HMG-CoA Reductase Inhibition Promotes Neurological Recovery, Peri-Lesional Tissue Remodeling, and Contralesional Pyramidal Tract Plasticity after Focal Cerebral Ischemia

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery.

Irisin – a myth rather than an exercise-inducible myokine

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand on NK Cells Protects From Hepatic Ischemia-Reperfusion Injury

BACKGROUND: Ischemia-reperfusion injury (IRI) significantly contributes to graft dysfunction after liver transplantation. Natural killer (NK) cells are crucial innate effector cells in the liver and express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent inducer of hepatocyte cell death. Here, we investigated if TRAIL expression on NK cells contributes to hepatic IRI. METHODS: The outcome after partial hepatic IRI was assessed in TRAIL-null mice and contrasted to C57BL/6J wild-type mice and after NK cell adoptive transfer in RAG2/common gamma-null mice that lack T, B, and NK cells. Liver IRI was assessed by histological analysis, alanine aminotransferase, hepatic neutrophil activation by myeloperoxidase activity, and cytokine secretion at specific time points. NK cell cytotoxicity and differentiation were assessed in vivo and in vitro. RESULTS: Twenty-four hours after reperfusion, TRAIL-null mice exhibited significantly higher serum transaminases, histological signs of necrosis, neutrophil infiltration, and serum levels of interleukin-6 compared to wild-type animals. Adoptive transfer of TRAIL-null NK cells into immunodeficient RAG2/common gamma-null mice was associated with significantly elevated liver damage compared to transfer of wild-type NK cells. In TRAIL-null mice, NK cells exhibit higher cytotoxicity and decreased differentiation compared to wild-type mice. In vitro, cytotoxicity against YAC-1 and secretion of interferon gamma by TRAIL-null NK cells were significantly increased compared to wild-type controls. CONCLUSIONS: These experiments reveal that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI through modulation of NK cell cytotoxicity and NK cell differentiation.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems

In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Postnatal Notch1 activation induces T‑cell malignancy in conditional and inducible mouse models

The Notch1 signaling pathway is essential for hematopoietic development. However, the effects of postnatal activation of Notch1 signaling on hematopoietic system is not yet fully understood. We previously generated ZEG‑IC‑Notch1 transgenic mice that have a floxed β‑geo/stop signal between a CMV promoter and intracellular domain of Notch1 (IC‑Notch1). Constitutively active IC‑Notch1 is silent until the introduction of Cre recombinase. In this study, endothelial/hematopoietic specific expression of IC‑Notch1 in double transgenic ZEG‑IC‑Notch1/Tie2‑Cre embryos induced embryonic lethality at E9.5 with defects in vascular system but not in hematopoietic system. Inducible IC‑Notch1 expression in adult mice was achieved by using tetracycline regulated Cre system. The ZEG‑IC‑Notch1/Tie2‑tTA/tet‑O‑Cre triple transgenic mice survived embryonic development when maintained on tetracycline. Post‑natal withdrawal of tetracycline induced expression of IC‑Notch1 transgene in hematopoietic cells of adult mice. The triple transgenic mice displayed extensive T‑cell infiltration in multiple organs and T‑cell malignancy of lymph nodes. In addition, the protein levels of p53 and alternative reading frame (ARF) were decreased in lymphoma‑like neoplasms from the triple transgenic mice while their mRNA expression remained unchanged, suggesting that IC‑Notch1 might repress ARF‑p53 pathway by a post‑transcriptional mechanism. This study demonstrated that activation of constitutive Notch1 signaling after embryonic development alters adult hematopoiesis and induces T‑cell malignancy.

