Targeted inhibition of thrombin attenuates murine neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and an orphan disease with no specific treatment. Most patients with confirmed NEC develop moderate-severe thrombocytopenia requiring one or more platelet transfusions. Here we used our neonatal murine model of NEC-related thrombocytopenia to investigate mechanisms of platelet depletion associated with this disease [K. Namachivayam, K. MohanKumar, L. Garg, B. A. Torres, A. Maheshwari, Pediatr. Res. 81, 817-824 (2017)]. In this model, enteral administration of immunogen trinitrobenzene sulfonate (TNBS) in 10-d-old mouse pups produces an acute necrotizing ileocolitis resembling human NEC within 24 h, and these mice developed thrombocytopenia at 12 to 15 h. We hypothesized that platelet activation and depletion occur during intestinal injury following exposure to bacterial products translocated across the damaged mucosa. Surprisingly, platelet activation began in our model 3 h after TNBS administration, antedating mucosal injury or endotoxinemia. Platelet activation was triggered by thrombin, which, in turn, was activated by tissue factor released from intestinal macrophages. Compared to adults, neonatal platelets showed enhanced sensitivity to thrombin due to higher expression of several downstream signaling mediators and the deficiency of endogenous thrombin antagonists. The expression of tissue factor in intestinal macrophages was also unique to the neonate. Targeted inhibition of thrombin by a nanomedicine-based approach was protective without increasing interstitial hemorrhages in the inflamed bowel or other organs. In support of these data, we detected increased circulating tissue factor and thrombin-antithrombin complexes in patients with NEC. Our findings show that platelet activation is an important pathophysiological event and a potential therapeutic target in NEC.

Peptide-Based Nanoparticles for Systemic Extrahepatic Delivery of Therapeutic Nucleotides.

Peptide-based nanoparticles (PBN) for nucleotide complexation and targeting of extrahepatic diseases are gaining recognition as potent pharmaceutical vehicles for fine-tuned control of protein production (up- and/or down-regulation) and for gene delivery. Herein, we review the principles and mechanisms underpinning self-assembled formation of PBN, cellular uptake, endosomal release, and delivery to extrahepatic disease sites after systemic administration. Selected examples of PBN that have demonstrated recent proof of concept in disease models in vivo are summarized to offer the reader a comparative view of the field and the possibilities for clinical application.

Simultaneous Inhibition of Thrombosis and Inflammation Is Beneficial in Treating Acute Myocardial Infarction.

Myocardial ischemia reperfusion injury (IRI) in acute coronary syndromes is a condition in which ischemic/hypoxic injury to cells subtended by the occluded vessel continues despite successful resolution of the thrombotic obstruction. For decades, most efforts to attenuate IRI have focused on interdicting singular molecular targets or pathways, but none have successfully transitioned to clinical use. In this work, we investigate a nanoparticle-based therapeutic strategy for profound but local thrombin inhibition that may simultaneously mitigate both thrombosis and inflammatory signaling pathways to limit myocardial IRI. Perfluorocarbon nanoparticles (PFC NP) were covalently coupled with an irreversible thrombin inhibitor, PPACK (Phe[D]-Pro-Arg-Chloromethylketone), and delivered intravenously to animals in a single dose prior to ischemia reperfusion injury. Fluorescent microscopy of tissue sections and 19F magnetic resonance images of whole hearts ex vivo demonstrated abundant delivery of PFC NP to the area at risk. Echocardiography at 24 h after reperfusion demonstrated preserved ventricular structure and improved function. Treatment reduced thrombin deposition, suppressed endothelial activation, inhibited inflammasome signaling pathways, and limited microvascular injury and vascular pruning in infarct border zones. Accordingly, thrombin inhibition with an extraordinarily potent but locally acting agent suggested a critical role for thrombin and a promising therapeutic strategy in cardiac IRI.

Rapamycin Perfluorocarbon Nanoparticle Mitigates Cisplatin-Induced Acute Kidney Injury.

