Emerging antenatal therapies for congenital diaphragmatic hernia-induced pulmonary hypertension in preclinical models.

Congenital diaphragmatic hernia (CDH)-related deaths are the largest contributor to in-hospital neonatal deaths in children with congenital malformations. Morbidity and mortality in CDH are directly related to the development of pulmonary hypertension (PH). Current treatment consists of supportive measures. To date, no pharmacotherapy has been shown to effectively reverse the hallmark finding of pulmonary vascular remodeling that is associated with pulmonary hypertension in CDH (CDH-PH). As such, there is a great need for novel therapies to effectively manage CDH-PH. Our review aims to evaluate emerging therapies, and specifically focuses on those that are still under investigation and not approved for clinical use by the Food and Drug Administration. Therapies were categorized into antenatal pharmacotherapies or antenatal regenerative therapies and assessed on their method of administration, safety profile, the effect on pulmonary vascular pathophysiology, and overall efficacy. In general, emerging antenatal pharmaceutical and regenerative treatments primarily aim to alleviate pulmonary vascular remodeling by restoring normal function and levels of key regulatory factors involved in pulmonary vascular development and/or in promoting angiogenesis. Overall, while these emerging therapies show great promise for the management of CDH-PH, most require further assessment of safety and efficacy in preclinical models before translation into the clinical setting. IMPACT: Emerging antenatal therapies for congenital diaphragmatic hernia-induced pulmonary hypertension (CDH-PH) show promise to effectively mitigate vascular remodeling in preclinical models. Further investigation is needed in preclinical and human studies to evaluate safety and efficacy prior to translation into the clinical arena. This review offers a comprehensive and up-to-date summary of emerging therapies currently under investigation in experimental animal models. There is no cure for CDH-PH. This review explores emerging therapeutic options for the treatment of CDH-PH and evaluates their impact on key molecular pathways and clinical markers of disease to determine efficacy in the preclinical stage.

A comparative study of a preclinical survival model of smoke inhalation injury in mice and rats.

Smoke inhalation injury increases morbidity and mortality. Clinically relevant animal models are necessary for the continued investigation of the pathophysiology of inhalation injury and the development of therapeutics. The goal of our research was threefold: 1) to develop a reproducible survival model of smoke inhalation injury in rats that closely resembled our previous mouse model, 2) to validate the rat smoke inhalation injury model using a variety of laboratory techniques, and 3) to compare and contrast our rat model with both the well-established mouse model and previously published rat models to highlight our improvements on smoke delivery and lung injury. Mice and rats were anesthetized, intubated, and placed in custom-built smoke chambers to passively inhale woodchip-generated smoke. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for confirmatory tests. Lung sections were hematoxylin and eosin stained, lung edema was assessed with wet-to-dry (W/D) ratio, and inflammatory cell infiltration and cytokine elevation were evaluated using flow cytometry, immunohistochemistry, and ELISA. We confirmed that our mouse and rat models of smoke inhalation injury mimic the injury seen after human burn inhalation injury with evidence of pulmonary edema, neutrophil infiltration, and inflammatory cytokine elevation. Interestingly, rats mounted a more severe immunological response compared with mice. In summary, we successfully validated a reliable and clinically translatable survival model of lung injury and immune response in rats and mice and characterized the extent of this injury. These animal models allow for the continued study of smoke inhalation pathophysiology to ultimately develop a better therapeutic.

Emerging therapies for smoke inhalation injury: a review.

