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Aims Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. Methods and results The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. Conclusion LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

Atherosclerosis remains the number one cause of death and disability worldwide. Atherosclerosis is treated by revascularization procedures to restore blood flow to distal tissue, but these procedures often fail due to restenosis secondary to neointimal hyperplasia. Diabetes mellitus is a metabolic disorder that accelerates both atherosclerosis development and onset of restenosis. Strategies to inhibit restenosis aim at reducing neointimal hyperplasia by inhibiting vascular smooth muscle cell (VSMC) proliferation and migration. Since increased production of reactive oxygen species promotes VSMC proliferation and migration, redox intervention to maintain vascular wall redox homeostasis holds the potential to inhibit arterial restenosis. Cinnamic aldehyde (CA) is an electrophilic Nrf2 activator that has shown therapeutic promise in diabetic rodent models. Nrf2 is a transcription factor that regulates the antioxidant response. Therefore, we hypothesized that CA would activate Nrf2 and would inhibit neointimal hyperplasia after carotid artery balloon injury in the Zucker Diabetic Fatty (ZDF) rat. In primary ZDF VSMC, CA inhibited cell growth by MTT with an EC50 of 118 ± 7 μM. At a therapeutic dose of 100 μM, CA inhibited proliferation of ZDF VSMC in vitro and reduced the proliferative index within the injured artery in vivo, as well as migration of ZDF VSMC in vitro. CA activated the Nrf2 pathway in both ZDF VSMC and injured carotid arteries while also increasing antioxidant defenses and reducing markers of redox dysfunction. Additionally, we noted a significant reduction of neutrophils (69%) and macrophages (78%) within the injured carotid arteries after CA treatment. Lastly, CA inhibited neointimal hyperplasia evidenced by a 53% reduction in the intima:media ratio and a 61% reduction in vessel occlusion compared to arteries treated with vehicle alone. Overall CA was capable of activating Nrf2, and inhibiting neointimal hyperplasia after balloon injury in a rat model of diabetic restenosis., Graphical abstract fx1, Highlights • CA inhibits ZDF VSMC migration and proliferation in vitro. • CA activates Nrf2 in VSMC in vitro and in injured diabetic carotid arteries. • CA increases antioxidants and reduces redox marker levels in VSMC. • CA reduces leukocytes and redox markers in injured diabetic carotid arteries. • CA reduces proliferation and intimal hyperplasia in ZDF rats after balloon injury.

Background Atherosclerosis remains the leading cause of death and disability worldwide. Atherosclerosis is treated by revascularization procedures that often fail due to neointimal hyperplasia (NH). NH results from vascular smooth muscle cell (VSMC) migration and proliferation from the media into the intima. Overproduction of reactive species drives NH formation. Furthermore, diabetes is a metabolic disorder that accelerates redox dysfunction and promotes both atherosclerosis and NH. We previously showed that the Nrf2 activator cinnamic aldehyde (CA) inhibits NH after carotid artery balloon injury in Zucker diabetic fatty (ZDF) rats. We hypothesize that CA inhibits NH by improving antioxidant defenses in VSMC via Nrf2 activation. Methods VSMC were isolated from the aortae of male ZDF rats. Nrf2 pathway activation was assessed by Western blot and confocal microscopy. Antioxidants were measured by Western blot and colorimetric assays. CA (100 µM) in Pluronic F127 (100 µL) or vehicle alone was applied to the periadventitial surface of the carotid after balloon injury in male ZDF rats. Arteries were harvested 3 days after surgery for measuring Nrf2 nuclear translocation and relative redox state. Dihydroethidium (DHE) and 3-nitrotyrosine staining were used to analyze redox dysfunction. Results 100 µM CA induced Nrf2 translocation to the nucleus in vitro and in vivo. CA activated the Nrf2 pathway in vitro as evidenced by increased levels (P Conclusion CA activated the Nrf2 pathway and improved antioxidant defenses in a diabetic rat model. Localized Nrf2 activation may provide a new therapeutic target for inhibiting NH.

Atherosclerosis, remains the leading cause of death and disability in Western countries. Current therapeutic modalities for the treatment of arterial disease include balloon angioplasty and stenting. However, the success rate of these procedures is limited due to the development of arterial restenosis secondary to neointimal hyperplasia. Redox intervention using nitric-oxide-based therapies or small electrophiles inhibit restenosis in various animal models. Whereas human studies using redox-based therapies have for the most part not shown differences in clinical outcomes, some studies using local delivery have shown promising results. Hence, the biggest challenge for successful clinical translation is the targeted delivery of the therapeutic in the right amount at the right site. My work focused on the development of local and/or targeted delivery systems with redox-based therapies. We have successfully inhibited restenosis with local periadventitial or intraluminal delivery of diazeniumdiolates, or cinnamic aldehyde. We developed S-nitrosated nano-scale fibers that administered systemically, locally target the injured arterial segment, and successfully inhibit restenosis. Targeted delivery is a promising approach to translate redox based therapies from the bench to the clinic.

Background Diabetes Mellitus (DM) accelerates the rate of atherosclerosis development. Balloon angioplasty is a common surgical intervention for the treatment of atherosclerotic disease. However, angioplasty often fails due to restenosis secondary to neointimal hyperplasia. Chronic inflammation and redox dysfunction in DM leads to higher restenosis rates at earlier time points. Neointimal hyperplasia is the result of vascular smooth muscle cells (VSMC) and adventitial fibroblasts proliferating and migrating into the intima, in a process driven by reactive species. Activation of the Nrf2-KEAP1 pathway, a regulator of the antioxidant response, inhibits neointimal hyperplasia in animal models of vascular disease. Therefore we hypothesize that a Nrf2 activator, cinnamic aldehyde (CA), will inhibit VSMC migration and proliferation in vitro and in vivo in a rat model of DM thus preventing neointimal hyperplasia. Methods VSMC were isolated from the aorta of male Zucker Diabetic Fatty (ZDF) rats. Cell viability was measured by flow cytometry and the colorimetric MTT assay. Migration was analyzed using the scratch assay. Nrf2 pathway activation was assessed by western blot and confocal microscopy. The balloon carotid injury model was performed in male ZDF rats after onset of diabetes. CA (100 µM) in Pluronic F127 (100 µl) or vehicle alone was applied to the periadventitial surface of the carotid after balloon injury. Arteries were harvested 3 days after surgery for BrdU incorporation, and Nrf2 localization analysis. Neointimal hyperplasia was assessed by morphometric analysis of H&E-stained carotid cross-sections 2 weeks after injury. Results CA inhibited VSMC proliferation by MTT (EC50 = 230 ± 19 µM). By flow cytometry, CA (100 µM) caused a 30% reduction in total cell number (P Conclusion CA effectively inhibited neointimal hyperplasia in a rat model of DM. Localized Nrf2 activation may provide a new therapeutic target for inhibiting restenosis and improving surgery outcomes.