For nearly five decades, cisplatin has played an important role as a standard chemotherapeutic agent and been prescribed to 10-20% of all cancer patients. Although nephrotoxicity associated with platinum-based agents is well recognized, treatment of cisplatin-induced acute kidney injury is mainly supportive and no specific mechanism-based prophylactic approach is available to date. Here, we postulated that systemically delivered rapamycin perfluorocarbon nanoparticles (PFC NP) could reach the injured kidneys at sufficient and sustained concentrations to mitigate cisplatin-induced acute kidney injury and preserve renal function. Using fluorescence microscopic imaging and fluorine magnetic resonance imaging/spectroscopy, we illustrated that rapamycin-loaded PFC NP permeated and were retained in injured kidneys. Histologic evaluation and blood urea nitrogen (BUN) confirmed that renal structure and function were preserved 48 h after cisplatin injury. Similarly, weight loss was slowed down. Using western blotting and immunofluorescence staining, mechanistic studies revealed that rapamycin PFC NP significantly enhanced autophagy in the kidney, reduced the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as decreased the expression of the apoptotic protein Bax, all of which contributed to the suppression of apoptosis that was confirmed with TUNEL staining. In summary, the delivery of an approved agent such as rapamycin in a PFC NP format enhances local delivery and offers a novel mechanism-based prophylactic therapy for cisplatin-induced acute kidney injury.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis.

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200 nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model in vivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

Sustained local inhibition of thrombin preserves renal microarchitecture and function after onset of acute kidney injury.

Acute kidney injury (AKI) management remains mainly supportive as no specific therapeutic agents directed at singular signaling pathways have succeeded in clinical trials. Here, we report that inhibition of thrombin-driven clotting and inflammatory signaling with use of locally-acting thrombin-targeted perfluorocarbon nanoparticles (PFC NP) protects renal vasculature and broadly modulates diverse inflammatory processes that cause renal ischemia reperfusion injury. Each PFC NP was complexed with ~13,650 copies of the direct thrombin inhibitor, PPACK (proline-phenylalanine-arginine-chloromethyl-ketone). Mice treated after the onset of AKI with PPACK PFC NP exhibited downregulated VCAM-1, ICAM-1, PGD2 prostanoid, M-CSF, IL-6, and mast cell infiltrates. Microvascular architecture, tubular basement membranes, and brush border components were better preserved. Non-reperfusion was reduced as indicated by reduced red blood cell trapping and non-heme iron. Kidney function and tubular necrosis improved at 24 hours versus the untreated control group, suggesting a benefit for dual inhibition of thrombosis and inflammation by PPACK PFC NP.

Division of labor in honey bees is associated with transcriptional regulatory plasticity in the brain.

Studies in evolutionary and developmental biology show that relationships between transcription factors (TFs) and their target genes can be altered to result in novel regulatory relationships that generate phenotypic plasticity. We hypothesized that context-dependent shifts in the nervous system associated with behavior may also be linked to changes in TF-target relationships over physiological time scales. We tested this hypothesis using honey bee (Apis mellifera) division of labor as a model system by performing bioinformatic analyses of previously published brain transcriptomic profiles together with new RNAi and behavioral experiments. The bioinformatic analyses identified five TFs that exhibited strong signatures of regulatory plasticity as a function of division of labor. RNAi targeting of one of these TFs (broad complex) and a related TF that did not exhibit plasticity (fushi tarazu transcription factor 1) was administered in conjunction with automated analyses of foraging behavior in the field, laboratory assays of aggression and brood care behavior, and endocrine treatments. The results showed that changes in the regulatory relationships of these TFs were associated with behavioral state, social context and endocrine state. These findings provide the first empirical evidence that TF-target relationships in the brain are altered in conjunction with behavior and social context. They also suggest that one mechanism for this plasticity involves pleiotropic TFs high up in regulatory hierarchies producing behavior-specific transcriptional responses by activating different downstream TFs to induce discrete context-dependent transcriptional cascades. These findings provide new insights into the dynamic nature of the transcriptional regulatory architecture underlying behavior in the brain.

Suppression of NF-κB activity via nanoparticle-based siRNA delivery alters early cartilage responses to injury.