BACKGROUND: Smoke inhalation injury increases overall burn mortality by up to 20 times. Current therapy remains supportive with a failure to identify an optimal or targeted treatment protocol for smoke inhalation injury. The goal of this review is to describe emerging therapies that are being developed to treat the pulmonary pathology induced by smoke inhalation injury with or without concurrent burn injury. MAIN BODY: A comprehensive literature search was performed using PubMed (1995-present) for therapies not approved by the U.S. Food and Drug Administration (FDA) for smoke inhalation injury with or without concurrent burn injury. Therapies were divided based on therapeutic strategy. Models included inhalation alone with or without concurrent burn injury. Specific animal model, mechanism of action of medication, route of administration, therapeutic benefit, safety, mortality benefit, and efficacy were reviewed. Multiple potential therapies for smoke inhalation injury with or without burn injury are currently under investigation. These include stem cell therapy, anticoagulation therapy, selectin inhibition, inflammatory pathway modulation, superoxide and peroxynitrite decomposition, selective nitric oxide synthase inhibition, hydrogen sulfide, HMG-CoA reductase inhibition, proton pump inhibition, and targeted nanotherapies. While each of these approaches shows a potential therapeutic benefit to treating inhalation injury in animal models, further research including mortality benefit is needed to ensure safety and efficacy in humans. CONCLUSIONS: Multiple novel therapies currently under active investigation to treat smoke inhalation injury show promising results. Much research remains to be conducted before these emerging therapies can be translated to the clinical arena.

Emerging Therapies for Prehospital Control of Hemorrhage.

BACKGROUND: The aim of this review was to describe emerging therapies that could serve as a prehospital intervention to slow or stop noncompressible torso hemorrhage in the civilian and military settings. Hemorrhage accounts for 90% of potentially survivable military deaths and 30%-40% of trauma deaths. There is a great need to develop novel therapies to slow or stop noncompressible torso hemorrhage at the scene of the injury. METHODS: A comprehensive literature search was performed using PubMed (1966 to present) for therapies not approved by the Food and Drug Administration for noncompressible torso hemorrhage in the prehospital setting. Therapies were divided into compressive versus intravascular injectable therapies. Ease of administration, skill required to use the therapy, safety profile, stability, shelf-life, mortality benefit, and efficacy were reviewed. RESULTS: Multiple potential therapies for noncompressible torso hemorrhage are currently under active investigation. These include (1) tamponade therapies, such as gas insufflation and polyurethane foam injection; (2) freeze-dried blood products and alternatives such as lyophilized platelets; (3) nanoscale injectable therapies such as polyethylene glycol nanospheres, polyethylenimine nanoparticles, SynthoPlate, and tissue factor-targeted nanofibers; and (4) other injectable therapies such as polySTAT and adenosine, lidocaine, and magnesium. Although each of these therapies shows great promise at slowing or stopping hemorrhage in animal models of noncompressible hemorrhage, further research is needed to ensure safety and efficacy in humans. CONCLUSIONS: Multiple novel therapies are currently under active investigation to slow or stop noncompressible torso hemorrhage in the prehospital setting and show promising results.

Nitric oxide differentially affects proteasome activator 28 after arterial injury in type 1 and type 2 diabetic rats.

BACKGROUND: Diabetic patients display aggressive restenosis after vascular interventions, likely because of proproliferative influences of hyperglycemia and hyperinsulinemia. We have shown that nitric oxide (NO) inhibits neointimal hyperplasia in type 2, but not in type 1, diabetic rats. Here, we examined proteasome activator 28 (PA28) after arterial injury in different diabetic environments, with or without NO. We hypothesize that NO differentially affects PA28 levels based on metabolic environment. MATERIALS AND METHODS: Vascular smooth muscle cell (VSMC) lysates from male, nondiabetic Lean Zucker (LZ) and Zucker Diabetic Fatty (ZDF) rats were assayed for 26S proteasome activity with or without PA28 and S-nitroso-N-acetylpenicillamine. LZ and ZDF VSMCs were treated with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate for 24 h. Balloon-injured carotid arteries from LZ, streptozotocin-injected LZ (STZ, type 1), and ZDF (type 2) rats treated with disodium 1-[2-(carboxylato)pyrrolidin-1-iyl]diazen-1-ium-1,2-diolate were harvested at 3 or 14 d. PA28α was assessed by Western blotting and immunofluorescent staining. RESULTS: S-nitroso-N-acetylpenicillamine reversed PA28-stimulated increases in 26S proteasome activity in LZ and ZDF VSMCs. Increased (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate lowered PA28α in LZ VSMCs but increased PA28α in ZDF VSMCs. At 3 d after injury, disodium 1-[2-(carboxylato)pyrrolidin-1-iyl]diazen-1-ium-1,2-diolate potentiated injury-induced PA28α decreases in LZ, STZ, and ZDF rats, suggesting VSMCs, depleted at this early time point, are major sources of PA28α. At 14 d after injury, total PA28α staining returned to baseline. However, although intimal and medial PA28α staining increased in injured STZ rats, adventitial PA28α staining increased in injured ZDF rats. CONCLUSIONS: PA28 dysregulation may explain the differential ability of NO to inhibit neointimal hyperplasia in type 1 versus type 2 diabetes.

