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Circulating red blood cell extracellular vesicles (RBC-EVs) are a promising biomarker for vascular health. However, generating, isolating, and characterizing physiologically relevant RBC-EVs with sufficient yield and purity for biological studies is non-trivial. Here, we present and rigorously characterize an in vitro model to mimic RBC-EV production during shear stress via mechanosensitive piezo1 ion channel stimulation. We optimize our RBC-EV isolation protocol to minimize hemolysis, maximize RBC-EV yield and purity, and improve the ease of EV characterization. RBC-EV purity was measured by quantifying protein (e.g., particles/μg), large particle (e.g., protein aggregates), and platelet EV contamination. This study compared RBC-EV isolation performance using membrane-based affinity (e.g., exoEasy), ultrafiltration (e.g., Amicon Ultra-15), and ultracentrifugation, with and without size exclusion chromatography purification. We found that treating 6% hematocrit with 10 μM piezo1-agonist yoda1 for 30 minutes and isolating RBC-EVs using ultracentrifugation minimized RBC hemolysis and maximized RBC-EV yield (~1012 particles/mL) and purity, provided the most consistent RBC-EV preparations, and improved ease of RBC-EV characterization. Our pressure myography experiments suggest that co-isolated protein contaminants, but not piezo1 RBC-EVs, induce rapid mouse carotid artery vasodilation. These results underscore the importance of characterizing EV purity for biological experiments. The standardized methods outlined here enable mechanistic studies of how RBC-EVs generated in physiological flow affect vascular response.

Atherosclerosis is a dynamic process starting with endothelial dysfunction and inflammation and eventually leading to life-threatening arterial plaques. Exercise generally improves endothelial function in a dose-dependent manner by altering hemodynamics, specifically by increased arterial pressure, pulsatility, and shear stress. However, athletes who regularly participate in high-intensity training can develop arterial plaques, suggesting alternative mechanisms through which excessive exercise promotes vascular disease. Understanding the mechanisms that drive atherosclerosis in sedentary versus exercise states may lead to novel rehabilitative methods aimed at improving exercise compliance and physical activity. Preclinical tools, including in vitro cell assays, in vivo animal models, and in silico computational methods, broaden our capabilities to study the mechanisms through which exercise impacts atherogenesis, from molecular maladaptation to vascular remodeling. Here, we describe how preclinical research tools have and can be used to study exercise effects on atherosclerosis. We then propose how advanced bioengineering techniques can be used to address gaps in our current understanding of vascular pathophysiology, including integrating in vitro, in vivo, and in silico studies across multiple tissue systems and size scales. Improving our understanding of the antiatherogenic exercise effects will enable engaging, targeted, and individualized exercise recommendations to promote cardiovascular health rather than treating cardiovascular disease that results from a sedentary lifestyle.

Current breast tumor margin detection methods are destructive, time-consuming, and result in significant reoperative rates. Dual-modality photoacoustic tomography (PAT) and ultrasound has the potential to enhance breast margin characterization by providing clinically relevant compositional information with high sensitivity and tissue penetration. However, quantitative methods that rigorously compare volumetric PAT and ultrasound images with gold-standard histology are lacking, thus limiting clinical validation and translation. Here, we present a quantitative multimodality workflow that uses inverted Selective Plane Illumination Microscopy (iSPIM) to facilitate image co-registration between volumetric PAT-ultrasound datasets with histology in human invasive ductal carcinoma breast tissue samples. Our ultrasound-PAT system consisted of a tunable Nd:YAG laser coupled with a 40 MHz central frequency ultrasound transducer. A linear stepper motor was used to acquire volumetric PAT and ultrasound breast biopsy datasets using 1100 nm light to identify hemoglobin-rich regions and 1210 nm light to identify lipid-rich regions. Our iSPIM system used 488 nm and 647 nm laser excitation combined with Eosin and DRAQ5, a cell-permeant nucleic acid binding dye, to produce high-resolution volumetric datasets comparable to histology. Image thresholding was applied to PAT and iSPIM images to extract, quantify, and topologically visualize breast biopsy lipid, stroma, hemoglobin, and nuclei distribution. Our lipid-weighted PAT and iSPIM images suggest that low lipid regions strongly correlate with malignant breast tissue. Hemoglobin-weighted PAT images, however, correlated poorly with cancerous regions determined by histology and interpreted by a board-certified pathologist. Nuclei-weighted iSPIM images revealed similar cellular content in cancerous and non-cancerous tissues, suggesting malignant cell migration from the breast ducts to the surrounding tissues. We demonstrate the utility of our nondestructive, volumetric, region-based quantitative method for comprehensive validation of 3D tomographic imaging methods suitable for bedside tumor margin detection.

