Janet M. Vercammen

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.

Targeted Nitric Oxide Delivery by Supramolecular Nanofibers for the Prevention of Restenosis After Arterial Injury

AIMS Cardiovascular interventions continue to fail as a result of arterial restenosis secondary to neointimal hyperplasia. We sought to develop and evaluate a systemically delivered nanostructure targeted to the site of arterial injury to prevent neointimal hyperplasia. Nanostructures were based on self-assembling biodegradable molecules known as peptide amphiphiles. The targeting motif was a collagen-binding peptide, and the therapeutic moiety was added by S-nitrosylation of cysteine residues. RESULTS Structure of the nanofibers was characterized by transmission electron microscopy and small-angle X-ray scattering. S-nitrosylation was confirmed by mass spectrometry, and nitric oxide (NO) release was assessed electrochemically and by chemiluminescent detection. The balloon carotid artery injury model was performed on 10-week-old male Sprague-Dawley rats. Immediately after injury, nanofibers were administered systemically via tail vein injection. S-nitrosylated (S-nitrosyl [SNO])-targeted nanofibers significantly reduced neointimal hyperplasia 2 weeks and 7 months following balloon angioplasty, with no change in inflammation. INNOVATION This is the first time that an S-nitrosothiol (RSNO)-based therapeutic was shown to have targeted local effects after systemic administration. This approach, combining supramolecular nanostructures with a therapeutic NO-based payload and a targeting moiety, overcomes the limitations of delivering NO to a site of interest, avoiding undesirable systemic side effects. CONCLUSION We successfully synthesized and characterized an RSNO-based therapy that when administered systemically, targets directly to the site of vascular injury. By integrating therapeutic and targeting chemistries, these targeted SNO nanofibers provided durable inhibition of neointimal hyperplasia in vivo and show great potential as a platform to treat cardiovascular diseases.

Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall

Superoxide (O2(•-)) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide ((•)NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether (•)NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether (•)NO exerts cell-specific, differential modulation of O2(•-) levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates (•)NO-dependent inhibition of neointimal hyperplasia. In vivo, (•)NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, (•)NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2(•-) levels. Lastly, to determine the role of SOD-1 in (•)NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without (•)NO. Interestingly, (•)NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2(•-) by (•)NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of (•)NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats

Oral all-trans retinoic acid (atRA) has been shown to reduce the formation of neointimal hyperplasia; however, the dose required was 30 times the chemotherapeutic dose, which already has reported side effects. As neointimal formation is a localized process, new approaches to localized delivery are required. This study assessed whether atRA within a citrate-based polyester, poly(1,8 octanediolcitrate) (POC), perivascular membrane would prevent neointimal hyperplasia following arterial injury. atRA-POC membranes were prepared and characterized for atRA release via high-performance liquid chromatography with mass spectrometry detection. Rat adventitial fibroblasts (AF) and vascular smooth muscle cells (VSMC) were exposed to various concentrations of atRA; proliferation, apoptosis, and necrosis were assessed in vitro. The rat carotid artery balloon injury model was used to evaluate the impact of the atRA-POC membranes on neointimal formation, cell proliferation, apoptosis, macrophage infiltration, and vascular cell adhesion molecule 1 (VCAM-1) expression in vivo. atRA-POC membranes released 12 μg of atRA over 2 wk, with 92% of the release occurring in the first week. At 24 h, atRA (200 μmol/l) inhibited [(3)H]-thymidine incorporation into AF and VSMC by 78% and 72%, respectively (*P = 0.001), with negligible apoptosis or necrosis. Histomorphometry analysis showed that atRA-POC membranes inhibited neointimal formation after balloon injury, with a 56%, 57%, and 50% decrease in the intimal area, intima-to-media area ratio, and percent stenosis, respectively (P = 0.001). atRA-POC membranes had no appreciable effect on apoptosis or proliferation at 2 wk. Regarding biocompatibility, we found a 76% decrease in macrophage infiltration in the intima layer (P < 0.003) in animals treated with atRA-POC membranes, with a coinciding 53% reduction in VCAM-1 staining (P < 0.001). In conclusion, perivascular delivery of atRA inhibited neointimal formation and restenosis. These data suggest that atRA-POC membranes may be suitable as localized therapy to inhibit neointimal hyperplasia following open cardiovascular procedures.

