Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles.

Introduction: Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. Methods: RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation. Results: Our results showed that treating RBCs at 6% hematocrit with 10 µM yoda1 for 30 min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. Conclusion: This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated in vivo under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Introduction: Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Methods: Male and female endothelial cells were treated with AngII for 24 h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Results: Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. Conclusions: These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

Graphene nanocomposites for real-time electrochemical sensing of nitric oxide in biological systems.

Nitric oxide (NO) signaling plays many pivotal roles impacting almost every organ function in mammalian physiology, most notably in cardiovascular homeostasis, inflammation, and neurological regulation. Consequently, the ability to make real-time and continuous measurements of NO is a prerequisite research tool to understand fundamental biology in health and disease. Despite considerable success in the electrochemical sensing of NO, challenges remain to optimize rapid and highly sensitive detection, without interference from other species, in both cultured cells and in vivo. Achieving these goals depends on the choice of electrode material and the electrode surface modification, with graphene nanostructures recently reported to enhance the electrocatalytic detection of NO. Due to its single-atom thickness, high specific surface area, and highest electron mobility, graphene holds promise for electrochemical sensing of NO with unprecedented sensitivity and specificity even at sub-nanomolar concentrations. The non-covalent functionalization of graphene through supermolecular interactions, including π-π stacking and electrostatic interaction, facilitates the successful immobilization of other high electrolytic materials and heme biomolecules on graphene while maintaining the structural integrity and morphology of graphene sheets. Such nanocomposites have been optimized for the highly sensitive and specific detection of NO under physiologically relevant conditions. In this review, we examine the building blocks of these graphene-based electrochemical sensors, including the conjugation of different electrolytic materials and biomolecules on graphene, and sensing mechanisms, by reflecting on the recent developments in materials and engineering for real-time detection of NO in biological systems.

Assessment of photoacoustic tomography contrast for breast tissue imaging using 3D correlative virtual histology.

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.

Preclinical techniques to investigate exercise training in vascular pathophysiology.

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.

Effects of Braiding Parameters on Tissue Engineered Vascular Graft Development.

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.

Label-free photoacoustic and ultrasound imaging for murine atherosclerosis characterization.

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.

Effects of Iliac Stenosis on Abdominal Aortic Aneurysm Formation in Mice and Humans.

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 TGFß1 (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.

Adjustable photoacoustic tomography probe improves light delivery and image quality.

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.

In vivo photoacoustic lipid imaging in mice using the second near-infrared window.

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.

Development of a Glycosaminoglycan Derived, Selectin Targeting Anti-Adhesive Coating to Treat Endothelial Cell Dysfunction.

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.

Imaging of small animal peripheral artery disease models: recent advancements and translational potential.

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.