Nitric oxide affects UbcH10 levels differently in type 1 and type 2 diabetic rats.

BACKGROUND: Nitric oxide (NO) more effectively inhibits neointimal hyperplasia in type 2 diabetic versus nondiabetic and type 1 diabetic rodents. NO also decreases the ubiquitin-conjugating enzyme UbcH10, which is critical to cell-cycle regulation. This study seeks to determine whether UbcH10 levels in the vasculature of diabetic animal models account for the differential efficacy of NO at inhibiting neointimal hyperplasia. MATERIALS AND METHODS: Vascular smooth muscle cells (VSMCs) harvested from nondiabetic lean Zucker (LZ) and type 2 diabetic Zucker diabetic fatty (ZDF) rats were exposed to high glucose (25 mM) and high insulin (24 nM) conditions to mimic the diabetic environment in vitro. LZ, streptozotocin-injected LZ (STZ, type 1 diabetic), and ZDF rats underwent carotid artery balloon injury (±10 mg PROLI/NO), and vessels were harvested at 3 and 14 d. UbcH10 was assessed by Western blotting and immunofluorescent staining. RESULTS: NO more effectively reduced UbcH10 levels in LZ versus ZDF VSMCs; however, addition of insulin and glucose dramatically potentiated the inhibitory effect of NO on UbcH10 in ZDF VSMCs. Three days after balloon injury, Western blotting showed NO decreased free UbcH10 and increased polyubiquitinated UbcH10 levels by 35% in both STZ and ZDF animals. Fourteen days after injury, immunofluorescent staining showed increased UbcH10 levels throughout the arterial wall in all animal models. NO decreased UbcH10 levels in LZ and STZ rats but not in ZDF. CONCLUSIONS: These data suggest a disconnect between UbcH10 levels and neointimal hyperplasia formation in type 2 diabetic models and contribute valuable insight regarding differential efficacy of NO in these models.

Nitric oxide may inhibit neointimal hyperplasia by decreasing isopeptidase T levels and activity in the vasculature.

OBJECTIVE: Isopeptidase T is a cysteine protease deubiquitinating enzyme that hydrolyzes unanchored polyubiquitin chains to free monoubiquitin. Nitric oxide (NO) decreases 26S proteasome activity in vascular smooth muscle cells (VSMCs) and inhibits neointimal hyperplasia in animal models. As NO can cause S-nitrosylation of active-site cysteines, we hypothesize that NO inhibits isopeptidase T activity through S-nitrosylation. Because accumulation of polyubiquitin chains inhibits the 26S proteasome, this may be one mechanism through which NO prevents neointimal hyperplasia. METHODS: To investigate our hypothesis, we examined the effect of NO on isopeptidase T activity, levels, and localization in VSMCs in vitro and in a rat carotid balloon injury model in vivo. RESULTS: NO inhibited recombinant isopeptidase T activity by 82.8% (t = 60 minutes, P < .001 vs control). Dithiothreitol and glutathione (5 mmol/L) both significantly reversed NO-mediated inhibition of isopeptidase T activity (P < .001). NO caused a time-dependent increase in S-nitrosylated isopeptidase T levels in VSMCs, which was reversible with dithiothreitol, indicating that isopeptidase T undergoes reversible S-nitrosylation on exposure to NO in vitro. Although NO did not affect isopeptidase T levels or subcellular localization in VSMCs in vitro, it decreased isopeptidase T levels and increased ubiquitinated proteins after balloon injury in vivo. CONCLUSIONS: Local administration of NO may prevent neointimal hyperplasia by inhibiting isopeptidase T levels and activity in the vasculature, thereby inhibiting the 26S proteasome in VSMCs. These data provide additional mechanistic insights into the ability of NO to prevent neointimal hyperplasia after vascular interventions.

Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.

Exogenous administration of nitric oxide (NO) markedly decreases neointimal hyperplasia following arterial injury in several animal models. However, the effect of NO on neointimal hyperplasia in hypertension remains unknown. Here, we employ the spontaneously hypertensive rat (SHR) strain, inbred from Wistar Kyoto (WKY) rats, and the carotid artery balloon injury model to assess the effects of NO on neointimal hyperplasia development. 2weeks after arterial injury, we showed that both rat strains developed similar levels of neointimal hyperplasia, but local administration of NO was less effective at inhibiting neointimal hyperplasia in the SHR compared to WKY rats (58% vs. 79%, P<0.001). Interestingly, local administration of NO did not affect systemic blood pressure in either rat strain. Compared to WKY, the SHR displayed more proliferation in the media and adventitia following balloon injury, as measured by BrdU incorporation. The SHR also showed more inflammation in the adventitia after injury, as well as more vasa vasorum, than WKY rats. NO treatment reduced the vasa vasorum in the SHR but not WKY rats. Finally, while NO decreased both injury-induced proliferation and inflammation in the SHR, it did not return these parameters to levels seen in WKY rats. We conclude that NO is less effective at inhibiting neointimal hyperplasia in the SHR than WKY rats. This may be due to increased scavenging of NO in the SHR, leading to diminished bioavailability of NO. These data will help to develop novel NO-based therapies that will be equally effective in both normotensive and hypertensive patient populations.

Nitric oxide delivery via a permeable balloon catheter inhibits neointimal growth after arterial injury.

BACKGROUND: Neointimal hyperplasia limits the longevity of vascular interventions. Nitric oxide (NO) is well known to inhibit neointimal hyperplasia. However, delivery of NO to the vasculature is challenging. Our study aims to evaluate the efficacy of delivering NO to the site of injury using a permeable balloon catheter. Our hypothesis is that ultra-short duration NO delivery using a permeable balloon catheter will inhibit neointimal hyperplasia. MATERIALS AND METHODS: Ten-week-old male Sprague-Dawley rats underwent carotid artery balloon injury. Groups included: (1) control, (2) injury, (3) injury + periadventitial NO, and (4) injury + endoluminal NO via permeable balloon catheter. The catheter was inflated to 5 atm pressure for 5 min. Arteries were harvested 2 wk following injury. Morphometric assessment for neointimal hyperplasia and immunohistochemical staining for inflammatory markers were performed. RESULTS: Injury increased neointimal hyperplasia compared with control (intima/media area [I/M] ratio 1.07 versus 0.11, respectively, P < 0.001). Periadventitial delivery of NO reduced the I/M area ratio compared with injury alone (55% decrease, P < 0.001). Endoluminal delivery of NO also reduced the I/M area ratio compared with injury alone (65% decrease; P < 0.001). Both endoluminal and periadventitial NO affected the I/M ratio by reducing the intimal area (64% and 46%, respectively, P < 0.001) whereas neither affected the medial area. Periadventitial NO delivery increased lumen area (P < 0.05), whereas endoluminal NO delivery increased circumference (P < 0.05). Periadventitial NO delivery inhibited macrophage intimal infiltration compared with injury alone (P < 0.05). CONCLUSIONS: These data demonstrate that short-duration endoluminal NO delivery via permeable balloon catheters inhibits neointimal hyperplasia following arterial interventions. Endoluminal delivery of NO could become a focus for future clinical interventions.

Systemic peptide amphiphile nanofiber delivery following subcutaneous injection.

