Advances and Challenges in Interventional Immuno-Oncology Locoregional Therapies.

Interventional immuno-oncology is making strides in locoregional therapies to address complex tumor microenvironments. Long-standing interventional radiology cancer therapies, such as tumor ablation and embolization, are being recharacterized in the context of immunotherapy. Intratumoral injections, such as those of genetically engineered or unaltered viruses, and the delivery of immune cells, antibodies, proteins, or cytokines into targeted tumors, along with advancements in delivery techniques, have produced promising results in preliminary studies, indicating their antitumor effectiveness. Emerging strategies using DNA scaffolding, polysaccharides, glycan, chitosan, and natural products are also showing promise in targeted cancer therapy. The future of interventional immuno-oncology lies in personalized immunotherapies that capitalize on individual immune profiles and tumor characteristics, along with the exploration of combination therapies. This study will review various interventional immuno-oncology strategies and emerging technologies to enhance delivery of therapeutics and response to immunotherapy.

Spinal Cord Inflammation: Molecular Imaging after Thoracic Aortic Ischemia Reperfusion Injury.

Purpose To evaluate whether noninvasive molecular imaging technologies targeting myeloperoxidase (MPO) can reveal early inflammation associated with spinal cord injury after thoracic aortic ischemia-reperfusion (TAR) in mice. Materials and Methods The study was approved by the institutional animal care and use committee. C57BL6 mice that were 8-10 weeks old underwent TAR (n = 55) or sham (n = 26) surgery. Magnetic resonance (MR) imaging (n = 6) or single photon emission computed tomography (SPECT)/computed tomography (CT) (n = 15) studies targeting MPO activity were performed after intravenous injection of MPO sensors (bis-5-hydroxytryptamide-tetraazacyclododecane [HT]-diethyneletriaminepentaacetic acid [DTPA]-gadolinium or indium 111-bis-5-HT-DTPA, respectively). Immunohistochemistry and flow cytometry were used to identify myeloid cells and neuronal loss. Proinflammatory cytokines, keratinocyte chemoattractant (KC), and interleukin 6 (IL-6) were measured with enzyme-linked immunosorbent assay. Statistical analyses were performed by using nonparametric tests and the Pearson correlation coefficient. P < .05 was considered to indicate a significant difference. Results Myeloid cells infiltrated into the injured cord at 6 and 24 hours after TAR. MR imaging confirmed the presence of ischemic lesions associated with mild MPO-mediated enhancement in the thoracolumbar spine at 24 hours compared with the sham procedure. SPECT/CT imaging of MPO activity showed marked MPO-sensor retention at 6 hours (P = .003) that continued to increase at 24 hours after TAR (P = .0001). The number of motor neurons decreased substantially at 24 hours after TAR (P < .01), which correlated inversely with in vivo inflammatory changes detected at molecular imaging (r = 0.64, P = .0099). MPO was primarily secreted by neutrophils, followed by lymphocyte antigen 6 complexhigh monocytes and/or macrophages. There were corresponding increased levels of proinflammatory cytokines KC (P = .0001) and IL-6 (P = .0001) that mirrored changes in MPO activity. Conclusion MPO is a suitable imaging biomarker for identifying and tracking inflammatory damage in the spinal cord after TAR in a mouse model. © RSNA, 2016 Online supplemental material is available for this article.

Ethyl pyruvate modulates delayed paralysis following thoracic aortic ischemia reperfusion in mice.