Development and Validation of 4 Different Rat Models of Uncontrolled Hemorrhage

IMPORTANCE Hemorrhage is the leading cause of death in military trauma and second leading cause of death in civilian trauma. Although many well-established animal models of hemorrhage exist in the trauma and anticoagulant literature, few focus on directly quantitating blood loss. OBJECTIVE To establish and validate a reproducible rodent model of uncontrolled hemorrhage to serve as the foundation for developing therapies for noncompressible torso trauma. DESIGN, SETTINGS, AND SUBJECTS We developed and evaluated 4 different hemorrhage models using male Sprague-Dawley rats (6 rats/model), aged 10 to 14 weeks and weighing 330 to 460 g, at the Department of Surgery, Northwestern University. INTERVENTIONS We used tail-cut (4 cm), liver punch biopsy (12 mm), liver laceration (3.0 × 1.5 cm), and spleen transection models. All animals underwent invasive hemodynamic monitoring. MAIN OUTCOMES AND MEASURES Blood loss, expressed as a percentage of total blood volume (TBV), mean arterial pressure, and heart rate, which were recorded at 2- to 5-minute intervals. RESULTS The tail-cut model resulted in a mean (SD) TBV loss of 15.4% (6.0%) with hemodynamics consistent with class I hemorrhagic shock. The liver punch biopsy model resulted in a mean (SD) TBV loss of 16.7% (3.3%) with hemodynamics consistent with class I hemorrhagic shock. The liver laceration model resulted in a mean (SD) TBV loss of 19.8% (3.0%) with hemodynamics consistent with class II hemorrhagic shock. The spleen transection model resulted in the greatest blood loss (P < .01), with a mean (SD) TBV loss of 27.9% (3.4%) and hemodynamics consistent with class II hemorrhagic shock. The liver laceration and punch biopsy models resulted in most of the blood loss within the first 2 minutes, whereas the spleen transection and tail-cut models resulted in a steady loss during 10 minutes. The liver laceration and spleen transection models resulted in the greatest degree of hemodynamic instability (mean [SD] arterial pressure decreases of 25 [1] and 41 [11] mm Hg, respectively). One-hour survival was 100% in all 4 models. CONCLUSIONS AND RELEVANCE We established and validated the reproducibility of 4 different rat models of uncontrolled hemorrhage. These models provide a foundation to design novel nonsurgical therapies to control hemorrhage, and the different degrees of hemorrhagic shock produced from these models allow for flexibility in experimental design.

Systemically administered collagen‐targeted gold nanoparticles bind to arterial injury following vascular interventions

Surgical and endovascular therapies for severe atherosclerosis often fail due to the development of neointimal hyperplasia and arterial restenosis. Our objective was to synthesize, characterize, and evaluate the targeting specificity and biocompatibility of a novel systemically injected nanoparticle. We hypothesize that surface‐functionalization of gold nanoparticles (AuNPs) with a collagen‐targeting peptide will be biocompatible and target specifically to vascular injury. 13 nm AuNPs were surface functionalized with a peptide‐molecular fluorophore and targeted to collagen (T‐AuNP) or a scrambled peptide sequence (S‐AuNP). After rat carotid artery balloon injury and systemic injection of T‐AuNP or S‐AuNP, arteries and organs were harvested and assessed for binding specificity and biocompatibility. The T‐AuNP bound with specificity to vascular injury for a minimum of 24 h. No significant inflammation was evident locally at arterial injury or systemically in major organs. The T‐AuNP did not impact endothelial cell viability or induce apoptosis at the site of injury in vivo. No major changes were evident in hepatic or renal blood chemistry profiles. Herein, we synthesized a biocompatible nanoparticle that targets to vascular injury following systemic administration. These studies demonstrate proof‐of‐principle and serve as the foundation for further T‐AuNP optimization to realize systemic, targeted delivery of therapeutics to the sites of vascular injury.