Tissue engineered vascular grafts (TEVGs) using scaffolds fabricated from braided poly(glycolic acid) (PGA) fibers coated with poly(glycerol sebacate) (PGS) are developed. The approach relies on in vivo tissue engineering by which neotissue forms solely within the body after a scaffold has been implanted. Herein, the impact of altering scaffold braid design and scaffold coating on neotissue formation is investigated. Several combinations of braiding parameters are manufactured and evaluated in a Beige mouse model in the infrarenal abdominal aorta. Animals are followed with 4D ultrasound analysis, and 12 week explanted vessels are evaluated for biaxial mechanical properties as well as histological composition. Results show that scaffold parameters (i.e., braiding angle, braiding density, and presence of a PGS coating) have interdependent effects on the resulting graft performance, namely, alteration of these parameters influences levels of inflammation, extracellular matrix production, graft dilation, neovessel distensibility, and overall survival. Coupling carefully designed in vivo experimentation with regression analysis, critical relationships between the scaffold design and the resulting neotissue that enable induction of favorable cellular and extracellular composition in a controlled manner are uncovered. Such an approach provides a potential for fabricating scaffolds with a broad range of features and the potential to manufacture optimized TEVGs.

Graphical abstract, One cause for suboptimal photoacoustic tomography (PAT) penetration depth is attenuation of incident light by soft tissue. To better understand this problem, we investigated the effects of illumination fiber optic bundle geometry on PAT penetration depth and signal-to-noise ratio. An adjustable, motorized PAT probe was used to reduce probe-skin reflection artifacts and improve light distribution in the image acquisition plane by tuning fiber orientation. We validated our motorized PAT probe through Monte Carlo simulations and ex vivo imaging of a tissue mimicking phantom, and in vivo imaging of murine periaortic fat. Overall, our ex vivo results showed a several millimeter improvement in penetration depth and in vivo results showed a >62% increase in lipid signal-to-noise ratio. Our PAT probe also utilized a 7-μm aluminum filter to block in vivo probe-skin reflection artifacts. Together, these findings showed the importance of optimizing illumination geometry to enhance PAT image quality.

Reduced lower-limb blood flow has been shown to lead to asymmetrical abdominal aortic aneurysms (AAAs) but the mechanism of action is not fully understood. Therefore, small animal ultrasound (Vevo2100, FUJIFILM VisualSonics) was used to longitudinally study mice that underwent standard porcine pancreatic elastase (PPE) infusion (n = 5), and PPE infusion with modified 20% iliac artery stenosis in the left (n = 4) and right (n = 5) iliac arteries. Human AAA computed tomography images were obtained from patients with normal (n = 9) or stenosed left (n = 2), right (n = 1), and bilateral (n = 1) iliac arteries. We observed rapid early growth and rightward expansion (8/9 mice) in the modified PPE groups (p < 0.05), leading to slightly larger and asymmetric AAAs compared to the standard PPE group. Further examination showed a significant increase in TGFβ1 (p < 0.05) and cellular infiltration (p < 0.05) in the modified PPE group versus standard PPE mice. Congruent, yet variable, observations were made in human AAA patients with reduced iliac outflow compared to those with normal iliac outflow. Our results suggest that arterial stenosis at the time of aneurysm induction leads to faster AAA growth with aneurysm asymmetry and increased vascular inflammation after 8 weeks, indicating that moderate iliac stenosis may have upstream effects on AAA progression.

Dual-modality photoacoustic tomography (PAT) and 4D ultrasound (4DUS) imaging have shown promise for cardiovascular applications, but their use in murine atherosclerosis imaging is limited. This study used PAT and 4DUS to correlate altered arterial strain and hemodynamics to morphological changes and lipid localization in a murine partial carotid ligation (PCL) model of atherosclerosis. Validation experiments showed a positive correlation between the PAT signal-to-noise ratio and plaque lipid composition obtained from oil-red O histology. Cross-sectional in situ PAT and longitudinal in vivo ultrasound imaging was performed using a 40 MHz transducer. Ultrasound timepoints included days 0, 1, 4, 7, 10, and 14 for hemodynamic and strain assessment, and 1100 nm and 1210 nm PAT was implemented at the study end point for hemoglobin and lipid characterization. These study groups were then separated into day 4 post-PCL with (n = 5) and without (n = 6) Western diet feeding, as well as days 7 (n = 8), 10 (n = 8), and 14 (n = 8) post-PCL, in addition to a sham control group on a Western diet (n = 5). Overall, our data revealed a substantial decrease in left carotid artery pulsatility by day 7. The hemodynamic results suggested greater disturbed flow in the caudal regions resulting in earlier vessel stenosis and greater lipid deposition than cranial regions. Morphological and compositional data revealed heterogeneous vascular remodeling between days 0 and 7, with a rapid decrease in the vessel volume/length and the presence of both intraplaque hematoma and lipid deposition at day 10 post-PCL. These results highlight the utility of utilizing dual-modality PAT and 4DUS to study atherosclerosis progression.