OBJECTIVE: Delayed paralysis is an unpredictable problem for patients undergoing complex repair of the thoracic/thoracoabdominal aorta. These experiments were designed to determine whether ethyl pyruvate (EP), a potent anti-inflammatory and antioxidant agent, might ameliorate delayed paralysis following thoracic aortic ischemia reperfusion (TAR). METHODS: C57BL6 mice were subjected to 5 minutes of thoracic aortic ischemia followed by reperfusion for up to 48 hours. Mice received either 300 mg/kg EP or lactated ringers (LR) at 30 minutes before ischemia and 3 hours after reperfusion. Neurologic function was assessed using an established rodent scale. Spinal cord tissue was analyzed for markers of inflammation (keratinocyte chemoattractant [KC], interleukin-6 [IL-6]), microglial activation (ionized calcium-binding adapter molecule-1 [Iba-1]), and apoptosis (Bcl-2, Bax, and terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] staining) at 24 and 48 hours after TAR. Nissl body stained motor neurons were counted in the anterior horns sections from L1-L5 segments. RESULTS: Ninety-three percent of the LR mice developed dense delayed paralysis between 40 and 48 hours after TAR, whereas only 39% of EP mice developed delayed paralysis (P < .01). Bcl-2 expression was higher (P < .05) and Iba-1 expression was lower (P < .05) in the EP group only at 24 hours reperfusion. At 48 hours, the number of motor neurons was higher (P < .01) and the number and TUNEL-positive cells was lower (P < .001) in the EP-treated mice. EP decreased the expression of KC (P < .01) and IL-6 (P < .001) at 48 hours after TAR. CONCLUSIONS: The protection provided by EP against delayed paralysis correlated with preservation of motor neurons, higher expression of antiapoptotic molecules, decreased microglial cell activation, and decreased spinal cord inflammation. EP may be a treatment for humans at risk for delayed paralysis.

Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice.

OBJECTIVE: Extracellular traps (ETs) consisting of DNA-protein complexes formed after tissue injury contribute to the inflammatory and thrombosis cascades, thereby exacerbating injury. Exogenous DNase I has been suggested as a therapeutic strategy to limit injury in the brain and myocardium. These studies were designed to evaluate the effects of exogenous DNase I treatment on skeletal muscle injury after acute hindlimb ischemia-reperfusion (IR) injury in mice and to determine whether neutrophils are a major source of ETs in postischemic muscle tissue. METHODS: C57BL6 mice were subjected to 1.5 hours of tourniquet ischemia and 24 hours of reperfusion with and without human recombinant DNase I treatment. A separate set of mice was subjected to neutrophil depletion (ND), followed by the same intervals of IR. Laser Doppler imaging and tissue harvesting were done at 24 hours for assessment of limb perfusion, muscle fiber injury, adenosine triphosphate (ATP) level, markers of inflammation, thrombosis, and formation of ETs. RESULTS: DNase I treatment significantly reduced detection of ETs in postischemic muscle but did not alter skeletal muscle fiber injury, levels of proinflammatory molecules, or ATP level. DNase I treatment did enhance postischemic hindlimb perfusion, decreased infiltrating inflammatory cells, and reduced the expression of thrombin-antithrombin III. ND resulted in a significant yet small reduction in ETs in the postischemic muscle. ND did not alter skeletal muscle fiber injury, hindlimb perfusion, or ATP levels. CONCLUSIONS: These data suggest that neither DNase I treatment nor ND was protective against IR injury, even though both decreased detection of ETs in skeletal muscle after IR. Neutrophils are not the only source of ETs after IR.

Revascularization and muscle adaptation to limb demand ischemia in diet-induced obese mice.

