Hemodynamic shear force is an important determinant of cardiovascular function and development. While mammalian models combined with next regeneration sequencing are essential for diagnosis and drug discovery, zebrafish has emerged as an important developmental model that combines in vivo analyses of cardiovascular phenotypes and the advantages of forward and reverse genomic engineering. The following studies in this thesis utilize an advanced imaging-based technique to characterize hemodynamic regulation in cardiovascular development and regeneration in embryonic zebrafish. Cardiogenesis involves a series of complex signaling pathways, while imaging cellular dynamics including cardiac trabeculation requires high spatiotemporal resolution. By using four-dimensional light-sheet fluorescent microscopy, we constructed four-dimensional moving domain models of the contracting myocardial wall to investigate hemodynamic regulation of endocardial Notch signaling in facilitating cardiac trabeculation. In vivo modulations of hematopoiesis and atrial contraction or ectopic expressions of Notch Intracellular Cytoplasmic Domain (NICD), suggested distinct flow patterns in myocardial geometry differentially activate endocardial Notch activity for trabecular organization and contractile function. Vascular disorders characterized by ischemic reperfusion injury, such as stroke, myocardial infarction, and peripheral vascular disease, remain the most frequent causes of incapacitating disease and death. Despite numerous efforts, structural and functional vessel recovery remains challenging, while molecular events underlying vascular injury and regeneration are largely unknown. A plethora of epidemiological studies consistently supports a link between redox active ultrafine particles (UFP, diameter < 0.1 um) in primary pollutants and cardiopulmonary disease. Due to the small size and the light weight of the particle, aspiration of UFP allows for penetration of pulmonary systems as well as the endothelial barrier. Once particles enter the circulatory system, they systematically affect endothelial homeostasis by promoting vascular oxidative and inflammatory responses. However, epigenetic and pathological effects underlying vascular regeneration upon particle exposure remains elusive. For the second part of this thesis, we investigated the importance of the Forkhead Box Sub-family O1 (FOXO1)/Notch activation complex upon UFP exposure in vascular regeneration. Finally, our vascular protective metabolomic profiles suggested hemodynamic shear forces is a central player in modulating the expression levels of glycolytic metabolites. In this context, we investigated the Vascular Endothelial Growth Factor Receptor (VEGFR)-Protein Kinase C isoform epsilon (PKCɛ) pathway and its role in promoting pro-glycolytic metabolites to help facilitate vascular regeneration.

Hemodynamic forces contribute to mechanotransduction and metabolomic pathways underlying microvascular development, remodeling, and atherogenesis. In micro-scale and developmental stages, microvascular adaptation, and remodeling in response to injury is modulated by spatiotemporal variations in wall shear stress (WSS); however, the underlying mechanisms and interplay leading to adaptation of microcirculation and remodeling at embryonic stage remain elusive. The understanding of the interplay between low Reynolds number (stokes flow) hemodynamic variations and microvascular adaptation provides translational implications in microvascular injury and repair for human diseases with associated vascular complications, such as diabetes and peripheral artery disease. In macro-scale and adult stage, disturbed flow and low WSS promote atherosclerosis which is mitigated as a result of exercise by changing the local hemodynamics and activating metabolomic transducers underlying vascular protection in the disturbed flow-prone vasculature. We used embryonic zebrafish tail as a microcirculation model and mouse aorta as a atherosclerosis model to investigate the role of flow-mediated microvascular remodeling and atherosclerosis development at different scales. In micro-scale, using computational fluid dynamics, we elucidated the interplay between WSS and vascular remodeling in zebrafish model of tail amputation and regeneration. The transgenic Tg (fli1:eGFP; Gata1:ds-red) zebrafish line was used to track the 3-D fluorescently-labeled vascular endothelium for post-image segmentation and reconstruction of fluid domain. Micro-particle image velocimetry was used to validate the blood flow. Following amputation to the dorsal aorta (DA) and posterior cardinal vein (PCV), vasoconstriction developed in the dorsal longitudinal anastomotic vessel (DLAV) along with increased WSS in the proximal segmental vessels (SV) from amputation. Angiogenesis ensued at the tips of the amputated DLAV and PCV where WSS was minimal. At 2 days post amputation (dpa), vasodilation occurred in a pair of SVs proximal to amputation, followed by increased blood flow and WSS, whereas in the SVs distal to amputation, WSS normalized to the baseline. At 3 dpa, the increase in blood flow in the arterial SV proximal to amputation promoted anastomosis with DLAV to form a loop with PCV. Thus, our in-silico modeling revealed the interplay between WSS and micro-vascular adaptation to changes in WSS and blood flow to restore micro-circulation following tail amputation. In macro-scale, we simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. Ultrasound-based 4-D+time moving boundary computational fluid dynamics analysis was performed on mouse aorta to simulate blood flow over several cardiac cycles. Exercise modulated the time-averaged shear stress (TAWSS) and oscillatory shear index (OSI), and upregulated SCD1 and attenuated well-known VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr-/- mice on high-fat diet but not in Ldlr-/-Scd1EC-/- mice. When human aortic endothelial cells (HAECs) were subjected to PSS (τ_ave = 50 dyne�cm−2, ∂τ/∂t = 71 dyne�cm−2�s−1, 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. SCD1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2, Acox1 and Adipor2 that modulate lipid metabolic pathways. We found inverse relation between Scd1 and Vcam1 gene expression indicating the atheroprotective metabolites generated through Scd1 pathway. Taken together, exercise modulates PSS (TAWSS and OSI) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

