Tzung Hsiai, M.d., Ph.D. - US grants

DESCRIPTION (provided by applicant): The focal nature of the atherosclerotic lesions in the arterial trees demonstrates the importance of hemodynamics; namely, shear stress, in regulating the biological activities of endothelial cells (EC). In vivo, velocity profiles are asymmetric in shape due to vessel geometry, as well as the time- and spatial-varying components of pulsatile flow. The emerging Micro Electro Mechanical Systems (MEMS) technology offers a new entry point to overcome the existing difficulties. Atherosclerosis is considered to be an inflammatory disease. We hypothesize that disturbed flow with fluctuating parameters such as frequency, direction, and amplitudes plays a distinct role in modulating the inflammatory responses in the arterial bifurcations. In contrast, unidirectional pulsatile flow, and the upstroke slopes or defined as slew rates, downregulate the inflammatory responses. To test our hypotheses, three specific aims are proposed. Specific Aim 1: To acquire real-time unsteady shear stress known to occur in the arterial bifurcations. Newly designed channel will be used to generate steady, pulsatile, or oscillatory flow to cultured ECs. We will develop and fabricate MEMS shear stress sensors to provide both the spatial and temporal resolution necessary to link shear stress with the inflammatory responses of cultured ECs. Specific Aim 2: To elucidate the molecular and, consequently, functional responses of ECs to pulsatile vs. oscillatory shear stress. In vitro, we will simulate EC and monocyte interactions in the lateral wall of arterial bifurcations where disturbed flow occurs. In parallel, we will investigate the dynamic relation between the inflammatory mediators such as monocyte chemoattractant protein-1 (MCP-1) and vasodilators such as nitric oxide (NO). Specific Aim 3: To demonstrate the significance of high vs. low shear stress slew rates known to occur during physical activities on ECs pretreated with oxidized lipid. We will isolate the effects of slew rates on ECs by investigating EC morphologic changes, interactions with monocytes, and inflammatory mediators. This proposed project is both design-directed and hypothesis-driven. By combining MEMS technology and vascular biology, this proposal will generate new insights into the mechanism of flow regulation at the arterial bifurcations.

oxidative stress; hypercholesterolemia; oxidized lipid; atherosclerosis; hemoprotein; monocyte chemoattractant protein 1; heme oxygenase; tocopherols; antioxidants; endotoxins; cytokine; macrophage; cytotoxic T lymphocyte; vascular endothelium; cholesterol esters; lipid peroxides; selenium; stress proteins; low density lipoprotein receptor; tissue /cell culture; laboratory rabbit; human tissue; western blottings;

[unreadable] DESCRIPTION (provided by applicant): Atherosclerosis is a systemic disease; however, its manifestations tend to be focal and eccentric. Shear stress is known to regulate NADPH oxidase activities as a source of endothelial superoxide production (O2-.). The Micro Electro Mechanical Systems (MEMS) provide a spatial resolution comparable to the individually elongated endothelial cells and temporal resolution at 71 kHz that permits investigation of the mechanisms whereby spatial and temporal variations of shear stress regulate the oxidant stress-mediated responses. Our working hypothesis is that at arterial bifurcations, the regions of moderate to high shear stress where flow remains unidirectional and axially aligned experience relatively little oxidative stress. In contrast, excess production of reactive oxygen species (ROS) develops largely in regions of relative low shear stress, flow separation, and departure from axjally aligned and unidirectional flow profiles. We propose that the spatial variations in shear stress at bifurcations regulate the relative production of 02-. or ROS and nitric oxide or reactive nitrogen species (RNS) production. At arterial bifurcations where oscillatory shear stress is prevalent, the increase in O2.- production relative to NO production likely limits NO bioavailability through formation of the potent oxidant, peroxynitrite (ONOO-). To interface the MEMS sensors with our hypothesis, we propose following three aims: Aim 1. Demonstrate that MEMS sensors provide spatial resolution to resolve circumferential variations in shear stress in a 3-D symmetric bifurcation model. Aim 2. Determine the effects of spatial variations in shear stress on specific regions of vascular oxidative stress in the aortas of New Zealand White (NZW) rabbits. Aim 3. Elucidate the mechanism(s) by which spatial and temporal variations in shear stress regulate endothelial .NO and O2-. production and subsequent atherogenic LDL modifications. The new shear stress sensing technology can be applied to in vivo measurements that are critical to validating the findings so far in cell systems and in vitro. Further development and application of MEMS technology to in vivo studies in rabbits will be a major goal of this project. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Atherosclerosis is a systemic disease;however, its manifestations tend to be focal and eccentric. Shear stress is known to regulate NADPH oxidase activities as a source of endothelial superoxide production (O2-.). The Micro Electro Mechanical Systems (MEMS) provide a spatial resolution comparable to the individually elongated endothelial cells and temporal resolution at 71 kHz that permits investigation of the mechanisms whereby spatial and temporal variations of shear stress regulate the oxidant stress-mediated responses. Our working hypothesis is that at arterial bifurcations, the regions of moderate to high shear stress where flow remains unidirectional and axially aligned experience relatively little oxidative stress. In contrast, excess production of reactive oxygen species (ROS) develops largely in regions of relative low shear stress, flow separation, and departure from axjally aligned and unidirectional flow profiles. We propose that the spatial variations in shear stress at bifurcations regulate the relative production of 02-. or ROS and nitric oxide or reactive nitrogen species (RNS) production. At arterial bifurcations where oscillatory shear stress is prevalent, the increase in O2.- production relative to NO production likely limits NO bioavailability through formation of the potent oxidant, peroxynitrite (ONOO-). To interface the MEMS sensors with our hypothesis, we propose following three aims: Aim 1. Demonstrate that MEMS sensors provide spatial resolution to resolve circumferential variations in shear stress in a 3-D symmetric bifurcation model. Aim 2. Determine the effects of spatial variations in shear stress on specific regions of vascular oxidative stress in the aortas of New Zealand White (NZW) rabbits. Aim 3. Elucidate the mechanism(s) by which spatial and temporal variations in shear stress regulate endothelial .NO and O2-. production and subsequent atherogenic LDL modifications. The new shear stress sensing technology can be applied to in vivo measurements that are critical to validating the findings so far in cell systems and in vitro. Further development and application of MEMS technology to in vivo studies in rabbits will be a major goal of this project.