BACKGROUND: Obesity and type 2 diabetes are major risk factors for peripheral arterial disease in humans, which can result in lower limb demand ischemia and exercise intolerance. Exercise triggers skeletal muscle adaptation including increased vasculogenesis. The goal of this study was to determine whether demand ischemia modulates revascularization, fiber size, and signaling pathways in the ischemic hind limb muscles of mice with diet-induced obesity (DIO). MATERIALS AND METHODS: DIO mice (n = 7) underwent unilateral femoral artery ligation and recovered for 2 wks followed by 4 wks with daily treadmill exercise to induce demand ischemia. A parallel sedentary ischemia (SI) group (n = 7) had femoral artery ligation without exercise. The contralateral limb muscles of SI served as control. Muscles were examined for capillary density, myofiber cross-sectional area, cytokine levels, and phosphorylation of STAT3 and ERK1/2. RESULTS: Exercise significantly enhanced capillary density (P < 0.01) and markedly lowered cross-sectional area (P < 0.001) in demand ischemia compared with SI. These findings coincided with a significant increase in granulocyte colony-stimulating factor (P < 0.001) and interleukin-7 (P < 0.01) levels. In addition, phosphorylation levels of STAT3 and ERK1/2 (P < 0.01) were increased, whereas UCP1 and monocyte chemoattractant protein-1 protein levels were lower (P < 0.05) without altering vascular endothelial growth factor and tumor necrosis factor alpha protein levels. Demand ischemia increased the PGC1α messenger RNA (P < 0.001) without augmenting PGC1α protein levels. CONCLUSIONS: Exercise-induced limb demand ischemia in the setting of DIO causes myofiber atrophy despite an increase in muscle capillary density. The combination of persistent increase in tumor necrosis factor alpha, lower vascular endothelial growth factor, and failure to increase PGC1α protein may reflect a deficient adaption to demand ischemia in DIO.

Effect of limb demand ischemia on autophagy and morphology in mice.

BACKGROUND: Obesity is a major risk factor for diabetes and peripheral arterial disease, which frequently leads to lower limb demand ischemia. Skeletal muscle autophagy and mitochondrial biogenesis are important processes for proper oxidative capacity and energy metabolism, which are compromised in diabetes. This study compares autophagy, mitochondrial biogenesis, energy metabolism, and morphology in the hind limbs of obese diabetic mice subjected to demand or sedentary ischemia. MATERIALS AND METHODS: Unilateral hind limb demand ischemia was created in a group of diet-induced obese mice after femoral artery ligation and 4 wk of daily exercise. A parallel group of mice underwent femoral artery ligation but remained sedentary for 4 wk. Hind limb muscles were analyzed for markers of autophagy, mitochondrial biogenesis, adenosine triphosphate, and muscle tissue morphology. RESULTS: At the end of the 4-wk exercise period, demand ischemia increased the autophagy mediator Beclin-1, but it did not alter the autophagy indicator, LC3B-II/I ratio, or markers of mitochondrial biogenesis, optic atrophy/dynamin-related protein. In contrast, exercise significantly increased the level of mitochondrial protein-succinate dehydrogenase subunit-A and reduced adipocyte accumulation and the percentage of centrally nucleated myofibers in the demand ischemia limb. In addition, demand ischemia resulted in decreased uncoupling protein-3 levels without altering muscle adenosine triphosphate or pS473-Akt levels. CONCLUSIONS: Limb demand ischemia markedly decreased adipocyte accumulation and enhanced muscle regeneration in obese mice, but it did not appear to enhance autophagy, mitochondrial biogenesis, energy metabolism, or insulin sensitivity. Future studies aimed at evaluating novel therapies that enhance autophagy and mitochondrial biogenesis in diabetes with peripheral arterial disease are warranted.

Syndecan-4 proteoliposomes enhance fibroblast growth factor-2 (FGF-2)-induced proliferation, migration, and neovascularization of ischemic muscle.

Ischemia of the myocardium and lower limbs is a common consequence of arterial disease and a major source of morbidity and mortality in modernized countries. Inducing neovascularization for the treatment of ischemia is an appealing therapeutic strategy for patients for whom traditional treatment modalities cannot be performed or are ineffective. In the past, the stimulation of blood vessel growth was pursued using direct delivery of growth factors, angiogenic gene therapy, or cellular therapy. Although therapeutic angiogenesis holds great promise for treating patients with ischemia, current methods have not found success in clinical trials. Fibroblast growth factor-2 (FGF-2) was one of the first growth factors to be tested for use in therapeutic angiogenesis. Here, we present a method for improving the biological activity of FGF-2 by codelivering the growth factor with a liposomally embedded coreceptor, syndecan-4. This technique was shown to increase FGF-2 cellular signaling, uptake, and nuclear localization in comparison with FGF-2 alone. Delivery of syndecan-4 proteoliposomes also increased endothelial proliferation, migration, and angiogenic tube formation in response to FGF-2. Using an animal model of limb ischemia, syndecan-4 proteoliposomes markedly improved the neovascularization following femoral artery ligation and recovery of perfusion of the ischemic limb. Taken together, these results support liposomal delivery of syndecan-4 as an effective means to improving the potential of using growth factors to achieve therapeutic neovascularization of ischemic tissue.