Current methods of cardiac functional assessment such as ejection fraction (EF), strain imaging, and ventricular inflow diastology have known limitations. We developed and applied a semi-automated, open-source software tool (DIAMOND) for quantitative assessment of segmental cardiac function with applicability to physiological and chemotherapy-induced cardiomyopathy conditions. Zebrafish embryos were imaged in vivo using a light-sheet fluorescence microscopy system, and the 4-D heart synchronized to address cardiac motion. Following re-orientation and isotropic resampling along the true ventricular short axis, resampled data were saved in different matrices. The ventricle was then divided based on a virtual centerline into 8 segments. To conform to the complexity of cardiac structures and deformation, we artificially created a group of rectangular parallelepipeds for 3-D rigid registration and derivation of a transformation matrix (Tm). Tm was utilized to align the diastolic myocardium to the systolic coordinate system, thereby maintaining their relative position. Therefore, DIAMOND allowed measurement of the 3-D displacement of segmental myocardial mass centroids in the same coordinate system. Following treatment of transgenic zebrafish lines with doxorubicin and by chemically and genetically manipulating the Notch signaling pathway, we demonstrate that global EF and focal DIAMOND displacement provide complementary information, with basal segments adjacent to the atrioventricular canal displaying the highest 3-D displacement, contributing the most to EF, and being the most susceptible to chemotherapy-induced cardiac injury. By utilizing a Tp1 Notch reporter line, FUCCI transgenic line, and DIAMOND, we established a temporal sequence of Dox-induced injury, Notch activation, cardiomyocyte proliferation, trabeculation restoration, and cardiac function normalization within two days post doxorubicin treatment. In conclusion, DIAMOND provides a novel approach for focal cardiac mechanics assessment to localize, track, and quantify segmental myocardial function in response to doxorubicin-induced cardiac injury and Notch signaling-medicated myocardial regeneration.

Over 1 million patients are implanted annually with a pacemaker for the treatment of cardiac arrhythmias and conduction disorders. However, despite numerous advancements in pacemaker technology, lead-related complications associated with these devices continue to compromise patient safety and survival. Wireless power transfer holds great promise for improving health outcomes in biomedical implants such as the pacemaker. However, efficient power transfer and effective operational range have remained a challenge within anatomical constraints. In this work, we demonstrated an intravascular, wirelessly-powered, batteryless, microscale pacer deployed to the anterior cardiac vein (ACV). First, we employed a novel intermittent transmission remote-control architecture with improved power efficiency to enable sufficient power transfer without compromising on Specific Absorption Rate (SAR). We then integrated this architecture into a 3-tiered, 2-sub-system, 4-coil design, which operates on two different frequencies. Our pacemaker was designed to achieve wireless power transfer over a 55 mm range from an external transmitter to an intravascular receiver. A charging sub-system was designed to transmit power over 25 mm using inductive power transfer to a subcutaneous receiver, which then charged a battery feeding the transmitter of a stimulation sub-system. We then introduced a stimulator circumferentially confined to a 3 mm diameter hollow-cylinder that delivered >5 V using wireless power transfer at 13.56 MHz, with over 20 mm transmitter-receiver displacement between the subcutaneous unit and pacer unit. Further validation was performed using Finite Element Method (FEM) simulation of the cardiac cycle, guided by anatomical variations established by Magnetic Resonance Images (MRI). Finally, we demonstrated the capacity for both ex vivo and in vivo pacing of pig hearts following pacer deployment in the ACV. This introduced, for the first time, the unprecedented capacity for wireless intravascular pacing with potential for multi-organ stimulation. Thus, the proposed system design has the potential to bypass the multitude of complications associated with pacemaker wires, repeated procedures for battery replacement, and mechanically burdened fixation mechanisms.