Osteoarthritis (OA) is a major cause of disability and morbidity in the aging population. Joint injury leads to cartilage damage, a known determinant for subsequent development of posttraumatic OA, which accounts for 12% of all OA. Understanding the early molecular and cellular responses postinjury may provide targets for therapeutic interventions that limit articular degeneration. Using a murine model of controlled knee joint impact injury that allows the examination of cartilage responses to injury at specific time points, we show that intraarticular delivery of a peptidic nanoparticle complexed to NF-κB siRNA significantly reduces early chondrocyte apoptosis and reactive synovitis. Our data suggest that NF-κB siRNA nanotherapy maintains cartilage homeostasis by enhancing AMPK signaling while suppressing mTORC1 and Wnt/ß-catenin activity. These findings delineate an extensive crosstalk between NF-κB and signaling pathways that govern cartilage responses postinjury and suggest that delivery of NF-κB siRNA nanotherapy to attenuate early inflammation may limit the chronic consequences of joint injury. Therapeutic benefits of siRNA nanotherapy may also apply to primary OA in which NF-κB activation mediates chondrocyte catabolic responses. Additionally, a critical barrier to the successful development of OA treatment includes ineffective delivery of therapeutic agents to the resident chondrocytes in the avascular cartilage. Here, we show that the peptide-siRNA nanocomplexes are nonimmunogenic, are freely and deeply penetrant to human OA cartilage, and persist in chondrocyte lacunae for at least 2 wk. The peptide-siRNA platform thus provides a clinically relevant and promising approach to overcoming the obstacles of drug delivery to the highly inaccessible chondrocytes.

Quantification of vascular damage in acute kidney injury with fluorine magnetic resonance imaging and spectroscopy.

PURPOSE: To design a fluorine MRI/MR spectroscopy approach to quantify renal vascular damage after ischemia-reperfusion injury, and the therapeutic response to antithrombin nanoparticles (NPs) to protect kidney function. METHODS: A total of 53 rats underwent 45 min of bilateral renal artery occlusion and were treated at reperfusion with either plain perfluorocarbon NPs or NPs functionalized with a direct thrombin inhibitor (PPACK:phenyalanine-proline-arginine-chloromethylketone). Three hours after reperfusion, kidneys underwent ex vivo fluorine MRI/MR spectroscopy at 4.7 T to quantify the extent and volume of trapped NPs, as an index of vascular damage and ischemia-reperfusion injury. Microscopic evaluation of structural damage and NP trapping in non-reperfused renal segments was performed. Serum creatinine was quantified serially over 7 days. RESULTS: The damaged renal cortico-medullary junction trapped a significant volume of NPs (P = 0.04), which correlated linearly (r = 0.64) with the severity of kidney injury 3 h after reperfusion. Despite global large vessel reperfusion, non-reperfusion in medullary peritubular capillaries was confirmed by MRI and microscopy, indicative of continuing hypoxia due to vascular compromise. Treatment of animals with PPACK NPs after acute kidney injury did not accelerate kidney functional recovery. CONCLUSIONS: Quantification of ischemia-reperfusion injury after acute kidney injury with fluorine MRI/MR spectroscopy of perfluorocarbon NPs objectively depicts the extent and severity of vascular injury and its linear relationship to renal dysfunction. The lack of kidney function improvement after early posttreatment thrombin inhibition confirms the rapid onset of ischemia-reperfusion injury as a consequence of vascular damage and non-reperfusion. The prolongation of medullary ischemia renders cortico-medullary tubular structures susceptible to continued necrosis despite restoration of large vessel flow, which suggests limitations to acute interventions after acute kidney injury, designed to interdict renal tubular damage. Magn Reson Med 79:3144-3153, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

NADPH oxidase 4 regulates vascular inflammation in aging and atherosclerosis.