Hind limb ischemia-reperfusion injury in diet-induced obese mice.

BACKGROUND: Obesity is a major risk factor for the development of diabetes. Limb ischemia-reperfusion injury (IR) is a common clinical problem in diabetics who have compromised lower extremity perfusion. This study compared the histologic, metabolic, and functional outcomes after hind limb IR in diet-induced obese (DIO) and non-diabetic (ND) mice during the acute and the regenerative phases of IR. METHODS: DIO and ND mice were subjected to 1.5 h unilateral hind limb ischemia followed by 1- or 28-d IR. Muscle morphology, metabolic, and genomic stress were evaluated at days 1 and 28 IR; Acute inflammation and thrombosis were only measured at day-1 IR. At day 28, IR, skeletal muscle contractility, and maturation were also assessed. RESULTS: At day-1 IR, similar levels of acute muscle fiber necrosis were seen in both groups. DIO mice demonstrated substantially greater inflammatory, prothrombotic, and genomic stress responses, which were also associated with a greater reduction in energy substrates and Akt phosphorylation. At 28d, there was no difference in the peak forces generated in the hind limbs for the two groups. DIO mice had reduced fatigue resistance compared with ND and larger areas of fat accumulation although there was no significant difference in muscle fiber maturation. CONCLUSIONS: DIO mice had an exacerbated acute response to IR with enhanced metabolic deficit, fat accumulation, and defective functional recovery during the regenerative phase of IR. These changes in fatigue resistance reflect compromised functional recovery after IR injury and have relevance for the functional recovery of patients with metabolic syndrome and insulin resistance.

Extracellular traps in lipid-rich lesions of carotid atherosclerotic plaques: implications for lipoprotein retention and lesion progression.

PURPOSE: To investigate the presence and location of extracellular traps (ETs) in atherosclerotic plaques and to determine whether they are spatially associated with inflammatory cells and the lipid core. MATERIALS AND METHODS: Human carotid atherosclerotic plaques were collected from seven patients after surgical endarterectomy. Sequential tissue sections were stained with hematoxylin-eosin or subjected to immunohistochemistry to detect ETs, neutrophils and macrophages or apolipoprotein B (ApoB). To demonstrate the specificity of the antibody used to detect ETs, the adjacent tissue section was pretreated with deoxyribonuclease-1 (DNase-1) before immunostaining for ETs. RESULTS: All seven carotid plaques demonstrated advanced atherosclerotic lesions. Extensive ET and ApoB immunostaining was detected predominantly within the acellular lipid core. Along the edges of the lipid core, confocal microscopy revealed areas suggestive of active release of ETs from MPO-positive cells. Pretreatment of tissue sections with DNase-1 abolished ET signal in the extracellular matrix, but not the signal within the cells along the margins of the core. CONCLUSIONS: The localization of ETs to the lipid core suggests a possible binding site for lipoproteins, which may further promote lesion progression and inflammation.

Biocompatible Liquid Embolic for the Treatment of Microvascular Hemorrhage.