Cardiovascular and correlated metabolomic diseases remain the leading cause of death in the United States and around the world. Three-dimensional (3-D) and real-time imaging techniques for investigating cardiovascular physiology and pathology mechanisms remain a significant challenge. In this thesis, I first introduce advanced light-sheet microscopy that enables multi-dimensional and multi-scale imaging via illuminating specimens with a thin sheet of the laser. This imaging strategy allows rapid data acquisition with a high spatiotemporal resolution, minimal photo-bleaching, and photo-toxicity. Its multiscale capability also empowers us to image and investigate the cardiovascular physiology and pathology at the scales from zebrafish embryos to adult mouse hearts and even human organs. Secondly, the custom-build light-sheet system was established and optimized to investigate the retinas in the embryonic mouse model. By combining light-sheet with tissue-clearing and computational quantification, we revealed the 3-D retinal microvascular network including primary (inner) and secondary (outer) plexuses as well as the vertical sprouts bridging the two plexuses. Whereas, in an oxygen-induced retinopathy (OIR) mouse model, we demonstrated preferential obliteration of the secondary plexus and bridging vessels with a relatively unscathed primary plexus. Using clustering coefficients and Euler numbers, we computed the local versus global vascular connectivity. While local connectivity was preserved, the global vascular connectivity in OIR retinas was significantly reduced. Overall, the application of 3-D LSFM images coupled with computational quantification provides vascular insights into OIR, with translational significance for developing therapeutic interventions to prevent visual impairment. Thirdly, we developed electrical impedance tomography (EIT) as a non-invasive and portable detection method for fatty infiltrate in the liver. Since non-alcoholic fatty liver disease (NAFLD) is still endemic in developed countries and is one of the most common causes of cardiometabolic diseases, there is an unmet clinical need for non-invasive and cost-effective monitoring of fatty liver disease. The current gold diagnosis standard is liver biopsy which is prone to bleeding and has the risk of complications and sampling errors. Using acquired voltage data and the reconstruction algorithm for the EIT imaging, we computed the absolute conductivity distribution of the abdomen in 2-D. We performed correlation analyses to compare the individual EIT conductivity vs. MRI PDFF. Our results demonstrated that EIT conductivity (S/m) is inversely correlated with the MRI proton-density fat fraction percentage (PDFF%) in the liver. This inverse correlation holds promises for developing non-invasive and portable liver EIT for early detection of fatty liver content in overweight individuals.

Hemodynamic forces such are intimately linked with cardiac morphogenesis. Defects in genetic programming during cardiac development result in functional and functional anomalies. During heart development, the trabeculae form a network of branching outgrowths from the ventricular wall, and both trabeculation and compaction are essential for normal cardiac contractile function. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), whereas hypertrabeculation (non-compaction) is closely associated with left ventricular non-compaction (LVNC). The former condition predisposes patients to embryonic heart failure and lethality, and the latter to arrhythmia and heart failure with preserved ejection fraction. Left ventricular non-compaction (LVNC) is the third most common cardiomyopathy after dilated and hypertrophic cardiomyopathy in the pediatric population. Its prevalence was estimated from 4.5 to 26 per 10,000 adult patients referred for echocardiographic diagnosis. 1, 2 Despite these important clinical observations over the past 3 decades, the etiology of LVNC is largely unknown. While the majority of clinical studies is based on the anatomical feature of LVNC in the adult patients, there remains a poor genotype-phenotype association in LVNC patients, and a paucity of knowledge on the mechano-signal transduction of trabeculation and compaction during cardiac development. Notch signaling, and Notch pathway mediates differentiation and proliferation of trabecular myocytes. However, the mechanotransduction mechanisms underlying endocardial shear stress and the initiation of trabeculation remain elusive.