Atherosclerosis accounts of 50% of the deaths in the western world leading to a plethora of diseases that include myocardial infarction, stroke, and peripheral artery disease. Currently available imaging modalities have inherent limitations, including ionizing radiation, lack of compositional information, and difficulty acquiring volumetric data that constrain their use in studying cardiovascular disease. Photoacoustic Tomography (PAT) has emerged as a promising modality that could address these limitations to improve the characterization and diagnosis of atherosclerosis-related conditions. Non-ionizing pulsed laser light is delivered to tissue leading to thermoelastic expansion followed by propagation of a pressure transient that can be detected with an ultrasound transducer. The magnitude of the ultrasonic PAT signal is proportional to the optical absorption at that location, revealing physiologically relevant compositional information of the tissue. The objective of this work is to therefore develop advanced volumetric imaging techniques to characterize disease progression in a murine model of atherosclerosis. The novelty of this work lies in the methodology and validation presented towards characterization of small animal vascular lipid accumulation with a high-resolution PAT system that utilizes the second near-infrared window (1100-1300nm). Additionally, we utilized in situ PAT to cross-sectionally assess lipid deposition and in vivoultrasound to longitudinally assess hemodynamic, kinematic, and morphological changes during atherosclerosis progression. Together, this dissertation lays the foundation towards utilizing dual-modality PAT and ultrasound for various applications including understanding atherosclerosis pathophysiology, evaluation of novel therapeutics, and translation of clinically relevant techniques.

One of the challenges in high-resolution in vivo lipid-based photoacoustic tomography (PAT) is improving penetration depth and signal-to-noise ratio (SNR) past subcutaneous fat absorbers. A potential solution is to create optical manipulation techniques to maximize the photon density within a region of interest. Here, we present a motorized PAT probe that is capable of tuning the depth in which light is focused, as well as substantially reducing probe-skin artifacts that can obscure image interpretation. Our PAT system consists of a Nd:YAG laser (Surelite EX, Continuum) coupled with a 40 MHz central frequency ultrasound transducer (Vevo2100, FUJIFILM Visual Sonics). This system allows us to deliver 10 Hz, 5 ns light pulses with fluence of 40 mJ/cm2 to the tissue interest and reconstruct PAT and ultrasound images with axial resolutions of 125 µm and 40 µm, respectively. The motorized PAT holder was validated by imaging a polyethylene-50 tubing embedded polyvinyl alcohol phantom and periaortic fat on apolipoprotein-E deficient mice. We used 1210 nm light for this study, as this wavelength generates PAT signal for both lipids and polyethylene-50 tubes. Ex vivo results showed a 2 mm improvement in penetration depth and in vivo experiments showed an increase in lipid SNR of at least 62%. Our PAT probe also utilizes a 7 μm aluminum filter to prevent in vivo probe-skin reflection artifacts that have been previously resolved using image post-processing techniques. Using this optimized PAT probe, we can direct light to various depths within tissue to improve image quality and prevent reflection artifacts.

Current methods for breast tumor margin detection are invasive, time consuming, and typically result in a reoperative rate of over 25%. This marks a clear clinical need to develop improved tools to intraoperatively differentiate negative versus positive tumor margins. Here, we utilize photoacoustic tomography (PAT), ultrasound (US), and inverted Selective Plane Illumination Microscopy (iSPIM) to assess breast tumor margins in eight human breast biopsies. Our PAT/US system consists of a tunable Nd:YAG laser (NT 300, EKSPLA) coupled with a 40MHz central frequency US probe (Vevo2100, FUJIFILM Visual Sonics). This system allows for the delivery of 10Hz, 5ns pulses with fluence of 40mJ/cm2 to the tissue with PAT and US axial resolutions of 125μm and 40μm, respectively. For this study, we used a linear stepper motor to acquire volumetric PAT/US images of the breast biopsies using 1100nm light to identify bloodrich “tumor” regions and 1210nm light to identify lipid-rich “healthy” regions. iSPIM (Applied Scientific Instrumentation) is an advanced microscopy technique with lateral resolution of 1.5μm and axial resolution of 7μm. We used 488nm laser excitation and acridine orange as a general comprehensive histology stain. Our results show that PAT/US can be used to identify lipid-rich regions, dense areas of arterioles and arteries, and other internal structures such as ducts. iSPIM images correlate well with histopathology slides and can verify nuclear features, cell type and density, stromal features, and microcalcifications. Together, this multimodality approach has the potential to improve tumor margin detection with a high degree of sensitivity and specificity.