Persistent or recurrent bleeding from microvessels inaccessible for direct endovascular intervention is a major problem in medicine today. Here, an innovative catheter-directed liquid embolic (P-LE) is bioengineered for rapid microvessel embolization to treat small vessel hemorrhage. Tested in rodent, porcine, and canine animal models under normal and coagulopathic conditions, P-LE outperformed clinically used embolic materials in both survival and non-survival experiments, effectively occluding vessels as small as 40 microns with no signs of recanalization. P-LE occlusion is independent of the coagulation cascade, and its resistance to displacement is ≈ 8 times greater than systolic blood pressure. P-LE is also found to be biocompatible and x-ray visible and does not require polymerization or a chemical reaction to embolize. To simulate the clinical scenario, acute microvascular hemorrhage is created in the pig kidney, liver, or stomach; these are successfully treated with P-LE achieving immediate hemostasis. Furthermore, P-LE is found to be bactericidal to highly resistant patient-derived bacteria, suggesting that P-LE may also protect against infectious complications that may occur following embolization procedures. P-LE is safe, easy to use, and effective in treating -microvessel hemorrhage.

Bioengineered Ionic Liquid for Catheter-Directed Tissue Ablation, Drug Delivery, and Embolization.

Delivery of therapeutics to solid tumors with high bioavailability remains a challenge and is likely the main contributor to the ineffectiveness of immunotherapy and chemotherapy. Here, a catheter-directed ionic liquid embolic (ILE) is bioengineered to achieve durable vascular embolization, uniform tissue ablation, and drug delivery in non-survival and survival porcine models of embolization, outperforming the clinically used embolic agents. To simulate the clinical scenario, rabbit VX2 orthotopic liver tumors are treated showing successful trans-arterial delivery of Nivolumab and effective tumor ablation. Furthermore, similar results are also observed in human ex vivo tumor tissue as well as significant susceptibility of highly resistant patient-derived bacteria is seen to ILE, suggesting that ILE can prevent abscess formation in embolized tissue. ILE represents a new class of liquid embolic agents that can treat tumors, improve the delivery of therapeutics, prevent infectious complications, and potentially increase chemo- and immunotherapy response in solid tumors.

Aging of deep venous thrombosis in-vivo using polarization sensitive optical coherence tomography.

Deep venous thrombosis (DVT) is a medical condition with significant post-event morbidity and mortality coupled with limited treatment options. Treatment strategy and efficacy are highly dependent on the structural composition of the thrombus, which evolves over time from initial formation and is currently unevaluable with standard clinical testing. Here, we investigate the use of intravascular polarization-sensitive optical coherence tomography (PS-OCT) to assess thrombus morphology and composition in a rat DVT model in-vivo, including changes that occur over the thrombus aging process. PS-OCT measures tissue birefringence, which provides contrast for collagen and smooth muscle cells that are present in older, chronic clots. Thrombi in the inferior vena cava of two cohorts of rats were imaged in-vivo with intravascular PS-OCT at 24 hours (acute, nrats = 3, 73 cross-sections) or 28 days (chronic, nrats = 4, 41 cross-sections) after thrombus formation. Co-registered histology was labelled by an independent pathologist to establish ground-truth clot composition. Automated analysis of OCT cross-sectional images differentiated acute and chronic thrombi with 97.6% sensitivity and 98.6% specificity using a linear discriminant model comprised of both polarization and conventional OCT metrics. These results support PS-OCT as a highly sensitive imaging modality for the assessment of DVT composition to differentiate acute and chronic thrombi. Intravascular PS-OCT imaging could be integrated with advanced catheter-based treatment strategies and serve to guide therapeutic decision-making and deployment, by offering an accurate assessment of DVT patients in real time.

Multi-Functional Biomaterial for the Treatment and Prevention of Central Line-Associated Bloodstream Infections.