The value of serial serum lactate measurements in predicting the extent of ischemic bowel and outcome of patients suffering acute mesenteric ischemia

BACKGROUND Acute mesenteric ischemia (AMI) is an emergency with a mortality rate up to 50 %. Detecting AMI continues to be a major challenge. This study assed the correlation of repeated preoperative serum lactate with bowel necrosis and to identify risk factors for a lethal outcome in patients with AMI. METHODS A retrospective study of 91 patients with clinically and pathologically confirmed AMI from January 2006 to December 2012 was performed. RESULTS In-hospital mortality rate was 42.9 %. Two hundred nine preoperative lactate measurements were analyzed (2.3 ± 1.1 values per patient). Less than or equal to six hours prior to surgery, the mean serum lactate level was significantly higher (4.97 ± 4.21 vs. 3.24 ± 3.05 mmol/L, p = 0.006) and the mean pH significantly lower (7.28 ± 0.12 vs. 7.37 ± 0.08, p = 0.001) compared to >6 h before surgery. Thirty-four patients had at least two lactate measurements within 24 h prior to surgery. In this subgroup, 17 patients (50 %) exhibited an increase, 17 patients (50 %) a decrease in lactate levels. Forward logistic regression analysis showed that length of necrotic bowel and the highest lactate value 24 h prior to surgery were independent risk factors for mortality (r (2)  = 0.329). CONCLUSION The value of serial lactate and pH measurements to predict the length of necrotic bowel is very limited. Length of necrotic bowel and lactate values are independent risk factors for mortality.

Ischemia/reperfusion injury: effect of simultaneous inhibition of plasma cascade systems versus specific complement inhibition.

Ischemia/reperfusion injury (IRI) may occur from ischemia due to thrombotic occlusion, trauma or surgical interventions, including transplantation, with subsequent reestablishment of circulation. Time-dependent molecular and structural changes result from the deprivation of blood and oxygen in the affected tissue during ischemia. Upon restoration of blood flow a multifaceted network of plasma cascades is activated, including the complement-, coagulation-, kinin-, and fibrinolytic system, which plays a major role in the reperfusion-triggered inflammatory process. The plasma cascade systems are therefore promising therapeutic targets for attenuation of IRI. Earlier studies showed beneficial effects through inhibition of the complement system using specific complement inhibitors. However, pivotal roles in IRI are also attributed to other cascades. This raises the question, whether drugs, such as C1 esterase inhibitor, which regulate more than one cascade at a time, have a higher therapeutic potential. The present review discusses different therapeutic approaches ranging from specific complement inhibition to simultaneous inhibition of plasma cascade systems for reduction of IRI, gives an overview of the plasma cascade systems in IRI as well as highlights recent findings in this field.

Drug-eluting balloon versus standard balloon angioplasty for infrapopliteal arterial revascularization in critical limb ischemia: 12-month results from the IN.PACT DEEP randomized trial.

BACKGROUND Drug-eluting balloons (DEB) may reduce infrapopliteal restenosis and reintervention rates versus percutaneous transluminal angioplasty (PTA) and improve wound healing/limb preservation. OBJECTIVES The goal of this clinical trial was to assess the efficacy and safety of IN.PACT Amphirion drug-eluting balloons (IA-DEB) compared to PTA for infrapopliteal arterial revascularization in patients with critical limb ischemia (CLI). METHODS Within a prospective, multicenter, randomized, controlled trial with independent clinical event adjudication and angiographic and wound core laboratories 358 CLI patients were randomized 2:1 to IA-DEB or PTA. The 2 coprimary efficacy endpoints through 12 months were clinically driven target lesion revascularization (CD-TLR) and late lumen loss (LLL). The primary safety endpoint through 6 months was a composite of all-cause mortality, major amputation, and CD-TLR. RESULTS Clinical characteristics were similar between the 2 groups. Significant baseline differences between the IA-DEB and PTA arms included mean lesion length (10.2 cm vs. 12.9 cm; p = 0.002), impaired inflow (40.7% vs. 28.8%; p = 0.035), and previous target limb revascularization (32.2% vs. 21.8%; p = 0.047). Primary efficacy results of IA-DEB versus PTA were CD-TLR of 9.2% versus 13.1% (p = 0.291) and LLL of 0.61 ± 0.78 mm versus 0.62 ± 0.78 mm (p = 0.950). Primary safety endpoints were 17.7% versus 15.8% (p = 0.021) and met the noninferiority hypothesis. A safety signal driven by major amputations through 12 months was observed in the IA-DEB arm versus the PTA arm (8.8% vs. 3.6%; p = 0.080). CONCLUSIONS In patients with CLI, IA-DEB had comparable efficacy to PTA. While primary safety was met, there was a trend towards an increased major amputation rate through 12 months compared to PTA. (Study of IN.PACT Amphirion™ Drug Eluting Balloon vs. Standard PTA for the Treatment of Below the Knee Critical Limb Ischemia [INPACT-DEEP]; NCT00941733).