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.

Introduction: Deep vein thrombosis (DVT) affects 300,000 to 600,000 people annually with a recurrence in 20-50% after 10 years. Treatment is managed through anticoagulant drugs, but side effects occur due to systemic administration. The venous endothelium is thought to perpetrate thrombosis, especially through endothelial cell surface adhesion receptors P-selectin and E-selectin. In this study, a selectin receptor-binding molecule (EC-SEAL), made with a selectin-binding peptide and dermatan sulfate backbone was used to evaluate thrombus prevention in the infrarenal vena cava (IVC) of a mouse over 24 hours using high frequency ultrasound. Purpose: P-selectin and E-selectin have exemplified a link between DVT, leukocyte accumulation and rolling, and platelet binding. Thus, the purpose was to evaluate the ability of EC-SEAL to reduce leukocyte and platelet binding, and subsequently DVT, in a mouse model by blocking the corresponding endothelial cell receptors. Methods: A surgical murine model of thrombosis was induced by IVC stenosis with non-absorbable 6-0 silk suture and a 30-gauge needle (male C57BL/6; 10.8 ± 0.2 weeks old). The stenosis led to a 90% reduction in blood flow through the vessel. Within 10 minutes of suture placement around the IVC, 100 μl of saline (n = 6), EC-SEAL (n = 4), or heparin (n = 6) were systemically injected via the tail vein. Thrombus was observed before surgery, 6 hours and 24 hours after surgery using the Vevo 2100 high frequency ultrasound (FUJIFILM VisualSonics Inc.). Thrombus volume and IVC volume were analyzed using 3D ultrasound images, while thrombus weight was measured ex vivo . Results and Conclusions: Six hours after post-ligation, the mean thrombus percentage (thrombus volume/total IVC volume) was 63.1±5.3% for saline, 25.8±11.2% for EC-SEAL, and 26.1±11.4% for heparin. EC-SEAL and heparin groups had significantly lower 6-hour mean thrombus percentage (p < 0.05; ANOVA with a Tukey HSD post-hoc). The 24-hour mean thrombus percentage and thrombus weight were not statistically significant. The data suggest EC-SEAL reduces thrombus at 6 hours while avoiding reduced clotting time side effects associated with heparin. Further work will be needed to determine the true potential for EC-SEAL as a DVT treatment.

Dual-modality photoacoustic tomography (PAT) and four-dimensional ultrasound (4DUS) imaging have recently been used study atherosclerosis progression in small animals. PAT uses pulsed laser light to induce acoustic waves and reconstruct lipid-specific compositional images of tissue. 4DUS captures dynamic volumetric information and can be used to estimate three-dimensional (3D) Green-Lagrange strain using a direct deformation estimation method. Here, we hypothesized that PAT/4DUS can be used to correlate changes in arterial strain and hemodynamics with lipid localization and density in animals that have undergone partial carotid ligation (PCL) induced-atherosclerosis. A 40 MHz transducer (Vevo2100, VisualSonics) and a ND:YAG pulsed laser (Surelite EX, Continuum) were used to image five apolipoprotein-E deficient mice that underwent PCL of the left carotid artery while being fed a Western diet. Animals were imaged using 4DUS at days 0, 1, 4, 7, 10, and 14 to obtain pulsed-wave Doppler for hemodynamic characterization and 4DUS images for strain mapping. At day 14 all animals were euthanized and 3D in situ PAT images of the left carotid artery were acquired using 1210 nm light. The results show that atherosclerotic lesions can be characterized via PAT to localize both lipid accumulation and density and 4DUS to identify regions of low strain.

Photoacoustic imaging has emerged as a promising technique to improve preclinical and clinical imaging by providing users with label-free optical contrast of tissue. Here, we present a proof-of-concept study for noninvasive in vivo murine lipid imaging using 1210 nm light to investigate differences in periaortic fat among mice of different gender, genotypes, and maturation. Acquired lipid signals suggest that adult male apoE-/- mice have greater periaortic fat accumulation compared to adolescent males, apoE-/- females, and wild-type mice. These results demonstrate the potential of photoacoustic tomography for studying vascular pathophysiology and improving the diagnosis of lipid-based diseases.