We recently reported that increased NADPH oxidase 4 (NOX4) expression and activity during aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. The goal of the present study was to elucidate the molecular mechanism(s) for these effects and determine the importance of NOX4 modulation of proinflammatory gene expression in mouse vascular smooth muscle cells (VSMCs). A novel peptide-mediated siRNA transfection approach was used to inhibit Nox4 expression with minimal cellular toxicity. Using melittin-derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs. 85% with Lipofectamine) and decreased toxicity (p<0.001 vs. Lipofectamine in MTT and p<0.0001 vs. Lipofectamine in LDH assays) in VSMCs. TGFß1 significantly upregulates Nox4 mRNA (p<0.01) and protein (p<0.01) expression in VSMCs. p5RHH-mediated Nox4 siRNA transfection greatly attenuated TGFß1-induced upregulation of Nox4 mRNA (p<0.01) and protein (p<0.0001) levels and decreased hydrogen peroxide production (p<0.0001). Expression of pro-inflammatory genes Ccl2, Ccl5, Il6, and Vcam1 was significantly upregulated in VSMCs in several settings cells isolated from aged vs. young wild-type mice, in atherosclerotic arteries of Apoe-/- mice, and atherosclerotic human carotid arteries and correlated with NOX4 expression. p5RHH-mediated Nox4 siRNA transfection significantly attenuated the expression of these pro-inflammatory genes in TGFß1-treated mouse VSMCs, with the highest degree of inhibition in the expression of Il6. p5RHH peptide-mediated knockdown of TGFß-activated kinase 1 (TAK1, also known as Map3k7), Jun, and Rela, but not Nfkb2, downregulated TGFß1-induced Nox4 expression in VSMCs. Together, these data demonstrate that increased expression and activation of NOX4, which might result from increased TGFß1 levels seen during aging, induces a proinflammatory phenotype in VSMCs, enhancing atherosclerosis.

Inhibition of Thrombin With PPACK-Nanoparticles Restores Disrupted Endothelial Barriers and Attenuates Thrombotic Risk in Experimental Atherosclerosis.

OBJECTIVE: A role for thrombin in the pathogenesis of atherosclerosis has been suggested through clinical and experimental studies revealing a critical link between the coagulation system and inflammation. Although approved drugs for inhibition of thrombin and thrombin-related signaling have demonstrated efficacy, their clinical application to this end may be limited because of significant potential for bleeding side effects. Thus, we sought to implement a plaque-localizing nanoparticle-based approach to interdict thrombin-induced inflammation and hypercoagulability in atherosclerosis. APPROACH AND RESULTS: We deployed a novel magnetic resonance spectroscopic method to quantify the severity of endothelial damage for correlation with traditional metrics of vessel procoagulant activity after dye-laser injury in fat-fed apolipoprotein E-null mice. We demonstrate that a 1-month course of treatment with antithrombin nanoparticles carrying the potent thrombin inhibitor PPACK (d-phenylalanyl-l-prolyl-l-arginyl chloromethylketone) nanoparticle (1) reduces the expression and secretion of proinflammatory and procoagulant molecules, (2) diminishes plaque procoagulant activity without the need for systemic anticoagulation, (3) rapidly restores disrupted vascular endothelial barriers, and (4) retards plaque progression in lesion-prone areas. CONCLUSIONS: These observations illustrate the role of thrombin as a pleiotropic atherogenic molecule under conditions of hypercholesterolemia and suggest the utility of its inhibition with locally acting antithrombin nanoparticle therapeutics as a rapid-acting anti-inflammatory strategy in atherosclerosis to reduce thrombotic risk.

Antithrombin nanoparticles inhibit stent thrombosis in ex vivo static and flow models.

OBJECTIVE: Despite significant advances in intravascular stent technology, safe prevention of stent thrombosis over prolonged periods after initial deployment persists as a medical need to decrease device failure. The objective of this project was to assess the potential of perfluorocarbon nanoparticles (NP) conjugated with the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-NP) to inhibit stent thrombosis. METHODS: In a static model of stent thrombosis, 3 × 3-mm pieces of stainless steel coronary stents were cut and adsorbed with thrombin to create a procoagulant surface that would facilitate thrombus development. After treatment with PPACK-NP or control NP, stents were exposed to platelet-poor plasma (PPP) or platelet-rich plasma (PRP) for set time points up to 60 minutes. Measurements of final clot weight in grams were used for assessing the effect of NP treatment on limiting thrombosis. Additionally, groups of stents were exposed to flowing plasma containing various treatments (saline, free PPACK, control NP, and PPACK-NP) and generated thrombi were stained and imaged to investigate the treatment effects of PPACK-NP under flow conditions. RESULTS: The static model of stent thrombosis used in this study indicated a significant reduction in thrombus deposition with PPACK-NP treatment (0.00067 ± 0.00026 g; n = 3) compared with control NP (0.0098 ± 0.0015 g; n = 3; P = .026) in PPP. Exposure to PRP demonstrated similar effects with PPACK-NP treatment (0.00033 ± 0.00012 g; n = 3) vs control NP treatment (0.0045 ± 0.00012 g; n = 3; P = .000017). In additional studies, stents were exposed to both PRP pretreated with vorapaxar and PPACK-NP, which illustrated adjunctive benefit to oral platelet inhibitors for prevention of stent thrombosis. Additionally, an in vitro model of stent thrombosis under flow conditions established that PPACK-NP treatment inhibited thrombus deposition on stents significantly. CONCLUSIONS: This study demonstrates that antithrombin perfluorocarbon NPs exert marked focal antithrombin activity to prevent intravascular stent thrombosis and occlusion.