Peptide amphiphile (PA) nanofibers have been shown to target and deliver drugs when administered via an intravenous (IV) injection. Subcutaneous administration can broaden the applicability of PA nanofibers in the medical field. The ability of PA nanofibers to be absorbed into systemic circulation after subcutaneous administration was investigated. Four PA molecules with different amino acid sequences were designed to understand the effect of nanofiber cohesion and charge on uptake. Solution small-angle X-ray scattering confirmed nanostructure morphology and provided characteristic lengths for co-assemblies. Circular dichroism and solution wide-angle X-ray scattering confirmed PA secondary structure and molecular order. PAs were co-assembled in a 95 %:5 % molar ratio of unlabeled PA to fluorescently labeled PA. Male and female Sprague Dawley rats were injected in the nape of the neck with PA co-assemblies. In vivo normalized abdominal fluorescence was measured 1-72 h after injection. PA nanofibers with a negative charge and low internal order showed the highest amount of systemic absorption at 1, 6, and 24 h. At 24 h after injection, white blood cell count decreased and glucose was elevated. Glucose began to decrease at 48 h. These data indicate that PA nanofibers can be absorbed into the systemic circulation after subcutaneous injection.

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature.

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, ß, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, ß and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, ß and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, ß and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and ß subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.

Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Nitric oxide increases lysine 48-linked ubiquitination following arterial injury.

BACKGROUND: Proteins are targeted for degradation by the addition of a polyubiquitin chain. Chains of ubiquitin linked via lysine 48 (K48) are associated with protein degradation while chains linked via lysine 63 (K63) are associated with intracellular signaling. We have previously shown that nitric oxide (NO) inhibits neointimal hyperplasia in association with increasing the ubiquitination and degradation of UbcH10. The aim of this study is to characterize the effect of arterial injury and NO on K48- or K63-linked ubiquitination of cellular proteins. METHODS: The rat carotid artery balloon injury model was performed. Treatment groups included uninjured, injury alone, injury + proline NONOate (PROLI/NO), and PROLI/NO alone. Arteries were harvested at designated time points and sectioned for immunohistochemical analysis of K48- and K63-linked ubiquitination or homogenized for protein analysis. Vascular smooth muscle cells (VSMC) harvested from rat aortae were exposed to the NO donor diethylenetriamine NONOate (DETA/NO). Protein expression was determined by Western blot analysis, or immunoprecipitation and Western blot analysis. RESULTS: Arterial injury increased K48-linked ubiquitination in vivo. The addition of PROLI/NO following injury caused a further increase in K48-linked ubiquitination at 1 and 3 d, however, levels returned to that of injury alone by 2 wk. Interestingly, treatment with PROLI/NO alone increased K48-linked ubiquitination in vivo to levels similar to injury alone. There were lesser or opposite changes in K63-linked ubiquitination in all three treatment groups. DETA/NO increased K48-linked ubiquitination in VSMC in vitro but had minimal effects on K63-linked ubiquitination. Low doses of DETA/NO decreased K48-linked ubiquitination of cyclin A and B, while high doses of DETA/NO increased K48-linked ubiquitination of cyclin A and B. Minimal changes were seen in K63-linked ubiquitination of cyclin A and B in vitro. CONCLUSIONS: Arterial injury and NO increased K48-linked ubiquitination in vivo and in vitro. Remarkably, minimal changes were seen in K63-linked ubiquitination. These novel findings provide important insights into the vascular biology of arterial injury and suggest that one mechanism by which NO may prevent neointimal hyperplasia is through regulation of protein ubiquitination.

Coating small-diameter ePTFE vascular grafts with tunable poly(diol-co-citrate-co-ascorbate) elastomers to reduce neointimal hyperplasia.