Central venous catheters are among the most used medical devices in hospitals today. Despite advances in modern medicine, catheter infections remain prevalent, causing significant morbidity and mortality worldwide. Here, SteriGel is reported, which is a multifunctional hydrogel engineered to prevent and treat central line-associated bloodstream infections (CLABSI). The mechanical properties of SteriGel are optimized to ensure appropriate gelation kinetics, bio-adhesiveness, stretchability, and recoverability to promote durability upon application and to provide persistent protection against infection. In vitro assays demonstrated that SteriGel exhibits long-term antimicrobial efficacy and has bactericidal effects against highly resistant patient-derived pathogens known to be frequently associated with CLABSI. SteriGel outperformed Biopatch, which is a clinically used device for CLABSI, in ex vivo cadaver studies that simulate clinical scenarios. Furthermore, SteriGel has biocompatible, pro-healing, and anti-inflammatory properties in vitro and in a rat subcutaneous injection model, suggesting a potential synergistic effect in the prevention and treatment of CLABSI. SteriGel is a multifunctional adherent biomaterial with potent antimicrobial effects for sustained sterility while promoting healing of the catheter incision site to protect against infection.

Percutaneous Delivery of Oncogel for Targeted Liver Tumor Ablation and Controlled Release of Therapeutics.

Advanced-stage liver cancers are associated with poor prognosis and have limited treatment options, often leading the patient to hospice care. Percutaneous intratumoral injection of anticancer agents has emerged as a potential alternative to systemic therapy to overcome tumor barriers, increase bioavailability, potentiate immunotherapy, and avoid systemic toxicity, which advanced-stage cancer patients cannot tolerate. Here, an injectable OncoGel (OG) comprising of a nanocomposite hydrogel loaded with an ionic liquid (IL) is developed for achieving a predictable and uniform tumor ablation and long-term slow release of anticancer agents into the ablation zone. Rigorous mechanical, physiochemical, drug release, cytotoxicity experiments, and ex vivo human tissue testing identify an injectable version of the OG with bactericidal properties against highly resistant bacteria. Intratumoral injection of OG loaded with Nivolumab (Nivo) and doxorubicin (Dox) into highly malignant tumor models in mice, rats, and rabbits demonstrates enhanced survival and tumor regression associated with robust tissue ablation and drug distribution throughout the tumor. Mass cytometry and proteomic studies in a mouse model of colorectal cancer that often metastasizes to the liver indicate an enhanced anticancer immune response following the intratumoral injection of OG. OG may augment immunotherapy and potentially improve outcomes in liver cancer patients.

Catheter-Directed Ionic Liquid Embolic Agent for Rapid Portal Vein Embolization, Segmentectomy, and Bile Duct Ablation.

Embolic materials currently in use for portal vein embolization (PVE) do not treat the tumor, which poses a risk for tumor progression during the interval between PVE and surgical resection. Here, is developed an ionic-liquid-based embolic material (LEAD) for portal vein embolization, liver ablation, and drug delivery. LEAD is optimized and characterized for diffusivity, X-ray visibility, and cytotoxicity. In the porcine renal embolization model, LEAD delivered from the main renal artery reached vasculature down to 10 microns with uniform tissue ablation and delivery of small and large therapeutics. In non-survival and survival porcine experiments, successful PVE is achieved in minutes, leading to the expected chemical segmentectomy, and delivery of a large protein drug (i.e., Nivolumab) with LEAD. In cholangiocarcinoma mouse tumor models and in ex vivo human tumors, LEAD consistently achieved an effective ablation and wide drug distribution. Furthermore, various strains of drug-resistant patient-derived bacteria showed significant susceptibility to LEAD, suggesting that LEAD may also prevent infectious complications resulting from tissue ablation. With its capabilities to embolize, ablate, and deliver therapeutics, ease of use, and a high safety profile demonstrated in animal studies, LEAD offers a potential alternative to tumor ablation with or without PVE for FLR growth.

Prostate tissue ablation and drug delivery by an image-guided injectable ionic liquid in ex vivo and in vivo models.