Quantifying progression and regression of thrombotic risk in experimental atherosclerosis.

Currently, there are no generally applicable noninvasive methods for defining the relationship between atherosclerotic vascular damage and risk of focal thrombosis. Herein, we demonstrate methods to delineate the progression and regression of vascular damage in response to an atherogenic diet by quantifying the in vivo accumulation of semipermeable 200-300 nm perfluorocarbon core nanoparticles (PFC-NP) in ApoE null mouse plaques with [(19)F] magnetic resonance spectroscopy (MRS). Permeability to PFC-NP remained minimal until 12 weeks on diet, then increased rapidly following 12 weeks, but regressed to baseline within 8 weeks after diet normalization. Markedly accelerated clotting (53.3% decrease in clotting time) was observed in carotid artery preparations of fat-fed mice subjected to photochemical injury as defined by the time to flow cessation. For all mice on and off diet, an inverse linear relationship was observed between the permeability to PFC-NP and accelerated thrombosis (P = 0.02). Translational feasibility for quantifying plaque permeability and vascular damage in vivo was demonstrated with clinical 3 T MRI of PFC-NP accumulating in plaques of atherosclerotic rabbits. These observations suggest that excessive permeability to PFC-NP may indicate prothrombotic risk in damaged atherosclerotic vasculature, which resolves within weeks after dietary therapy.

Dual-therapy with αvß3-targeted Sn2 lipase-labile fumagillin-prodrug nanoparticles and zoledronic acid in the Vx2 rabbit tumor model.

Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvß3-Fum-PD NP). Dual anti-angiogenic therapy combining αvß3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvß3-Fum-PD NP reduced (P<0.05) endothelial cell viability without impacting macrophage viability. ZA suppressed (P<0.05) macrophage viability at high dosages but not endothelial cell proliferation. 3D MR neovascular imaging of rabbit Vx2 tumors showed no effect with ZA, whereas αvß3-Fum-PD NP alone and with ZA decreased angiogenesis (P<0.05). Immunohistochemistry revealed decreased (P<0.05) microvascularity with αvß3-Fum-PD NP and ZA and further microvascular reduction (P<0.05) with dual-therapy. In vivo, ZA did not decrease tumor macrophage numbers nor cancer cell proliferation, whereas αvß3-Fum-PD-NPs reduced both measures. Dual-therapy with ZA and αvß3-Fum-PD-NP may provide enhanced neo-adjuvant utility if macrophage ZA uptake is increased. From the Clinical Editor: Although anti-angiogenesis is one of the treatment modalities in the fight against cancer, many cancers become resistant to VEGF pathway inhibitors. In this article, the authors investigated the use of dual therapy using fumagillin, integrin-targeted lipid-encapsulated nanoparticles (αvß3- Fum-PD NP) and zoledronic acid (ZA), in both in-vitro and in-vivo experiments. This combination approach may provide an insight to the design of future drugs against cancers.

RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee.

Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing.

Antithrombin nanoparticles improve kidney reperfusion and protect kidney function after ischemia-reperfusion injury.