Vibrational Photoacoustic Tomography (VPAT) is an emerging imaging modality that utilizes pulsed laser light to induce acoustic waves and obtain tissue specific compositional information. When used in combination with ultrasound, VPAT has the potential to identify the location, size, and distribution of lipids in vivo . In our study, we hypothesized that VPAT can be used to distinguish perivascular aortic fat accumulation between apolipoprotein E-deficient (apoE -/- ) and wild-type (WT) mice. A 40 MHz central frequency transducer (Vevo2100, VisualSonics) and a ND:YAG pulsed laser (NT352C, Ekspla) were used to simultaneously obtain both long axis in vivo B-mode and VPAT images of the infrarenal aorta in three apoE -/- (8.7±0 months old) and WT controls (2.9±0 months old) mice. Pulsed laser light was specifically tuned to 1100nm and 1210nm to image blood and lipid, respectively. We then measure perivascular lipid thickness in three distinct locations along the anterior wall of the aorta. These measurements were then averaged to obtain a final perivascular fat thickness score for both the apoE -\- and WT groups. We observed greater 1210 nm signal in apoE -/- (0.47±0.14 mm) mice versus WT mice (0.32±0.11 mm; p-/- mice have greater perivascular lipid accumulation than WT controls. These findings are significant as they show that VPAT can be used to image and track lipid accumulation in vivo . Future work will focus on monitoring luminal plaque progression in atherosclerotic murine models, including hypercholesterolemic mice on a high fat diet or rodent models of severe carotid stenosis. If successful, VPAT has the potential to be used in the clinic to 1) study atherosclerosis plaque development, 2) evaluate the effectiveness of prospective therapeutics, and 3) improve the diagnosis of atherosclerosis or related vascular diseases.

Vibrational Photoacoustic Tomography (VPAT) is a novel imaging modality that uses pulsed laser light to generate an acoustic signal, making it ideal to obtain structural and compositional information. These characteristics allow detection of lipid distribution in diseased aortae by overlaying ultrasound (40 MHz transducer) and VPAT lipid signal (Fig. 1A). We used VPAT to test the hypothesis that arterial remodeling of suprarenal AAAs cause less lipid accumulation in the false lumen compared to other regions prone to atherosclerosis. To induce AAAs, osmotic minipumps were subcutaneously implanted in the backs of apolipoprotein E-deficient mice (n=4; 25±4.7 weeks old) to deliver 1000 ng/kg/min angiotensin II for 28 days. Aortae were imaged in situ, 21 days after AAA diagnosis, using VPAT at 1100 nm for blood (Fig. 1B) and 1210 nm for lipid (Fig. 1C) in a long-axis view. We quantified lipid and blood signal in the false lumen and the aortic wall proximal and distal to the AAAs. We observed a two-fold increase in lipid signal distal to the AAA (p0.05), likely due to atherosclerosis in these regions (AAA: 34.1±8.4 AU; distal: 68.5±19.4 AU; proximal: 56.7±9.7 AU). Blood signal was relatively constant along the vessel (AAA: 43.5±17.4 AU; distal: 40.2±28.2 AU; proximal: 27.8±17 AU). The aortae of these animals are prone to atherosclerosis, consistent with VPAT images and previous histological data. Interestingly, our findings show that while blood signal did not vary with location, lipid signal was reduced in the false lumen of these dissecting AAAs compared to other regions. These results suggest that VPAT may be useful to quantify atherosclerosis development, a disease becoming more prevalent with the current increase in obesity and diabetes.

Endothelial cell (EC) dysfunction is associated with many disease states including deep vein thrombosis (DVT), chronic kidney disease, sepsis and diabetes. Loss of the glycocalyx, a thin glycosaminoglycan (GAG)-rich layer on the EC surface, is a key feature of endothelial dysfunction and increases exposure of EC adhesion molecules such as selectins, which are involved in platelet binding to ECs. Once bound, platelets cause thrombus formation and an increased inflammatory response. We have developed a GAG derived, selectin targeting anti-adhesive coating (termed EC-SEAL) consisting of a dermatan sulfate backbone and multiple selectin-binding peptides designed to bind to inflamed endothelium and prevent platelet binding to create a more quiescent endothelial state. Multiple EC-SEAL variants were evaluated and the lead variant was found to preferentially bind to selectin-expressing ECs and smooth muscle cells (SMCs) and inhibit platelet binding and activation in a dose-dependent manner. In an in vivo model of DVT, treatment with the lead variant resulted in reduced thrombus formation. These results indicate that EC-SEAL has promise as a potential therapeutic in the treatment of endothelial dysfunction.