Benign prostatic hyperplasia and prostate cancer are often associated with lower urinary tract symptoms, which can severely affect patient quality of life. To address this challenge, we developed and optimized an injectable compound, prostate ablation and drug delivery agent (PADA), for percutaneous prostate tissue ablation and concurrently delivered therapeutic agents. PADA is an ionic liquid composed of choline and geranic acid mixed with anticancer therapeutics and a contrast agent. The PADA formulation was optimized for mechanical properties compatible with hand injection, diffusion capability, cytotoxicity against prostate cells, and visibility of an x-ray contrast agent. PADA also exhibited antibacterial properties against highly resistant clinically isolated bacteria in vitro. Ultrasound-guided injection, dispersion of PADA in the tissue, and tissue ablation were tested ex vivo in healthy porcine, canine, and human prostates and in freshly resected human tumors. In vivo testing was conducted in a murine subcutaneous tumor model and in the canine prostate. In all models, PADA decreased the number of viable cells in the region of dispersion and supported the delivery of nivolumab throughout a portion of the tissue. In canine survival experiments, there were no adverse events and no impact on urination. The injection approach was easy to perform under ultrasound guidance and produced a localized effect with a favorable safety profile. These findings suggest that PADA is a promising therapeutic prostate ablation strategy to treat lower urinary tract symptoms.

Poly-ADP-ribose-polymerase inhibition ameliorates hind limb ischemia reperfusion injury in a murine model of type 2 diabetes.

INTRODUCTION: Diabetes is known to increase poly-ADP-ribose-polymerase (PARP) activity and posttranslational poly-ADP-ribosylation of several regulatory proteins involved in inflammation and energy metabolism. These experiments test the hypothesis that PARP inhibition will modulate hind limb ischemia reperfusion (IR) in a mouse model of type-II diabetes and ameliorate the ribosylation and the activity/transnuclear localization of the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). METHODS: db/db mice underwent 1.5 hours of hind limb ischemia followed by 1, 7, or 24 hours of reperfusion. The treatment group received the PARP inhibitor PJ34 (PJ34) over a 24-hour period; the untreated group received Lactated Ringer (LR) at the same time points. IR muscles were analyzed for indices of PARP activity, fiber injury, metabolic activity, inflammation, GAPDH activity/intracellular localization, and poly-ADP-ribosylation of GAPDH. RESULTS: PARP activity was significantly lower in the PJ34-treated groups than in the Lactated Ringer group at 7 and 24 hours of reperfusion. There was significantly less muscle fiber injury in the PJ34-treated group than in the Lactated Ringer-treated mice at 24 hours of reperfusion. PJ34 lowered levels of select proinflammatory molecules at 7 hours and 24 hours of IR. There were significant increases in metabolic activity only at 24 hours of IR in the PJ34 group, which temporally correlated with increase in GAPDH activity, decreased GAPDH poly-ADP-ribosylation, and nuclear translocation of GAPDH. CONCLUSIONS: PJ34 reduced PARP activity, GAPDH ribosylation, and GAPDH translocation; ameliorated muscle fiber injury; and increased metabolic activity after hind limb IR injury in a murine model of type-II diabetes. PARP inhibition might be a therapeutic strategy after IR in diabetic humans.

Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps.