In the extension phase of acute kidney injury, microvascular thrombosis, inflammation, vasoconstriction, and vascular endothelial cell dysfunction promote progressive damage to renal parenchyma after reperfusion. In this study, we hypothesized that direct targeting and pharmaceutical knockdown of activated thrombin at the sites of injury with a selective nanoparticle (NP)-based thrombin inhibitor, PPACK (phenylalanine-proline-arginine-chloromethylketone), would improve kidney reperfusion and protect renal function after transient warm ischemia in rodent models. Saline- or plain NP-treated animals were employed as controls. In vivo 19F magnetic resonance imaging revealed that kidney nonreperfusion was evident within 3 h after global kidney reperfusion at 34 ± 13% area in the saline group and 43 ± 12% area in the plain NP group and substantially reduced to 17 ± 4% (∼50% decrease, P < 0.05) in the PPACK NP pretreatment group. PPACK NP pretreatment prevented an increase in serum creatinine concentration within 24 h after ischemia-reperfusion, reflecting preserved renal function. Histologic analysis illustrated substantially reduced intrarenal thrombin accumulation within 24 h after reperfusion for PPACK NP-treated kidneys (0.11% ± 0.06%) compared with saline-treated kidneys (0.58 ± 0.37%). These results suggest a direct role for thrombin in the pathophysiology of AKI and a nanomedicine-based preventative strategy for improving kidney reperfusion after transient warm ischemia.

Angiogenesis and airway reactivity in asthmatic Brown Norway rats.

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 µg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvß3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.

Magnetic resonance imaging of melanoma exosomes in lymph nodes.

PURPOSE: Exosomes are cell derived extracellular nanovesicles that relay molecular signals pertinent to both normal physiologic and disease processes. The ability to modify and track exosomes in vivo is essential to understanding exosome pathogenesis, and for utilizing exosomes as effective diagnostic and therapeutic nanocarriers to treat diseases. METHODS: We recently reported a new electroporation method that allow exosomes to be loaded with superparamagnetic iron oxide nanoparticles for magnetic resonance tracking. RESULTS: Building on this approach, we now demonstrate for the first time using a C57BL/6 mouse model that melanoma exosomes can be imaged in vitro, and within lymph nodes in vivo with the use of standard MRI approaches. CONCLUSION: These findings demonstrate proof of principle that exosome biology can be followed in vivo and pave the way for the development of future diagnostic and therapeutic applications. Magn Reson Med 74:266-271, 2015. © 2014 Wiley Periodicals, Inc.

Balanced UTE-SSFP for 19F MR imaging of complex spectra.

PURPOSE: A novel technique for highly sensitive detection of multiresonant fluorine imaging agents was designed and tested with the use of dual-frequency 19F/1H ultrashort echo times (UTE) sampled with a balanced steady-state free precession (SSFP) pulse sequence and three-dimensional (3D) radial readout. METHODS: Feasibility of 3D radial balanced UTE-SSFP imaging was demonstrated for a phantom comprising liquid perfluorooctyl bromide (PFOB). Sensitivity of the pulse sequence was measured and compared with other sequences imaging the PFOB (CF2 )6 line group including UTE radial gradient-echo (GRE) at α = 30°, as well as Cartesian GRE, balanced SSFP, and fast spin-echo (FSE). The PFOB CF3 peak was also sampled with FSE. RESULTS: The proposed balanced UTE-SSFP technique exhibited a relative detection sensitivity of 51 µmolPFOB(-1) min(-1/2) (α = 30°), at least twice that of other sequence types with either 3D radial (UTE GRE: 20 µmolPFOB(-1) min(-1/2) ) or Cartesian k-space filling (GRE: 12 µmolPFOB(-1) min(-1/2) ; FSE: 16 µmolPFOB(-1) min(-1/2) ; balanced SSFP: 23 µmolPFOB(-1) min(-1/2) ). In vivo imaging of angiogenesis-targeted PFOB nanoparticles was demonstrated in a rabbit model of cancer on a clinical 3 Tesla scanner. CONCLUSION: A new dual 19F/1H balanced UTE-SSFP sequence manifests high SNR, with detection sensitivity more than two-fold better than traditional techniques, and alleviates imaging problems caused by dephasing in complex spectra.

Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 µM) and cytotoxic (IC50: 2.4 µM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 µM) and cytotoxicity (IC50: > 100 µM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 µM) and cytotoxicity (IC50: 8.1 µM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.