Lack of long-term patency has hindered the clinical use of small-diameter prosthetic vascular grafts with the majority of these failures due to the development of neointimal hyperplasia. Previous studies by our laboratory revealed that small-diameter expanded polytetrafluoroethylene (ePTFE) grafts coated with antioxidant elastomers are a promising localized therapy to inhibit neointimal hyperplasia. This work is focused on the development of poly(diol-co-citrate-co-ascorbate) (POCA) elastomers with tunable properties for coating ePTFE vascular grafts. A bioactive POCA elastomer (@20 : 20 : 8, [citrate] : [diol] : [ascorbate]) coating was applied on a 1.5 mm diameter ePTFE vascular graft as the most promising therapeutic candidate for reducing neointimal hyperplasia. Surface ascorbate density on the POCA elastomer was increased to 67.5 ± 7.3 ng mg-1 cm-2. The mechanical, antioxidant, biodegradable, and biocompatible properties of POCA demonstrated desirable performance for in vivo use, inhibiting human aortic smooth muscle cell proliferation, while supporting human aortic endothelial cells. POCA elastomer coating number was adjusted by a modified spin-coating method to prepare small-diameter ePTFE vascular grafts similar to natural vessels. A significant reduction in neointimal hyperplasia was observed after implanting POCA-coated ePTFE vascular grafts in a guinea pig aortic interposition bypass graft model. POCA elastomer thus offers a new avenue that shows promise for use in vascular engineering to improve long-term patency rates by coating small-diameter ePTFE vascular grafts.

Development of novel nanofibers targeted to smoke-injured lungs.

Smoke inhalation injury is associated with significant mortality and current therapies remain supportive. The purpose of our study was to identify proteins upregulated in the lung after smoke inhalation injury and develop peptide amphiphile nanofibers that target these proteins. We hypothesize that nanofibers targeted to angiotensin-converting enzyme or receptor for advanced glycation end products will localize to smoke-injured lungs. METHODS: Five targeting sequences were incorporated into peptide amphiphile monomers methodically to optimize nanofiber formation. Nanofiber formation was assessed by conventional transmission electron microscopy. Rats received 8 min of wood smoke. Levels of angiotensin-converting enzyme and receptor for advanced glycation end products were evaluated by immunofluorescence. Rats received the targeted nanofiber 23 h after injury via tail vein injection. Nanofiber localization was determined by fluorescence quantification. RESULTS: Peptide amphiphile purity (>95%) and nanofiber formation were confirmed. Target proteins were increased in smoke inhalation versus sham (p < 0.001). After smoke inhalation and injection of targeted nanofibers, we found a 10-fold increase in angiotensin-converting enzyme-targeted nanofiber localization to lung (p < 0.001) versus sham with minimal localization of non-targeted nanofiber (p < 0.001). CONCLUSIONS: We synthesized, characterized, and evaluated systemically delivered targeted nanofibers that localized to the site of smoke inhalation injury in vivo. Angiotensin-converting enzyme-targeted nanofibers serve as the foundation for developing a novel nanotherapeutic that treats smoke inhalation lung injury.

Self-Assembled Peptide Amphiphile Nanofibers for Controlled Therapeutic Delivery to the Atherosclerotic Niche.

Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers were developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs released LXR in response to physiological levels of ROS and reducing agents and could be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers targeted plaque within an atherosclerotic mouse model in vivo and activated ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.

Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice.

Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non-specific biodistribution. Self-assembled peptide amphiphile (PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off-target toxicity. Here, PA nanofibers that target the angiotensin I-converting enzyme and the receptor for advanced glycation end-products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE-targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia-induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off-target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE-targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.

Isopropylamine NONOate (IPA/NO) moderates neointimal hyperplasia following vascular injury.