The Importance of Patency in Patients with Critical Limb Ischemia Undergoing Endovascular Revascularization for Infrapopliteal Arterial Disease.

Critical limb ischemia (CLI) represents the most severe form of peripheral arterial disease (PAD) and frequently occurs in medically frail patients. CLI patients frequently exhibit multi-segmental PAD commonly including the tibial arterial segment. Endovascular therapy has been established as first-line revascularization strategy for most CLI patients. Restenosis was reported to occur in up to more than two-thirds of CLI patients undergoing angioplasty of complex tibial arterial obstructions. Nevertheless, favorable clinical outcomes were observed for infrapopliteal angioplasty when compared with bypass surgery, despite higher patency rates for the latter. Based on these observations, infrapopliteal patency was considered to be only of secondary importance upon clinical outcomes in CLI patients. In contrast to these earlier observations, however, recent findings from two randomized clinical trials indicate that infrapopliteal patency does impact on clinical outcomes in CLI patients. The purpose of the present manuscript is to provide a critical reappraisal of the present literature on the clinical importance of tibial arterial patency in CLI patients undergoing endovascular revascularization and to discuss utility and limitations of currently available anti-restenosis technologies.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Interventional management of recurrent paroxysmal atrial fibrillation despite isolated pulmonary veins: impact of an ablation strategy targeting inducible atrial tachyarrhythmias

AIMS Pulmonary vein isolation (PVI) is an effective treatment option for paroxysmal atrial fibrillation (PAF). Reconnection of pulmonary veins (PVs) is the predominant cause for recurrence of PAF. However, treatment of patients with recurrence of PAF despite isolated PV in the absence of extra-PV foci remains challenging. METHODS AND RESULTS Of 265 patients undergoing repeat catheter ablation (CA) for recurrence of PAF 21 (8%) patients (14 men, age 58 ± 14 years) showed no reconnection of PV. Therefore, inducibility of sustained atrial arrhythmias was tested. If sustained atrial fibrillation (AF) or sustained atrial tachycardia (AT) was induced, patients underwent CA. During follow-up (FU), Holter- and Tele-electrocardiogram were performed. In 19 (91%) of 21 patients, sustained atrial arrhythmias [16 (84%) AF; 3 (15%) patients AT] were induced. One patient showed PAF. Eighteen patients underwent CA aiming for termination of induced arrhythmia. In 14 (77%) patients, termination into sinus rhythm was achieved. Despite extensive CA, three (16%) patients were externally cardioverted. No periprocedural complications occurred. During 21.2 ± 6.8-month FU, 10 (53%) patients were free of any arrhythmia. Paroxysmal atrial fibrillation recurred in 4 (21%) and AT in 5 (26%) patients. One patient showed persistent AF. Repeat CA was scheduled and successfully performed for these patients. CONCLUSION In patients with recurrence of PAF despite isolated PV, termination of induced atrial arrhythmias can be achieved in most patients by defragmentation and AT ablation. Moreover, this ablation strategy results in favourable mid-term outcome results.

Herbivore intoxication as a potential primary function of an inducible volatile plant signal

Plants release herbivore-induced volatiles (HIPVs), which can be used as cues by plants, herbivores and natural enemies. Theory predicts that HIPVs may initially have evolved because of their direct benefits for the emitter and were subsequently adopted as infochemicals. Here, we investigated the potential direct benefits of indole, a major HIPV constituent of many plant species and a key defence priming signal in maize. We used indole-deficient maize mutants and synthetic indole at physiologically relevant doses to document the impact of the volatile on the generalist herbivore Spodoptera littoralis. Our experiments demonstrate that indole directly decreases food consumption, plant damage and survival of S. littoralis caterpillars. Surprisingly, exposure to volatile indole increased caterpillar growth. Furthermore, we show that S. littoralis caterpillars and adults consistently avoid indole-producing plants in olfactometer experiments, feeding assays and oviposition trials. Synthesis. Together, these results provide a potential evolutionary trajectory by which the release of a HIPV as a direct defence precedes its use as a cue by herbivores and an alert signal by plants. Furthermore, our experiments show that the effects of a plant secondary metabolite on weight gain and food consumption can diverge in a counterintuitive manner, which implies that larval growth can be a poor proxy for herbivore fitness and plant resistance.