The goal of my work was to study how endocardial WSS activates Notch signaling to promote the initiation of trabeculation. Zebrafish was utilized as our model organism to study the changes in Notch signaling during trabeculation since they provide many advantages in studying these developmental phenomena, such as rapid organ development, high fecundity, and are optically transparent during early embryonic stages. Furthermore, the genes related to congenital heart disease genes in humans are conserved in zebrafish. This study will provide a deeper understanding of the mechanisms that drive of cardiac morphogenesis in zebrafish model and contribute to developing therapies for humans with CHD.

Zebrafish cardiac development can be visually assessed in transgenic zebrafish with fluorescently labeled proteins to visualize the heart or endocardium. Through a series of acquired image, we computationally reconstructed 4-dimensional (x,y,z,t) in vivo cardiac images using Selective Plane Illumination Microscopy (SPIM) using a custom synchronization algorithm. These 4-D SPIM images provided the visual assessment of zebrafish cardiac mechanics and cardiac flow, which allowed for the quantification of endocardial WSS by Computational Fluid Dynamics (CFD). Zebrafish blood viscosity, a key parameter in calculating endocardial WSS, has not been previously measured due to technical challenges with small sample size and extremely limited blood volumes. To address this issue, we have developed a capillary pressure-driven microfluidic channel to measure viscosity across a range of shear rates. This allowed us to also determine the relationship between zebrafish hematocrit and blood viscosity in our studies. We then modulated zebrafish endocardial WSS by genetically reducing blood viscosity and hematopoeisis via gata1a-MO injection to examine changes in trabeculation. We observed changes in Notch signaling related genes expressions which was supported by Tg(tp1:gfp) which provided spatial Notch signaling expression levels. Furthermore, co-injection of Nrg1 mRNA with gata1a-MO was applied to rescue Notch activation by promoting cardiac contractility induced by trabeculation. This multidisciplinary approach to study ventricular morphogenesis via Notch signaling clarifies the mechanistic understanding of NC.

Furthermore, we utilized adult zebrafish to study cardiac regeneration in addition to the embryonic model. Unlike the human heart, zebrafish hearts naturally regenerate and undergo rapid scar degradation. To study the electrical and mechanical changes in the zebrafish heart, we developed an ECG gated pulse-wave Doppler ultrasound device to monitor changes in cardiac electrical-mechanical properties during regeneration. Although zebrafish hearts become fully regenerated 90 days after injury, their cardiac electrical conduction remains damaged, as indicated by longer QT intervals in injured fish. This synchronous ECG gated pulse-wave Doppler provides synergistic information that would otherwise be undetected. This integrated approach can be further applied to assess changes in electrophysiological cardiac diseases, including arrhythmias and atrial fibrillation.

Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Fluid shear stress plays a major role in regulating endothelial homeostasis, dysfunction, and vasculogenesis. First study we demonstrated atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•−) production via NADPH oxidase and c-Jun NH2-terminal kinase (JNK) signaling. Next we revealed that OSS-mediated oxidative stress and JNK activation induced autophagy but impaired autophagic flux to promote mtO2•- production, mtDNA damage, and mitochondrial dysfunction in the disturbed flow-exposed regions. Then we demonstrated rapid and irregular pacing reduced intravascular shear stress (ISS) with implications to endothelial response modulation. Lastly, we investigated whether shear stress activated angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with implications in vascular endothelial repair. Collectively, we characterized the mechanisms whereby shear stress modulates these effects.

Hemodynamic shear stress is intimately linked with endothelial metabolic effects, regulating key mechanisms in endothelial function, homeostasis, and repair. In-vivo modulation of shear stress signaling pathways in the zebrafish model allows for identification of mechanisms with potential therapeutic implications. The following studies combine in-vivo zebrafish models with in-vitro shear stress studies to characterize the mechanisms whereby shear stress regulates vascular development and repair.

We investigated shear stress-modulated genes via microarray, and identified Angiopoietin-2 (Ang-2), a well known regulator of angiogenesis and vascular development, as a potential target for further mechanistic study. Oscillatory shear stress (OSS) induced Ang-2 mRNA expression in a Wnt-signaling dependent manner. Inhibition of Wnt signaling or Ang-2 expression supressed endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2 treatment. These results were recapitulated in the embryonic zebrafish model using heat-shock inducible transgenic Tg(hsp70l:dkk1-GFP) and Tg(kdrl:GFP) embryos injected with Ang-2 morpholino. Inhibition of Wnt signaling with IWR-1 also impaired vascular repair after tail amputation, which was rescued by injection of zAng-2 mRNA. Taken together, this data demonstrated shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and recapitulated shear stress-Wnt-Ang-2 signaling vascular repair in the zebrafish model.

Shear stress-modulated gene- and protein-based mechanisms result in significant changes at the metabolomic level. In the second part of my thesis, we examined the role of emerging mechano-sensitive metabolic pathways in vascular repair. Metabolomic analysis revealed both pulsatile shear stress (PSS) and OSS significantly increased endothelial glycolytic metabolites, but decreased gluconeogenic metabolites. Additionally, both OSS and PSS up-regulated the expression of PKCɛ. We therefore tested whether shear stress modulates endothelial metabolomics to promote vascular repair via PKCɛ-mediated glycolytic metabolites. Treatment with pro-glyocolytic metabolites in-vitro rescued tube formation following treatment with siPKCɛ. To recapitulate vascular repair in transgenic Tg(flk1:GFP) zebrafish embryos, we decreased viscosity and fluid shear stress by micro-injection of GATA-1a morpholino oligonucleotide (MO). In the zebrafish tail amputation model, GATA-1a MO impaired and delayed vascular repair. Co-injection of PKCɛ mRNA with GATA-1a MO rescued this phenomenon. Injection of epo mRNA to increase viscosity resulted in enhanced tail repair. Overall, our studies revealed that shear responsive VEGFR- PKCɛ-metabolomic signaling modulates glycolytic metabolites to influence vascular repair.

Mitochondria are the metabolic center of the cell, and mitochondrial state is intimately linked to endothelial function. To further investigate the effect of shear stress on endothelial metabolic function, we examined the effect of physiological pulsatile shear stress (PSS) on mitochondrial membrane potential (ΔΨm) and the role of Mn-SOD expression on ΔΨm. PSS induced a dynamic increase in ΔΨm, while silencing Mn-SOD attenuated PSS-mediated ΔΨm increase. Mn-SOD mimetic MnTMPyP increased ΔΨm to the similar extent as induced by PSS. Our findings suggest that PSS modulates mitochondrial function by increasing mitochondrial ΔΨm, in part, via Mn-SOD up-regulation.

In addition to using the embryonic zebrafish model to investigate vascular repair, we worked to develop technologies enabling the use of the adult zebrafish as a model for genetic and chemically-induced cardiomyopathies. To address the confounding effects from sedation of fish and removal from the aquatic habitat for micro-electrocardiogram (?ECG) measurements, we developed waterproof and wearable flexible electronic sensors to uncover the circadian variation in heart rate (HR) and heart rate variability (HRV). Innovations including an ultra-soft silicone integrated jacket, matching Young’s modulus to the fish surface, and embedded micro-glass spheres to reduce the effective density enabled physiological ECG telemetry in the fish’s natural habitat without the need for sedation. The novel features of the flexible silicon jacket for ?ECG telemetry unraveled the biological clock and normalization of QT intervals at following ventricular resection at 26 days post ventricular amputation, providing the first evidence of new physiological phenomena during cardiac injury and repair. Additionally, we revealed Amiodarone-mediated QTc prolongation, HR reduction and HRV increase otherwise masked by sedation. This light weight and waterproof design holds promise to advance the next generation of mobile health and drug discovery.

Light-sheet fluorescence microscopy stands out in the field of bioimaging as its rapid imaging speed, deep penetration depth, and reduced phototoxicity and photobleaching. However, an essential step in imaging large tissues is optical clearing. This process removes lipids, pigments, and calcium phosphate, which are not conducive to clear imaging, and matches the specimen's refractive index with the imaging medium to achieve high transparency. Numerous optical clearing strategies are employed, categorized into three main types: hydrophobic, hydrophilic, and hydrogel-based, each with distinct refractive indices. Typically, biologists design an optical system tailored to a specific sample and clearing method, restricting it to one fixed refractive index.

This thesis hereby proposes a light-sheet system compatible with various tissue clearing methods with large field of view. The system also integrates multi-view reconstruction algorithm to maintain stable high resolution. Its performance is assessed by measuring the point spread function and imaging different specimens: Tg(flk1:mcherry) zebrafish in water, mouse retina cleared by CLARITY, αMHCCre; R26VT2/GK mouse rainbow heart cleared by CUBIC, and mouse descending aorta cleared by iDISCO+. The system demonstrated robustness across multiple optical clearing approaches while maintaining high resolution and a large field of view. It offers the possibility for biologists to explore diverse tissue clearing strategies and study various tissues using a single system.

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.