Inhibiting intimal hyperplasia in prosthetic vascular grafts via immobilized all-trans retinoic acid

&NA; Peripheral arterial disease is a leading cause of morbidity and mortality. The most commonly utilized prosthetic material for peripheral bypass grafting is expanded polytetrafluoroethylene (ePTFE) yet it continues to exhibit poor performance from restenosis due to neointimal hyperplasia, especially in femoral distal bypass procedures. Recently, we demonstrated that periadventitial delivery of all‐trans retinoic acid (atRA) immobilized throughout porous poly(1,8 octamethylene citrate) (POC) membranes inhibited neointimal formation in a rat arterial injury model. Thus, the objective of this study was to investigate whether atRA immobilized throughout the lumen of ePTFE vascular grafts would inhibit intimal formation following arterial bypass grafting. Utilizing standard ePTFE, two types of atRA‐containing ePTFE vascular grafts were fabricated and evaluated: grafts whereby all‐trans retinoic acid was directly immobilized on ePTFE (atRA‐ePTFE) and grafts where all‐trans retinoic acid was immobilized onto ePTFE grafts coated with POC (atRA‐POC‐ePTFE). All grafts were characterized by SEM, HPLC, and FTIR and physical characteristics were evaluated in vitro. Modification of these grafts, did not significantly alter their physical characteristics or biocompatibility, and resulted in inhibition of intimal formation in a rat aortic bypass model, with atRA‐POC‐ePTFE inhibiting intimal formation at both the proximal and distal graft sections. In addition, treatment with atRA‐POC‐ePTFE resulted in increased graft endothelialization and decreased inflammation when compared to the other treatment groups. This work further confirms the biocompatibility and efficacy of locally delivered atRA to inhibit intimal formation in a bypass setting. Thus, atRA‐POC‐ePTFE grafts have the potential to improve patency rates in small diameter bypass grafts and warrant further investigation. Graphical abstract Figure. No caption available.

Long-term effect of PROLI/NO on cellular proliferation and phenotype after arterial injury.

Vascular interventions are associated with high failure rates from restenosis secondary to negative remodeling and neointimal hyperplasia. Periadventitial delivery of nitric oxide (NO) inhibits neointimal hyperplasia, preserving lumen patency. With the development of new localized delivery vehicles, NO-based therapies remain a promising therapeutic avenue for the prevention of restenosis. While the time course of events during neointimal development has been well established, a full characterization of the impact of NO donors on the cells that comprise the arterial wall has not been performed. Thus, the aim of our study was to perform a detailed assessment of proliferation, cellularity, inflammation, and phenotypic cellular modulation in injured arteries treated with the short-lived NO donor, PROLI/NO. PROLI/NO provided durable inhibition of neointimal hyperplasia for 6 months after arterial injury. PROLI/NO inhibited proliferation and cellularity in the media and intima at all of the time points studied. However, PROLI/NO caused an increase in adventitial proliferation at 2 weeks, resulting in increased cellularity at 2 and 8 weeks compared to injury alone. PROLI/NO promoted local protein S-nitrosation and increased local tyrosine nitration, without measurable systemic effects. PROLI/NO predominantly inhibited contractile smooth muscle cells in the intima and media, and had little to no effect on vascular smooth muscle cells or myofibroblasts in the adventitia. Finally, PROLI/NO caused a delayed and decreased leukocyte infiltration response after injury. Our results show that a short-lived NO donor exerts durable effects on proliferation, phenotype modulation, and inflammation that result in long-term inhibition of neointimal hyperplasia.

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.

Novel large animal model of xanthoma formation.

0923-1811/ ã 2015 Japanese Society for Investigative Dermatology. Published by Elsev presentation, underlying etiology, and histological features. Initial treatment for xanthomas relies on treating the underlying dyslipidemia. Surgical excision or local destruction may be appropriate for lesions that do not respond to lipid-lowering therapy and impair function or are disfiguring [1]. To better understand the underlying pathogenesis of xanthomas and develop new medical therapeutic approaches, a reproducible animal model of xanthoma formation is needed. Currently, the exact mechanism of formation is unknown, but xanthomas are believed to result from low density lipoprotein infiltration of the dermis and subsequent phagocytosis by resident macrophages, which then become foam cells. Xanthomas have been reported to occur in mice, rats, and rabbits with hyperlipidemia [2–4], but major differences between their skin and human skin in terms of structure, mechanism of healing, and immune