sheet fluorescent microscopy and pulsed-wave Doppler ultrasound to unravel the 3D architecture and electromechanical coupling of doxorubicin-induced cardiac injury and regeneration in the adult zebrafish model. 3-month old zebrafish were injected intraperitoneally with doxorubicin followed by imaging at 3, 30, and 60 days post-injection. We observed an initial decrease in myocardial and endocardial cavity volume at day 3, followed by ventricular remodeling and hypertrabeculation at day 30, and normalization at day 60. Doxorubicin-injected fish developed ventricular diastolic dysfunction evidenced by elevated E/A ratios at day 30, normalizing at day 60. Myocardial performance indexes were also elevated at day 30 in the doxorubicin group, indicating worsening of global cardiac function, followed by normalization at day 60. qRT-PCR to investigate the pathways involved revealed up-regulation of Notch signaling genes, particularly the ligand Jagged1 and target gene HEY2 at days 30 and 60. Treatment with the γ-secretase inhibitor DAPT to inhibit Notch signaling attenuated restoration of ventricular function, demonstrated by persistence of abnormal E/A ratios and myocardial performance indexes at day 60, thereby implicating Notch pathways in the cardiac regeneration process. Our results suggest that doxorubicin-induced cardiac injury leads to ventricular remodeling, followed by activation of Notch signaling to promote hypertrabeculation and restoration of cardiac function.

Next, we developed and studied invasive, and non-invasive imaging approaches to characterize atherosclerotic plaques. Four-point electrode systems are commonly used for electric impedance measurements of biomaterials and tissues. We introduced a 2-point system to reduce electrode polarization for heterogeneous measurements of vascular wall. Presence of endoluminal oxidized low density lipoprotein (oxLDL) and lipids alters the electrochemical impedance that can be measured by electrochemical impedance spectroscopy (EIS). We developed a catheter-based 2-point micro-electrode configuration for intravascular deployment in New Zealand White rabbits. An array of 2 flexible round electrodes, 240 �m in diameter and separated by 400 �m was microfabricated and mounted on an inflatable balloon catheter for EIS measurement of the oxLDL-rich lesions developed as a result of high-fat diet-induced hyperlipidemia. Upon balloon inflation, the 2-point electrode array conformed to the arterial wall to allow deep intraplaque penetration via alternating current. The frequency sweep from 10 – 300 kHz generated an increase in capacitance, providing distinct changes in both impedance (Ohm) and phase (degree) in relation to varying degrees of intraplaque lipid burden in the aorta. Aortic endoluminal EIS measurements were compared with epicardial fat tissue and validated by intravascular ultrasound and immunohistochemistry for plaque lipids and foam cells. Thus, we demonstrate a new approach to quantify endoluminal EIS via a 2-point stretchable electrode strategy.

In the next study, a new absolute quantitation of myocardial blood flow (MBF) method with the novel positron emission tomography (PET) 18F-Flurpiridaz radiopharmaceutical was developed, taking advantage of its early kinetics and high first-pass extraction by the myocardium. We performed the first in human measurements of MBF in 7 normal subjects and 8 patients with documented coronary artery disease (CAD). PET images with time-activity curves were acquired at rest and during adenosine stress. In normal subjects, regional MBF between coronary artery territories did not differ significantly, leading to a mean global MBF of 0.73 mL/min/g at rest and 2.53 mL/min/g during stress, with a mean global myocardial flow reserve (MFR) of 3.70. CAD vascular territories with <50% stenosis demonstrated a mean MBF of 0.73 at rest and 2.02 during stress, leading to a mean MFR of 2.97. CAD vascular territories with ≥50% stenosis exhibited a mean MBF of 0.86 at rest and 1.43 during stress, leading to a mean MFR of 1.86. Differences in stress MBF and MFR between normal and CAD territories, as well as between <50% and ≥50% stenosis vascular territories, were significant. The significant decrease in stress MBF and ensuing MFR in CAD territories allows a clear distinction between vascular territories exhibiting stress-inducible myocardial ischemia and those with normal perfusion using 18F-Flurpiridaz PET.

In the last study, we studied the ability of fractional flow reserve by computerized tomography (FFR-CT) to predict subsequent coronary revascularization. FFR-CT provides non-invasive functional assessment of the hemodynamic significance of coronary artery stenosis. We determined the FFR-CT values, receiver operator characteristic curves and predictive ability of FFR-CT for actual standard of care guided coronary revascularization. Consecutive outpatients who underwent coronary CT angiography (coronary CTA) followed by invasive angiography over a 24-month period from 2012-2014 were identified, sent for FFR-CT analysis, and results stratified by coronary artery calcium (CAC) scores. Coronary CTA studies were re-interpreted in a blinded manner, and baseline FFR-CT values obtained retrospectively. Therefore, results did not interfere with clinical decision-making. Median FFR-CT values were 0.70 in revascularized (n=69) and 0.86 in not revascularized (n=138) coronary arteries. Using clinically established significance cutoffs of FFR-CT ≤0.80 and coronary CTA ≥70% stenosis for the prediction of clinical decision making and subsequent coronary revascularization, the positive predictive values were 74% and 88%, and negative predictive values 96% and 84%, respectively. The area under the curve (AUC) for all studied territories was 0.904 for coronary CTA, 0.920 for FFR-CT, and 0.941 for coronary CTA combined with FFR-CT. With increasing CAC scores, the AUC decreased for coronary CTA but remained higher for FFR-CT. The addition of FFR-CT provides a complementary role to coronary CTA and increases the ability of a CT-based approach to identify subsequent standard of care guided coronary revascularization.

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