DESCRIPTION (provided by applicant): Atherosclerosis is a systemic disease;however, its manifestations tend to be focal and eccentric. Hemodynamics, specifically, fluid shear stress, is intimately involved in vascular oxidative stress. Oxidative stress induces molecular signaling regulates the development of intimal calcification that has been identified as a distinct, but relevant process to atherosclerosis. The vascular cells that calcify, previously termed calcifying vascular cells (CVC), are multipotent, with the capacity for chondrogenic, leiomyogenic (smooth muscle), and stromogenic (marrow stromal) lineages. Whether vascular calcification stabilizes atherosclerotic plaques or promotes plaque rupture remains undefined. We propose to assess vascular oxidative stress in non-obstructive, albeit inflammatory, lesions in explants of human coronary arteries and New Zealand White (NZW) rabbits. The development of Micro Electro Mechanical Systems (MEMS) shear stress and oxidative stress sensors in our lab has provided a means to undertake study of atherogenic hemodynamics and vascular oxidative stress. We hypothesize that flow disturbance as assessed by the micro-scale sensors in non-obstructive plaques is associated with oxidative stress relevant for initiation of the arterial plaque. This hypothesis will be tested by three Aims: Aim 1: Assess vascular oxidative stress in arterial regions exposed to atherogenic hemodynamics. The level of vascular oxidative stress will be determined from explants of arterial bifurcations of human coronary arteries using the MEMS oxidative stress sensors. Immunohistochemistry will validate oxidative content, including oxidized low density lipoprotein (oxLDL), foam cells and intimal calcification, in the regions exposed to atherogenic hemodynamics. Aim 2: Determine shear stress and oxidative stress in non-obstructive plaque. Intravascular sensors will be deployed into the aortas of NZW rabbits. Athero-prone regions will be localized by shear stress sensors and vascular oxidative stress will be assessed prior to and after hypercholesterolemic diet. The rabbit aorta will be dissected for immuno-staining for regions that harbor oxidative stress. Aim 3: Study whether vascular calcification stabilizes a mechanically unstable plaque. Vascular mesenchymal stem cell (MSC)-derived plaque that harbors oxLDL, foam cells, and calcification will be used in an in vitro model. Atherogenic hemodynamics;namely, low and oscillatory shear stress, will be delivered and plaque rupture will be captured in the context of vascular oxidative stress and calcification. Our proposal represents a concerted effort between two labs (Tzung Hsiai and Linda Demer) to test hypothesis, to establish causality, and to assess mechanically unstable plaque in the presence of calcification. Our research is relevant to public health because a better understanding of the biomechanics of rupture-prone plaques has the potential to reduce the morbidity and mortality associated with atherothrombotic disease. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because a better understanding of the biomechanics of rupture-prone plaques has the potential to reduce the morbidity and mortality associated with atherothrombotic disease.

DESCRIPTION (provided by applicant): Fluid shear stress imparts both metabolic and mechanical effects on vascular endothelial function. The spatial ( D/ x) and temporal ( D/ t) components of shear stress largely determine the focal nature of vascular oxidative stress, leading to pro-inflammatory states. The focus of the previous grant period was a paradigm shift in the approach from one of the static models (oxidative biology) to the dynamic models of investigation (vascular oxidative stress) that combined biophysical and biochemical approaches of pathophysiological significance. We demonstrated that variations in D/ x and D/ t differentially regulated the endothelial production of O2.- and .NO, leading to low density lipoprotein (LDL) oxidative modifications relevant for the initiation of atherosclerotic lesions. We developed microelectromechanical systems (MEMS) sensors to measure in real-time intravascular shear stress in the New Zealand White (NZW) rabbits on a hypercholesterolemic diet, and applied our intravascular methodology to the swine model. We gained new insights into the mechanisms whereby atheroprotective hemodynamics increased mitochondrial membrane potential ( (m) accompanied by a decrease in mitochondrial O2.- production via an up-regulation in Mn-SOD activities. In contrast, atherogenic hemodynamics and oxidized LDL induced mitochondrial O2.- production, leading to apoptosis via c-Jun NH2 terminal kinase (JNK)-induced Mn-SOD ubiquitination and protein degradation. Our finding led to a novel observation that active lipid and macrophages in the vessel wall cause electrochemical modifications that can be measured by electrochemical impedance spectroscopy (EIS). In this context, we hypothesize that shear stress regulates mitochondrial redox status, modulating vascular oxidative stress to cause distinct changes in electrochemical impedance in regions of non-obstructive, albeit inflammatory lesions. In the new Aim 1, we will provide an ex vivo model of EIS; specifically, the frequency-dependent electrical and dielectrical properties between concentric bipolar microelectrodes and endoluminal surface of explants of human arteries and NZW rabbit aortas. In Aim 2, we will establish an in vivo model of EIS measurements using fat-fed NZW rabbits; specifically, microfabrication and deployment of the electrodes for intravascular EIS measurements. In Aim 3, we will provide molecular and genetic models to demonstrate redox signaling as a requite factor underlying changes in electrochemical modifications. The focus in the next grant period will integrate electrochemical, redox signaling, and genetic approaches to establish specific EIS that occur in response to local pro- inflammatory states during angiograms with the possibility of identifying unstable plaque. In summary, the publication record (30 corresponding authors) of our laboratory in the previous funding cycle is a testimony of our commitment and productivity in mechanobiology and vascular oxidative stress research.