OBJECTIVE: Isopropylamine NONOate (IPA/NO) is a nitroxyl (HNO) donor at physiologic pH. HNO is a positive inotrope and vasodilator, but little is known about its effect on neointimal hyperplasia. The aims of this study are to determine the effect of IPA/NO on endothelial and vascular smooth muscle cells (VSMC) in vitro and to determine if IPA/NO inhibits neointimal hyperplasia in vivo. METHODS: VSMC were harvested from the abdominal aortas of male Sprague Dawley rats, and human umbilical vein endothelial cells were purchased from ATCC. In vitro, cellular proliferation was assessed by (3)H-thymidine incorporation, cell migration was assessed using the scrape assay, and cell death was assessed using Guava personal cell analysis (PCA). Cell cycle analysis was performed using propidium iodide staining and flow cytometry analysis. Protein expression was assessed using Western blot analysis. Phosphorylated proteins were assessed using immunoprecipitation and Western blot analysis. In vivo, the carotid artery injury model was performed on male Sprague Dawley rats treated with (n = 12) or without (n = 6) periadventitial IPA/NO (10 mg). Arteries harvested at 2 weeks were assessed for morphometrics using ImageJ. Inflammation was assessed using immunohistochemistry. Endothelialization was assessed by Evans blue staining of carotid arteries harvested 7 days after balloon injury from rats treated with (n = 6) or without (n = 3) periadventitial IPA/NO (10 mg). RESULTS: In vitro, 1000 micromol/L IPA/NO inhibited both VSMC (38.7 +/- 4.5% inhibition vs control, P = .003) and endothelial cell proliferation (54.0 +/- 2.9% inhibition vs control, P < or = 0.001) without inducing cell death or inhibiting migration. In VSMC, this inhibition was associated with an S-phase cell cycle arrest and increased expression of cyclin A, cyclin D1, and the cyclin-dependent kinase inhibitor p21. No change was noted in the phosphorylation status of cdk2, cdk4, or cdk6 by IPA/NO. In rodents subjected to the carotid artery balloon injury model, IPA/NO caused significant reductions in neointimal area (298 +/- 20 vs 422 +/- 30, P < or = .001) and medial area (311 +/- 14 vs 449 +/- 16, P < or = .001) compared with injury alone, and reduced macrophage infiltration to 1.7 +/- 0.8 from 16.1 +/- 3.5 cells per high power field (P < or = .001). IPA/NO also prevented re-endothelialization compared with injury alone (55.9 +/- 0.5% nonendothelialized vs 21 +/- 4.4%, respectively, P = .001). Lastly, a 50% mortality rate was observed in the IPA/NO-treated groups. CONCLUSIONS: In summary, while IPA/NO modestly inhibited neointimal hyperplasia by inhibiting VSMC proliferation and macrophage infiltration, it also inhibited endothelial cell proliferation and induced significant mortality in our animal model. Since HNO is being investigated as a treatment for congestive heart failure, our results raise some concerns about the use of IPA/NO in the vasculature and suggest that further studies be conducted on the safety of HNO donors in the cardiovascular system.

Development of Poly(1,8-octanediol-co-citrate-co-ascorbate) Elastomers with Enhanced Ascorbate Performance for Use as a Graft Coating to Prevent Neointimal Hyperplasia.

Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg-1 cm-2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg-1 cm-2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young's modulus (2.15-2.61 MPa), elongation (189.5-214.6%) and ultimate tensile strength (2.73-3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.

Development of Fractalkine-Targeted Nanofibers that Localize to Sites of Arterial Injury.

Atherosclerosis is the leading cause of death and disability around the world, with current treatments limited by neointimal hyperplasia. Our goal was to synthesize, characterize, and evaluate an injectable, targeted nanomaterial that will specifically bind to the site of arterial injury. Our target protein is fractalkine, a chemokine involved in both neointimal hyperplasia and atherosclerosis. We showed increased fractalkine staining in rat carotid arteries 24 h following arterial injury and in the aorta of low-density lipoprotein receptor knockout (LDLR-/-) mice fed a high-fat diet for 16 weeks. Three peptide amphiphiles (PAs) were synthesized: fractalkine-targeted, scrambled, and a backbone PA. PAs were ≥90% pure on liquid chromatography/mass spectrometry (LCMS) and showed nanofiber formation on transmission electron microscopy (TEM). Rats systemically injected with fractalkine-targeted nanofibers 24 h after carotid artery balloon injury exhibited a 4.2-fold increase in fluorescence in the injured artery compared to the scrambled nanofiber (p < 0.001). No localization was observed in the non-injured artery or with the backbone nanofiber. Fluorescence of the fractalkine-targeted nanofiber increased in a dose dependent manner and was observed for up to 48 h. These data demonstrate the presence of fractalkine after arterial injury and the localization of our fractalkine-targeted nanofiber to the site of injury and serve as the foundation to develop this technology further.