Inhalation anesthesia of rats: influence of the fraction of inspired oxygen on limb ischemia/reperfusion injury

Inhalation anesthesia with isoflurane is a well-established and safe method used in small laboratory animals. In most cases oxygen is used as a carrier gas for isoflurane, but room air or mixtures of oxygen with air or nitrous oxide are also being used. Anesthesia is therefore administered using different fractions of inspired oxygen (FiO2), and this may have consequences for the outcome of experiments. The aim of the present study was to investigate the influence of FiO2 on rat hind limb ischemia/reperfusion injury and to refine the used inhalation anesthesia. Male Wistar rats were subjected to 3.5 h of ischemia and 2 h of reperfusion, and divided into three groups according to FiO2 in the O2/air/isoflurane anesthesia gas mixture: 40%, 60%, and 100% O2. Normal, healthy rats were used as controls. Muscle edema and creatine kinase MM, a marker for myocyte necrosis, were significantly increased with 40% FiO2 as compared with 100% FiO2 (P<0.05). Partial pressure of oxygen, oxygen saturation, and oxyhemoglobin were significantly higher in the 100% O2 group as compared with 40% O2. No significant differences were detected for other parameters, such as the oxidative stress markers malondialdehyde and superoxide dismutase. We conclude that a refined inhalation anesthesia setting using 40% FiO2, reflecting more or less the clinical situation, leads to a more severe and more physiologically relevant reperfusion injury than higher FiO2. Oxidative stress did not correlate with FiO2 and seemed to have no influence on reperfusion injury.

An analysis of IN.PACT DEEP randomized trial on the limitations of the societal guidelines-recommended hemodynamic parameters to diagnose critical limb ischemia.

OBJECTIVE Recent small single-center data indicate that the current hemodynamic parameters used to diagnose critical limb ischemia are insensitive. We investigated the validity of the societal guidelines-recommended hemodynamic parameters against core laboratory-adjudicated angiographic data from the multicenter IN.PACT DEEP (RandomIzed AmPhirion DEEP DEB vs StAndard PTA for the treatment of below the knee Critical limb ischemia) Trial. METHODS Of the 358 patients in the IN.PACT DEEP Trial to assess drug-eluting balloon vs standard balloon angioplasty for infrapopliteal disease, 237 had isolated infrapopliteal disease with an available ankle-brachial index (ABI), and only 40 of the latter had available toe pressure measurements. The associations between ABI, ankle pressure, and toe pressure with tibial runoff, Rutherford category, and plantar arch were examined according to the cutoff points recommended by the societal guidelines. Abnormal tibial runoff was defined as severely stenotic (≥70%) or occluded and scored as one-, two-, or three-vessel disease. A stenotic or occluded plantar arch was considered abnormal. RESULTS Only 14 of 237 patients (6%) had an ABI <0.4. Abnormal ankle pressure, defined as <50 mm Hg if Rutherford category 4 and <70 mm Hg if Rutherford category 5 or 6, was found only in 37 patients (16%). Abnormal toe pressure, defined as <30 mm Hg if Rutherford category 4 and <50 mm Hg if Rutherford category 5 or 6, was found in 24 of 40 patients (60%) with available measurements. Importantly, 29% of these 24 patients had an ABI within normal reference ranges. A univariate multinomial logistic regression found no association between the above hemodynamic parameters and the number of diseased infrapopliteal vessels. However, there was a significant paradoxic association where patients with Rutherford category 6 had higher ABI and ankle pressure than those with Rutherford category 5. Similarly, there was no association between ABI and pedal arch patency. CONCLUSIONS The current recommended hemodynamic parameters fail to identify a significant portion of patients with lower extremity ulcers and angiographically proven severe disease. Toe pressure has better sensitivity and should be considered in all patients with critical limb ischemia.