DESCRIPTION (provided by applicant): Despite current treatment regimens, heart failure remains the leading cause of morbidity and mortality in the US and developed world due to failure to adequately replace lost ventricular myocardium from ischemia- induced infarct. Adult mammalian ventricular cardiomyocytes have a limited capacity to divide, and this proliferation is insufficient to overcome the significant loss of myocardium from ventricular injury. However, zebrafish (Danio rerio) possess the remarkable capacity to regenerate a significant amount of myocardium in injured hearts, and thus, represent an emerging vertebrate model for regenerative medicine and cardiovascular research. While the small size of zebrafish system allows for high-throughput research, the small heart size (1-2 mm in length) renders it challenging to perform functional physiological analyses. Toward this end, our collaborated efforts have enabled the applications of the micro-electrical cardiogram (ECG) and high-frequency ultrasonic transducers (>45 MHz) to further investigate the electrical and mechanical attributes of regenerating myocardium in injured zebrafish hearts. We have observed that ventricular repolarization (ST intervals and T waves) failed to normalize despite fully regenerated myocardium at 60 days post ventricular amputation, suggesting further cardiac remodeling may be required to fully integrate regenerating myocardium with host myocardium. We hypothesize that early regenerating cardiomyocytes may lack the electrical and mechanical cardiac phenotypes, and thus may require additional cardiac cellular remodeling for full electrical and mechanical integration into injured hearts. To assess the restoration of cardiac function during cardiac regeneration, we propose to interface implantable flexible micro-electrode arrays with high- frequency ultrasonic transducers and optical voltage mapping to test the conduction and mechanical phenotypes, followed by mechanistic assessment by conditionally blocking or activating Wnt/2-catenin and FGF signaling pathways. The development and application of implantable and flexible micro-electrode arrays, high frequency ultrasonic transducers hold a great promise in the era of stem cell and regenerative medicine. In sum, our concerted efforts will likely provide both novel technology and new mechanistic insights into cardiac conduction and mechanical phenotypes in response to genetic, epigenetic, and pharmaceutical perturbations with relevance to regenerative medicine.

DESCRIPTION (provided by applicant): Electrochemical Impedance to Assess Metabolically Active Plaque Atherosclerosis is a systemic disease; however, its manifestations tend to be focal and eccentric, and rupture of individual plaques is the primary underlying mechanism for myocardial infarction and stroke. Plaques prone to rupture contain high levels of inflammatory activity, due to oxidized lipids and foam cells. Fluid shear stress, in addition to its mechanical effects on vascular endothelial cells, promotes oxidative stress and inflammatory responses in plaque. However, real-time detection of the atherosclerotic lesions prone to rupture remains an unmet clinical challenge. Encouraging results from our previous exploratory R21 funding period demonstrated that integration of intravascular shear stress (ISS) and endoluminal electrochemical impedance spectroscopy (EIS) distinguishes pre-atherogenic lesions associated with oxidative stress in fat-fed New Zealand White (NZW) rabbits. Specifically, vessel walls harboring oxidized low density lipoprotein (oxLDL) exhibit distinct electrochemical impedance spectroscopy (EIS) magnitude, and that monocytes and oxLDL together destabilize calcific vascular nodules via induction of matrix metalloproteinase (MMP). In this context, we seek to develop an electrochemical strategy to identify culprit (albeit non-obstructive) lesions containing oxLDL-laden monocyte- macrophages (foam cells), during diagnostic angiography or percutaneous coronary intervention. We hypothesize that oxLDL-rich lesions harbor distinct electrochemical properties in the vessel wall that can be measured by frequency-dependent electrochemical impedance to identify metabolically active atherosclerotic lesions. Our hypothesis will be tested in three Specific Aims. Aim 1: Determine the mechanism by which oxLDL-rich lesions increase electrochemical impedance. EIS will be obtained in plaque from LDL receptor-knockout (LDLR-/-) mice. We hypothesize that it is the oxidant stress in the lesions that increases EIS magnitude. Aim 2: Determine in vivo sensitivity and specificity of EIS for oxLDL-laden, foam cell-rich lesions in fat-fed NZW rabbits as an established model of atherosclerosis with plaques accessible to catheter interrogation. We will also integrate three intravascular sensing modalities, shear stress (ISS), ultrasound (IVUS), and electrochemical impedance (EIS), for early detection of metabolically unstable lesions. Aim 3: Determine in vivo risk of rupture in high EIS plaque in a swine model. We will test whether high EIS lesions are prone to rupture and embolization, and we will assess whether the combination of high impedance and high shear predict lesion predisposition to embolization. Overall, our cross-disciplinary efforts aim to integrate electrochemical properties of active lipid-laden lesions with three animal models and three sensing modalities to establish early detection of unstable lesions for patient-specific intervention.

? DESCRIPTION (provided by applicant): Hemodynamic forces such as shear stress are intimately linked with cardiac morphogenesis. Mutations in Notch signaling components result in congenital heart defects in humans and mice. 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. Hemodynamic shear stress induces vascular endothelial Notch signaling, and Notch pathway mediates differentiation and proliferation of trabecular myocytes. However, the mechanotransduction mechanisms underlying endocardial shear stress (ESS) and the initiation of trabeculation remain elusive. Our multi-disciplinary approach has demonstrated that peristaltic contraction of the embryonic heart tube produces time-varying shear stress (??/?t) and pressure gradients (?P) across the atrioventricular (AV) canal in a zebrafish model of cardiac development. The advent of zebrafish genetic system has enabled the application of fli1 promoter to drive expression of enhanced green fluorescent protein (EGFP) in all vasculature throughout embryogenesis (Tg(fli1a:EGFP)y1); thereby, allowing for 3-D visualization of the moving boundary conditions (2D + time) for computational fluid dynamics (CFD) simulation. However, 4-D (3-D + time) imaging to recapitulate the endocardium throughout the cardiac cycle requires fast tissue scanning and deep axial penetration. In this context, we seek to develop fluorescent Super-Resolution Light-Sheet Microscopy (SRLSM) to capture the 4-D endocardial trabecular network. We hypothesize that spatial (??/?x) and temporal ??/?t) variations in shear stress differentially modulate endocardial Notch signaling to initiate trabeculation. To test our hypothesis, we propose the following three aims. In Aim 1, we will develop a fast 4-D cardiac imaging technique for moving boundary conditions. Our goal is to image endocardiac morphogenesis throughout the cardiac cycle. In Aim 2, we will establish a link between shear stress and endocardial trabeculation. Our goal is to determine the effects of temporal (??/?t) and spatial (??/?x) variations in endocadial shear stress on the initiation of trabeculation from 20 to 120 hours post fertilization. In Aim 3, e will elucidate the mechanotransduction mechanisms underlying trabeculation via Notch signaling. Our goal is to demonstrate that the shear stress-mediated endocardial Notch signaling pathway initiates differentiation and proliferation of cardiac trabeculation. Overall, ou team approach aims to establish a fundamental direction at the interface of hemodynamic forces and cardiac development with pathophysiological significance to non- compaction cardiomopathy.

DESCRIPTION (provided by applicant): Electrochemical Impedance to Assess Metabolically Active Plaque Atherosclerosis is a systemic disease; however, its manifestations tend to be focal and eccentric, and rupture of individual plaques is the primary underlying mechanism for myocardial infarction and stroke. Plaques prone to rupture contain high levels of inflammatory activity, due to oxidized lipids and foam cells. Fluid shear stress, in addition to its mechanical effects on vascular endothelial cells, promotes oxidative stress and inflammatory responses in plaque. However, real-time detection of the atherosclerotic lesions prone to rupture remains an unmet clinical challenge. Encouraging results from our previous exploratory R21 funding period demonstrated that integration of intravascular shear stress (ISS) and endoluminal electrochemical impedance spectroscopy (EIS) distinguishes pre-atherogenic lesions associated with oxidative stress in fat-fed New Zealand White (NZW) rabbits. Specifically, vessel walls harboring oxidized low density lipoprotein (oxLDL) exhibit distinct electrochemical impedance spectroscopy (EIS) magnitude, and that monocytes and oxLDL together destabilize calcific vascular nodules via induction of matrix metalloproteinase (MMP). In this context, we seek to develop an electrochemical strategy to identify culprit (albeit non-obstructive) lesions containing oxLDL-laden monocyte- macrophages (foam cells), during diagnostic angiography or percutaneous coronary intervention. We hypothesize that oxLDL-rich lesions harbor distinct electrochemical properties in the vessel wall that can be measured by frequency-dependent electrochemical impedance to identify metabolically active atherosclerotic lesions. Our hypothesis will be tested in three Specific Aims. Aim 1: Determine the mechanism by which oxLDL-rich lesions increase electrochemical impedance. EIS will be obtained in plaque from LDL receptor-knockout (LDLR-/-) mice. We hypothesize that it is the oxidant stress in the lesions that increases EIS magnitude. Aim 2: Determine in vivo sensitivity and specificity of EIS for oxLDL-laden, foam cell-rich lesions in fat-fed NZW rabbits as an established model of atherosclerosis with plaques accessible to catheter interrogation. We will also integrate three intravascular sensing modalities, shear stress (ISS), ultrasound (IVUS), and electrochemical impedance (EIS), for early detection of metabolically unstable lesions. Aim 3: Determine in vivo risk of rupture in high EIS plaque in a swine model. We will test whether high EIS lesions are prone to rupture and embolization, and we will assess whether the combination of high impedance and high shear predict lesion predisposition to embolization. Overall, our cross-disciplinary efforts aim to integrate electrochemical properties of active lipid-laden lesions with three animal models and three sensing modalities to establish early detection of unstable lesions for patient-specific intervention.

ABSTRACT Heart failure remains the leading cause of morbidity and mortality in the US, afflicting nearly 5 million people. Recently, adult Zebrafish (Danio rerio) have been utilized to model different types of heart failure, and to search for genetic modifiers via mutagenesis screening. However, the small size of the zebrafish heart hinders precise electrical and mechanical assessments following genetic modifications. During the previous funding cycle, we integrated a flexible micro-electrode array with high-frequency ultrasonic transducers to demonstrate that early regenerating cardiomyocytes lack the electrical phenotypes needed to integrate into injured hearts. We further showed that the pressure gradient across the atrioventricular valve is greater than that across the ventriculobulbar valve following ventricular cryo-injury. However, the initial rise and subsequent normalization of ventricular passive (E) and active (A) filling waves (E/A ratios) indicate recovery of diastolic function. In the next funding cycle, we will combine our micro-sensing capacity with novel genetic models of cardiomyopathy to elucidate electromechanical coupling following chemotherapy-induced injury and genetic models of cardiomyopathy. Our multi-disciplinary team established an adult zebrafish model of doxorubicin (Dox)-induced cardiomyopathy (CM) as a conserved vertebrate model to investigate myocardial injury and regeneration in response to the breast cancer chemotherapy targeting ErbB2 (HER2)/NEU. Our team has further developed three murine genetic models of CM; namely, bag3 knockout (KO), mBAG3 overexpression (OE), and Imna KO. We have further developed a forward-genetic approach to identify genetic modifiers of Dox-induced CM. A pilot screen of >500 gene-breaking transposon (GBT) mutants has identified four GBT lines, of which GBT419/rxraa (retinoid X receptor alpha a) resembles mTOR to improve zebrafish survival following Dox-induced CM. Our goal is to integrate micro-sensors with advanced imaging to study electrical conduction and mechanical function of the injured myocardium in response to Dox-induced and 3 genetic models of CM. Our hypothesis is that genetic modifiers such as GBT419/rxraa promotes electromechanical coupling in Dox-induced and genetic models of CM to restore contractile function. To test our hypothesis, we have three aims: In Aim 1, we will determine electrical conduction in our Dox-induced and genetic models. In Aim 2, we will demonstrate mechanical function in our Dox-induced and genetic models. In Aim 3, we will assess electromechanical coupling following treatments with CM modifying genes. Overall, these aims will provide new insights into electromechanical coupling in cardiomyopathy using forward-genetics to discover therapeutic modifiers capable of restoring heart function.

ABSTRACT Cardiometabolic disorders, including hyperlipidemia, obesity, and pre-diabetes, constitute the rising epidemic in the US. These silent disorders progress to chronic diseases, including atherosclerosis. Metabolically active plaques prone to rupture contain high levels of oxidized lipids and M1 macrophages. While rupture of individual plaques is the primary underlying mechanism of myocardial infarction and stroke, real-time detection of the vulnerable plaques prone to rupture remains an unmet clinical challenge. During the previous funding cycle, we demonstrated the sensitivity and specificity of electrochemical impedance spectroscopy for oxidized low density lipoprotein (oxLDL)-laden macrophages (foam cells) in the subendothelial layers of plaques in fat-fed New Zealand White (NZW) rabbits, based on integration of 3 intravascular sensing modalities; namely, shear stress sensor (SSS), ultrasound (IVUS), and electrochemical impedance spectroscopy (EIS). This strategy allowed initial detection in area of disturbed flow, then visualization by IVUS, and then electrochemical characterization by EIS. Vessel walls harboring oxLDL in the macrophages or foam cells exhibit a significant increase in the frequency-dependent EIS magnitude, and these macrophages induce matrix metalloproteinase (MMP) which destabilizes the calcified fibrous cap. We further deployed 3-D EIS sensors in Yucatan mini-pigs undergoing right carotid artery ligation to establish the changes in EIS parameters caused by 12 weeks of high- fat diet. For the next funding cycle, we seek to demonstrate that high 3-D EIS lesions are prone to rupture and embolization. The routine measurement of Fraction Flow Reserve (FFR), defined as the ratio of pressure across the stenotic lesions (Pdownstream/Pupstream) during coronary catheterization, determines the indication for intervention in the significant, ischemia-causing coronary stenoses. For FFR ? 0.8, patients are treated with medical therapy; for FFR ? 0.8, patients are referred for coronary revascularization. However, the predictors for metabolically active, albeit non-obstructive, lesions prone to rupture remain undefined. In this context, our multi-disciplinary team aims to make the fundamental translation of electrochemical impedance spectroscopy (EIS) in the pre-clinical swine models and to test the hypothesis that 3-D EIS mapping of endoluminal oxLDL- laden macrophages advances our ability to detect human atherosclerotic lesions prone to embolization. To test our hypothesis, we have three Specific Aims. In Aim 1, we will determine in vivo 3-D electrochemical properties to enhance detection of oxLDL-laden plaque. In Aim 2, we will establish 3-D EIS mapping in rupture-prone plaque in swine. In Aim 3, we will compare EIS with near-infrared spectroscopy for oxLDL- laden plaque. Overall, establishing 3-D electrochemical mapping of lipid-laden lesions in a swine model of plaque rupture provides a pre-clinical strategy to identify metabolically active, albeit non-obstructive, lesions, and improve the accuracy of personalized intervention for cardiometabolic disorders.

ABSTRACT Cumulative epidemiological and experimental data have shown that exposure to ambient particulate matter (PM) leads to increased cardiovascular morbidity and mortality. A causal association between PM exposure and atherosclerosis has been established. Unfortunately, the pathogenic mechanisms remain unknown preventing the development of effective therapeutic strategies. We have found that exposures to ultrafine particles (UFP, PM with an aerodynamic diameter < 0.2 µm) and diesel exhaust lead to increased lipid peroxidation in the lungs and systemic tissues, accompanied by dyslipidemia and a proatherogenic plasma lipoprotein profile, consisting of LDL particles more susceptible to oxidation and dysfunctional HDL particles with loss of their vascular protective properties. However, the mechanisms by which inhalation of UFP lead to effects in the systemic vasculature remain unknown. We and others have shown that exposure to PM lead to marked changes in the gut microbiome, which is known to modulate host metabolism, immunity, and inflammatory responses resulting in pathological conditions, including cardiovascular diseases. This project will evaluate whether a novel microbome-mediated gastrointestinal (GI) pathway mediates PM-induced dyslipidemia and atherosclerosis. Our preliminary data indicate that oral administration of UFP or inhaled diesel exhaust induces changes in gut microbiota diversity, which associates with lipid oxidation in the intestines and blood, dyslipidemia, and liver steatosis together with decreased expression of hepatic PPAR?, which may mediate some of the UFP-mediated cardiometabolic actions. Our central hypothesis is that inhalation exposure to ambient UFP induces dyslipidemia and atherosclerosis partly due to changes in gut microbiota composition that lead to dysregulation of PPAR? in the liver. We will test this hypothesis via three specific aims: 1) To determine the changes in gut microbiota composition following pulmonary exposure to ultrafine PM. We will perform both UFP inhalation and oral gavage studies to characterize the relative changes in microbiota in Ldlr KO and C57BL/6 mice. 2) To examine whether UFP-induced dyslipidemia and atherosclerosis are mediated by the gut microbiome. The microbiota of UFP-exposed mice will be transferred into germ-free and antibiotic-treated Ldlr KO and C57BL/6 recipients to establish a causal link between UFP- induced gut microbiota effects, lipid metabolism, and atherosclerosis. 3) To determine whether UFP-mediated changes in gut microbiota promote lipid metabolic effects and atherosclerosis via modulation of PPAR? expression in the liver. We will determine if UFP-induced changes in hepatic PPAR? mediate effects induced by UFP exposure on lipid and atherosclerosis using PPAR? KO mice. The results are expected to enhance our understanding of a novel gut microbiome-mediated pathway by which UFP induce adverse systemic effects. If successful, results derived from this project are expected to have a significant impact in developing preventive and therapeutic efforts to ameliorate the health impact of air pollution.

Abstract Despite recent advances in implantable biomedical devices, the utilization of wireless power delivery continues to be a challenge due to anatomical size constraints that limit sufficient power transfer. In addition to pacemakers, implantable stimulators, including neuromodulation devices used for spinal cord, deep brain, and peripheral nerve stimulation, are confined by the same lead-based architecture. Thus, developing wireless power transfer for implantable devices, including the pacemaker, has the potential to mitigate a host of device- related complications. A primary challenge in inductively powered biomedical devices remains in developing a micro-scale receiver antenna with sufficient power output while minimizing transmitter power consumption over an anatomically and wirelessly feasible range. Eliminating the pacing leads, bulky batteries, fixation-associated mechanical burden, and repeated procedures for battery replacement and device retraction remains an unmet clinical need. In this context, we seek to advance a long-range inductively powered wireless and batteryless micro (µ)-system with sufficient power for pacing functionality. Our encouraging preliminary results support the feasibility of a pacing system with a subcutaneous unit and micro-scale pacer unit to induce sufficient power transfer for ex vivo pacing to a porcine heart. We hereby address the fundamental constraints of in vivo long- range pacing using an intravascular micro-pacing system. Our objective is to integrate advanced antenna and circuit design into a pacer system to enable intravascular deployment of wirelessly powered µ-pacer to the anterior cardiac vein (ACV) for pacing. Our goal is to eliminate the device fixation- and lead-related mechanical complications for optimal power transfer efficiency. To deliver our objective, we have three aims. In Aim 1, we will demonstrate the fundamental µ-antenna design and fabrication to enhance power transfer efficiency. In Aim 2, we will integrate CMOS technology and the novel parylene-on-oil encapsulation to enable intravascular deployment. In Aim 3, we will demonstrate the µ-pacer for real-time intravascular pacing in our pre-clinical model. Successful deployment of this wireless power transmission system provides the theoretical and experimental framework to overcome the anatomical size constraints that limit sufficient power transfer with translational implications for both cardiac and non-cardiac stimulation.

ABSTRACT The California Institute of Technology (Caltech) and the University of California at Los Angeles (UCLA) have partnered to integrate advanced imaging and sensing coupled with computing needed to translate technological innovation to address the global cardiometabolic disease. The UCLA/Caltech integrated Theranostic Engineering to Advance Metabolic Medicine (iTEAM) Program represents a new paradigm that will be formalized into a 2-year, structured curriculum with an emphasis on recruiting the under-represented post-doctoral engineers or physical scientists into leadership roles in academia and industry. The convergence of the fundamental strengths of Caltech and clinical strengths of UCLA is conducive to individualize training in 1) advanced sensing or 2) imaging coupled with computing to address 3) cardiometabolic disease. The iTEAM program is partnering with industry leaders (Amgen, Johnson & Johnson, Medtronic, and Edwards Lifesciences) for internship, mentorship, and leadership programs. Both the Caltech Diversity Center and UCLA Faculty Diversity & Development Office have supported workshops on Science Technology Engineering & Mathematics (STEM) for women and underrepresented minorities. To implement this UCLA/Caltech iTEAM program, we have developed a mentoring and self-evaluating structure in the inclusion of 21 primary and co-mentors, 13 consulting mentors, and 10 industry leaders (42% female). Each iTEAM scholar will have co-mentorships: a primary mentor from enabling technologies and a secondary from cardiometabolic medicine and/or industry. In Year 1, iTEAM scholars will: 1) Participate in an initial two- day workshop including mentors, program leaders, clinicians, physician-scientists, and industry leaders to explore projects, expectations, mentorship, and goals; 2) Meet one-to-one with the Program Director(s) to finalize a primary (imaging or sensing) and a co-mentor (cardiometabolic disease or industry); 3) Develop an Individualized Development Plans (IDP) with the Advising & Training Committee to finalize the project; and 4) Strengthen fundamental knowledge in advanced imaging, sensors, or computation and didactic training for ethics in biomedical research and publications. In Year 2, iTEAM scholars will be afforded 1) the opportunity to present work-in-progress and provide feedback in quarterly meetings with a primary mentor and with a co- mentor, 2) an option to participate in a certificate in Pathways in Clinical and Translational Research from the UCLA Clinical Translational Science Institute (CTSI) or Law and Technology for FDA regulatory science (BE188/299); and/or 3) to participate in UCLA CTSI-sponsored professional development in preparation for an academic or industry career. Both Caltech and UCLA Deans have committed matching funds for each iTEAM scholar. UCLA Vice Provost for Graduate Education and has also committed supplemental trainee support to enhance the diversity of trainees. Overall, this program infuses the scientific workforce with the next generation of theranostic bioengineers prepared to solve the worldwide threat of cardiometabolic disease.

ABSTRACT This T32 program represents a new paradigm for training biophysical scientists and engineers to traverse cardiovascular medicine. The convergence of engineering with medicine is transforming clinical and patient care via advances in flexible electronics for sensing and imaging, coupled with machine learning, which has introduced new innovations to confront the rising endemic of cardiometabolic disorders. The University of California at Los Angeles (UCLA) and the California Institute of Technology (Caltech) are partnering to develop the UCLA/Caltech integrated Cardiovascular Medicine for Bioengineers (iCMB) Program, that will be formalized into a 2-year, structured curriculum with an emphasis on recruiting the under-represented post- doctoral engineers or biophysical scientists into leadership roles in academia and industry. The approach is to strengthen individualized training in 1) advanced sensing or 2) imaging coupled with machine learning to address 3) cardiometabolic disease. The iCMB program will be built upon two successful Caltech and UCLA joint training programs: the Medical Scientist Training Program (MSTP) for MD-PhD students and the unique Subspecialty Training and Advanced Research Program (STAR) for fellowship-level physicians to obtain a PhD. The partnerships with the industry leaders from Amgen, Johnson & Johnson, Medtronic, and Edwards Lifesciences have enriched our internship, mentorship, and leadership programs. Both the Caltech Diversity Center and UCLA Faculty Diversity & Development Office have supported workshops on Science Technology Engineering & Mathematics (STEM) for women and under-represented minorities. To implement this iCMB Program, we have developed a novel mentoring and self-evaluating structure in the inclusion of 24 seasoned mentors, 4 emerging faculty, 22 Clinical Faculty (42% female), and 10 industry leaders. Each iCMB scholar will have co-mentorships: a primary mentor from cardiometabolic medicine and a secondary from enabling technologies or industry. In Year 1, iCMB scholars will develop fundamental knowledge in advanced imaging, sensors, or computation and didactic training for ethics in biomedical research and publications. In Year 2, iCMB scholar will have an option 1) to participate in a certificate in Pathways in Clinical and Translational Research from the UCLA Clinical Translational Science Institute (CTSI) or Law and Technology for FDA regulatory science (BE188/299); and/or 2) to participate in UCLA CTSI-sponsored professional development in preparation for an academic or industry career. Both Caltech and UCLA Deans have committed matching funds for each iCMB scholar. Our Vice Provost for UCLA Graduate Education has also committed supplemental trainee support to enhance the diversity of trainees. Therefore, the complementary and synergistic strengths of the Caltech/UCLA iCMB Program provide a new infusion of workforce prepared to solve the worldwide endemic of cardiometabolic disease.

Shear Stress and Light-Field to Elucidate the Initiation of Cardiac Outflow Tract Biomechanical forces modulate cardiac morphogenesis, and mutations in mechano-sensitive signaling pathways result in congenital heart defects. During the previous funding cycle, our team custom-built a Light- Sheet Fluorescence Microscopy (LSFM) with sub-voxel resolution to enhance axial resolution needed to provide a large field-of-view. This laser optical system allowed for imaging pulsatile vs. oscillatory shear stress- mediated Notch signaling to initiate endocardial trabeculation. We demonstrated that spatial (??/?x) and temporal (??/?t) variations in shear stress modulates Notch-EphrinB2-Neureguilin-1 signaling in the endocardium to activate erb-B2 receptor tyrosine kinase (ErbB2) that promotes proliferation of trabeculation. By integrating LSFM, computation, and transgenic models, we further established that trabeculation dissipates intracardiac shear stress-generated kinetic energy; thus, mitigating ventricular remodeling. However, it remains unclear what would be the consequences of reduced myocardial contractility or altered intracardiac flow dynamics on valve morphogenesis. Thus, we seek to integrate light-sheet (Bessel-Gaussian beam arrays) with a new 2) light-field (microlens array). The former provides non-diffracting illumination, and the latter provides volumetric detection as a paradigm shift to image both myocardial contractility and intracardiac flow dynamics in the outflow tract (OFT). Our preliminary study reveals that shear-mediated Notch1b expression in the endocardium of OFT regulates endothelial-to-mesenchymal transition (EndoMT); however, the mechanotransduction causation whereby myocardial contractility and intracardiac shear stress reciprocally interact to form bicuspid valves and subsequent remodeling to multi-cuspid valves remains elusive. Thus, our hypothesis is that integration of the new light-field system with imaging computation enhances spatiotemporal resolution needed to decouple myocardial contraction from intracardiac flow dynamics that modulates Notch1b-EndoMT to mediate valve morphogenesis in the OFT. In Aim 1, we plan to integrate light-sheet with the new light-field system for 4-D volumetric imaging of valve formation in the OFT. Our goal is to capture myocardial contractility and intracardiac shear stress at one snapshot. In Aim 2, we will demonstrate the interaction between intracardiac shear stress and myocardial contractility underlying valve morphogenesis. Our goal is to decouple hemodynamic shear from contractile forces that mediate Notch1b-mediated EndoMT. In Aim 3, we will determine the relative role of shear stress and contractility underlying Notch1b-mediated EndoMT. Our goal is to elucidate the relative role of contractility and intracardiac stress to transmit Notch1b- EndoMT signaling underlying bicuspid-valve formation. Overall, our team aims to establish the micro- environment in which intracardiac flow dynamics and myocardial contractility interact to modulate OFT valve formation, with clinical significance to aortic valvular disease.

ABSTRACT Integrating Volumetric Light-Field with Computational Fluid Dynamics to Study Myocardial Trabeculation and Function Non-compaction cardiomyopathy (NCC) is a disease of endomyocardial trabeculation or known as spongy myocardium. NCC carries a high risk of malignant arrhythmias, thromboembolic events, and ventricular dysfunction in association with congenital heart defects or skeletal myopathy. Studies have linked left ventricular non-compaction with autosomal dominant inherited disorders, and mutations in Notch pathways are implicated in defective trabeculation and ventricular NCC. Biomechanical force is intimately connected with mechanotransduction and cardiac morphogenesis. During development, the myocardium differentiates into an outer compact zone and an inner trabeculated zone. Notch receptor- ligand interaction induces EphrinB2-Nrg-ErbB2 signaling to initiate trabecular formation. Our in silico analysis (Alison Marsden, Stanford) revealed elevated oscillatory shear index (OSI) in trabecular ridges, leading to increased viscous dissipation, which was associated with changes in ventricular contractile function and remodeling. However, uncoupling myocardial contraction from intracardiac flow dynamics to elucidate Notch-mediated trabecular organization and subsequent associated changes in local hemodynamics remains an unmet biomechanical challenge. In this context, we hypothesize that hemodynamic shear and myocardial contractile forces coordinate trabecular organization needed to preserve the ventricular structure and contractile efficiency. In combination of laser light-sheet and light- field for super resolution and volumetric imaging, we simultaneously captured myocardial contraction and intracardiac flow dynamics. In collaboration with Stanford Cardiac Mechanics, we integrated fluid structure interaction (FSI) with super resolution imaging to demonstrate 4-D endocardial shear stress in the trabecular ridges and grooves as possible developmental modulator. To test our hypothesis, we propose three specific aims. In Aim 1, we will demonstrate that intracardiac shear stress activates endocardial Delta-Notch signaling to promote trabecular ridge formation. In Aim 2, we will demonstrate that ventricular contraction activates myocardial Jagged-Notch signaling to organize trabecular groove formation. In Aim 3, we will demonstrate that the combination of trabecular ridge and groove formation leads to optimal local hemodynamics and ventricular energetics. The integration of advanced imaging, fluid structure interaction, and zebrafish genetics is uniquely suitable to unveil trabecular organization in relation to kinetic energy dissipation. Our multi-disciplinary approach provides new biomechanical insights into non-compaction cardiomyopathy with pathophysiological significance to ventricular remodeling and function.