OBJECTIVE: Ischemia-reperfusion (IR) injury is a significant problem in the management of patients with acute limb ischemia. Despite rapid restoration of blood flow after technically successful open and endovascular revascularization, complications secondary to IR injury continue to occur and limit clinical success. Our aim was to create a murine model of hind limb IR injury to examine the role of Toll-like receptor-4 (TLR4) and to determine whether inactive TLR4 led to a decrease in the detection of neutrophil extracellular traps (NETs), which are known to be highly thrombogenic and may mediate microvascular injury. METHODS: A calibrated tension tourniquet was applied to unilateral hind limb of wild-type (WT) and TLR4 receptor mutant (TLR4m) mice for 1.5 hours to induce ischemia and then removed to initiate reperfusion. At the end of 48 hours of reperfusion, mice were euthanized and hind limb tissue and serum specimens were collected for analysis. Hematoxylin and eosin-stained sections of hind limb skeletal muscle tissue were examined for fiber injury. For immunohistochemistry, mouse monoclonal antihistone H2A/H2B/DNA complex antibody to detect NETs and rabbit polyclonal antimyeloperoxidase antibody were used to identify infiltrating cells containing myeloperoxidase. Muscle adenosine triphosphate levels, nuclear factor (NF)-κB activity, the α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, poly (adenosine diphosphate-ribose) polymerase activity, and inducible nitric oxide synthase expression were measured. Systemic levels of keratinocyte-derived chemokine, monocyte chemotactic protein-1, and vascular endothelial growth factor in the serum samples were also examined. RESULTS: IR injury in the hind limb of WT mice demonstrated significant levels of muscle fiber injury, decreased energy substrates, increased NF-κB activation, decreased levels of α-subunit of inhibitor of NF-κB light polypeptide gene enhancer, increased inducible nitric oxide synthase expression, and increased poly (adenosine diphosphate-ribose) polymerase activity levels compared with the TLR4m samples. Additionally, there was marked decrease in the level of neutrophil and monocyte infiltration in the TLR4m mice, which corresponded to similar levels of decreased NET detection in the interstitial space and in microvascular thrombi. In situ nuclease treatment of WT tissue sections significantly diminished the level of NET immunostaining, demonstrating the specificity of the antibody to detect NETs and suggesting a potential role for nuclease treatment in IR injury. CONCLUSIONS: These results suggest a pivotal role for TLR4 in mediating hind limb IR injury and suggest that NETs may contribute to muscle fiber injury.

Divergent systemic and local inflammatory response to hind limb demand ischemia in wild-type and ApoE-/- mice.

BACKGROUND: We designed studies to determine whether the ApoE-/- phenotype modulates the local skeletal muscle and systemic inflammatory (plasma) responses to lower extremity demand ischemia. The ApoE-/- phenotype is an experimental model for atherosclerosis in humans. METHODS: Aged female ApoE-/- and C57BL6 mice underwent femoral artery ligation, then were divided into sedentary and demand ischemia (exercise) groups on day 14. We assessed baseline and postexercise limb perfusion and hind limb function. On day 14, animals in the demand ischemia group underwent daily treadmill exercise through day 28. Sedentary mice were not exercised. On day 28, we harvested plasma and skeletal muscle from ischemic limbs from sedentary and exercised mice. We assayed muscle for angiogenic and proinflammatory proteins, markers of skeletal muscle regeneration, and evidence of skeletal muscle fiber maturation. RESULTS: Hind limb ischemia was similar in ApoE-/- and C57 mice before the onset of exercise. Under sedentary conditions, plasma vascular endothelial cell growth factor and interleukin-6, but not keratinocyte chemoattractant factor (KC) or macrophage inflammatory protein-2 (MIP-2), were higher in ApoE (P < 0.0001). After exercise, plasma levels of vascular endothelial cell growth factor, KC, and MIP-2, but not IL-6, were lower in ApoE (P < 0.004). The cytokines KC and MIP-2 in muscle were greater in exercised ApoE-/- mice compared with C57BL6 mice (P = 0.01). Increased poly-ADP-ribose activity and mature muscle regeneration were associated with demand ischemia in the C57BL6 mice, compared with the ApoE-/- mice (P = 0.01). CONCLUSIONS: Demand limb ischemia in the ApoE-/- phenotype exacerbated the expression of select systemic cytokines in plasma and blunted indices of muscle regeneration.

Differential effect of zoledronic acid on human vascular smooth muscle cells.

INTRODUCTION: The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether zoledronic acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating versus quiescent cells, and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. METHODS: Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity, and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly-adenosine diphosphate ribose polymerase enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly adenosine diphosphate-ribosylated protein, respectively. RESULTS: There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating versus quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of activity. CONCLUSIONS: These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion, and migration, which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation.