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.

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.

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.

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.

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.

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.

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.

Pharmacokinetics and biodistribution of a collagen-targeted peptide amphiphile for cardiovascular applications.

Atherosclerosis remains a leading cause of death and disability around the world and a major driver of health care spending. Nanomaterials have gained widespread attention due to their promising potential for clinical translation and use. We have developed a collagen-targeted peptide amphiphile (PA)-based nanofiber for the prevention of neointimal hyperplasia after arterial injury. Our goal was to characterize the pharmacokinetics and biodistribution of the collagen-targeted PA to further its advancement into clinical trials. Collagen-targeted PA was injected into the internal jugular vein of Sprague Dawley rats. PA concentrations in plasma collected at various times after injection (0 to 72 hours) were measured by liquid chromatography-tandem mass spectrometry. Pharmacokinetics of the collagen-targeted PA were characterized by a three-compartment model, with an extremely rapid apparent elimination clearance resulting in a plasma concentration decrease of more than two orders of magnitude within the first hour after injection. This rapid initial decline in plasma concentration was not due to degradation by plasma components, as collagen-targeted PA was stable in plasma ex vivo for up to 3 hours. Indeed, cellular blood components appear to be partly responsible, as only 15% of collagen-targeted PA were recovered following incubation with whole blood. Nanofibers in whole blood also adhered to red blood cells (RBCs) and were engulfed by mononuclear cells. This work highlights the unique pharmacokinetics of our collagen-targeted PA, which differ from pharmacokinetics of small molecules. Because of their targeted nature, these nanomaterials should not require sustained elevated plasma concentrations to achieve a therapeutic effect the way small molecules typically do.

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.

Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

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.

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.

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.

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.

Peptide Amphiphile Supramolecular Nanostructures as a Targeted Therapy for Atherosclerosis.

The rising prevalence of cardiovascular disease worldwide necessitates novel therapeutic approaches to manage atherosclerosis. Intravenously administered nanostructures are a promising noninvasive approach to deliver therapeutics that reduce plaque burden. The drug liver X receptor agonist GW3965 (LXR) can reduce atherosclerosis by promoting cholesterol efflux from plaque but causes liver toxicity when administered systemically at effective doses, thus preventing its clinical use. The ability of peptide amphiphile nanofibers containing apolipoprotein A1-derived targeting peptide 4F to serve as nanocarriers for LXR delivery (ApoA1-LXR PA) in vivo is investigated here. These nanostructures are found to successfully target atherosclerotic lesions in a mouse model within 24 h of injection. After 8 weeks of intravenous administration, the nanostructures significantly reduce plaque burden in both male and female mice to a similar extent as LXR alone in comparison to saline-treated controls. Furthermore, they do not cause increased liver toxicity in comparison to LXR treatments, which may be related to more controlled release by the nanostructure. These findings demonstrate the potential of supramolecular nanostructures as safe, effective drug nanocarriers to manage atherosclerosis.

Atheroma Niche-Responsive Nanocarriers for Immunotherapeutic Delivery.

Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.

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.

Tissue-Factor Targeted Peptide Amphiphile Nanofibers as an Injectable Therapy To Control Hemorrhage.

Noncompressible torso hemorrhage is a leading cause of mortality in civilian and battlefield trauma. We sought to develop an i.v.-injectable, tissue factor (TF)-targeted nanotherapy to stop hemorrhage. Tissue factor was chosen as a target because it is only exposed to the intravascular space upon vessel disruption. Peptide amphiphile (PA) monomers that self-assemble into nanofibers were chosen as the delivery vehicle. Three TF-binding sequences were identified (EGR, RLM, and RTL), covalently incorporated into the PA backbone, and shown to self-assemble into nanofibers by cryo-transmission electron microscopy. Both the RLM and RTL peptides bound recombinant TF in vitro. All three TF-targeted nanofibers bound to the site of punch biopsy-induced liver hemorrhage in vivo, but only RTL nanofibers reduced blood loss versus sham (53% reduction, p < 0.05). Increasing the targeting ligand density of RTL nanofibers yielded qualitatively better binding to the site of injury and greater reductions in blood loss in vivo (p < 0.05). In fact, 100% RTL nanofiber reduced overall blood loss by 60% versus sham (p < 0.05). Evaluation of the biocompatibility of the RTL nanofiber revealed that it did not induce RBC hemolysis, did not induce neutrophil or macrophage inflammation at the site of liver injury, and 70% remained intact in plasma after 30 min. In summary, these studies demonstrate successful binding of peptides to TF in vitro and successful homing of a TF-targeted PA nanofiber to the site of hemorrhage with an associated decrease in blood loss in vivo. Thus, this therapeutic may potentially treat noncompressible hemorrhage.

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.