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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.

Atherosclerotic vascular disease is the leading cause of death world-wide with few novel therapies available in spite of the ongoing health burden. Oxidative stress is a well-established driver of atherosclerotic progression; however the clinical translation of redox-based therapies is lacking. One of the challenges facing redox-based therapies is their targeted delivery to cellular domains of redox dysregulation. In the current study we sought to develop NPs encapsulating redox-based interventions that exploit passive means of targeting to selectively accumulate in atherosclerotic plaque with the aim of enhancing the intra-plaque bioavailability of interventions. Herein we present two types of nanoparticles (NPs): (i) We have employed flash nanoprecipitation to synthesize polymeric NPs encapsulating the hydrophobic Nrf2 activator drug, CDDO-Methyl, (ii) we have generated LDL-like NPs encapsulating the anti-inflammatory compound, oleic acid (OA). Nrf2-activators are a promising class of redox-active drug molecules whereby activation of Nrf2 results in the expression of several antioxidant and cyto-protective enzymes. Moreover, local activation of Nrf2 within the atherosclerotic plaque can be athero-protective. In this study we characterize the physiochemical properties of these NPs as well as confirm in vitro association of NPs with murine macrophages. In vitro drug release of CDDO-Me from polymeric NPs was determined by Nrf2-ARE-driven GFP fluorescence. In vivo localization was assessed through immunofluorescence of histological sections as well as whole-tissue light sheet fluorescence microscopy. We show that CDDO-Me-NPs and LDL-OA-NPs selectively accumulate in atherosclerotic plaque of two widely-used murine models of atherosclerosis: ApoE-/-and LDLr-/-mice. Overall, these studies underline that targeting of atherosclerotic plaque is an effective means to enhance delivery of redox-based interventions. Future work will assess the therapeutic efficacy of intra-plaque Nrf2 activation or anti-inflammatory actions with CDDO-Me-NPs or LDL-OA-NPs, respectively.

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.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Reactive oxygen and nitrogen species are indispensable in cellular physiology and signaling. Overproduction of these reactive species or failure to maintain their levels within the physiological range results in cellular redox dysfunction, often termed cellular oxidative stress. Redox dysfunction in turn is at the molecular basis of disease etiology and progression. Accordingly, antioxidant intervention to restore redox homeostasis has been pursued as a therapeutic strategy for cardiovascular disease, cancer, and neurodegenerative disorders among many others. Despite preliminary success in cellular and animal models, redox-based interventions have virtually been ineffective in clinical trials. We propose the fundamental reason for their failure is a flawed delivery approach. Namely, systemic delivery for a geographically local disease limits the effectiveness of the antioxidant. We take a critical look at the literature and evaluate successful and unsuccessful approaches to translation of redox intervention to the clinical arena, including dose, patient selection, and delivery approach. We argue that when interpreting a failed antioxidant-based clinical trial, it is crucial to take into account these variables and importantly, whether the drug had an effect on the redox status. Finally, we propose that local and targeted delivery hold promise to translate redox-based therapies from the bench to the bedside.

Exposure to elevated levels of ambient ozone in photochemical smog is associated with eosinophilic airway inflammation and nonatopic asthma in children. In the present study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced nonatopic asthma by using lymphoid cell-sufficient C57BL/6 mice, ILC-sufficient Rag2−/− mice (devoid of T and B cells), and ILC-deficient Rag2−/−Il2rg−/− mice (depleted of all lymphoid cells including ILCs). Mice were exposed to 0 or 0.8 parts per million ozone for 1 day or 9 consecutive weekdays (4 hr/day). A single exposure to ozone caused neutrophilic inflammation, airway epithelial injury, and reparative DNA synthesis in all strains of mice, irrespective of the presence or absence of ILCs. In contrast, 9-day exposures induced eosinophilic inflammation and mucous cell metaplasia only in the lungs of ILC-sufficient mice. Repeated ozone exposures also elicited increased messenger RNA expression of transcripts associated with type 2 immunity and airway mucus production in ILC-sufficient mice. ILC-deficient mice repeatedly exposed to ozone had no pulmonary pathology or increased gene expression related to type 2 immunity. These results suggest a new paradigm for the biologic mechanisms underlying the development of a phenotype of childhood nonatopic asthma that has been linked to ambient ozone exposures.

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.

Epidemiological studies have demonstrated the exacerbation of respiratory diseases following sandstorm-derived particulate matter (PM) exposure. The presence of anthropogenic and biological agents on the sandstorm PM and the escalation of PM

Exposure to airborne particulate matter (PM) is a major risk factor for allergic airway inflammation such as asthma. Many of the PM components (i.e., polycyclic aromatic hydrocarbons and metals) are redox-active and capable of inducing cellular oxidative stress and injuries including inflammation and cell death. Airway epithelial cells and antigen-presenting dendritic cells (DC) are the major and direct targets of inhaled PM. The epithelial cells can further enhance the DC response to allergen and PM through several immune regulatory cytokines including thymic stromal lymphopoietin (TSLP), IL-33, and IL-25. Among these cytokines TSLP is particularly relevant to the mechanisms by which particulate air pollutants contribute to asthma pathogenesis. Studies have found that TSLP released by PM-exposed human airway epithelial cells could polarize the DC towards a T-helper 2 immune response, which is one of the key immunological mechanisms in asthma pathogenesis. The convergence of regulatory signals generated by PM-induced oxidative stress in DC and the interactions among them may be one of the major mechanisms that are specifically related to the contribution of PM towards asthma pathogenesis.

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.