Mitochondrial elongation confers protection against doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX)-induced cardiac damage remains a leading cause of death amongst cancer survivors. DOX-induced cardiotoxicity (DIC) is mediated by disturbed mitochondrial dynamics, but it remains debated that the mechanisms by which DOX disrupted equilibrium between mitochondrial fission and fusion. In the present study, we observed that DOX induced mitochondrial elongation in multiple cardiovascular cell lines. Mechanically, DOX not only downregulated the mitochondrial fusion proteins including Mitofusin 1/2 (MFN1/2) and Optic atrophy 1 (OPA1), but also induced lower motility of dynamin-related protein 1(Drp1) and its phosphorylation on 637 serine, which could inhibit mitochondrial fission. Interestingly, DOX failed to induce mitochondrial elongation in cardiomyocytes co-treated with protein kinase A (PKA) inhibitor H89 or expressing phosphodeficient Drp1-S637A variants. Besides, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was able to blocked the mitochondrial elongation induced by DOX treatment, which could be phenocopied by OPA1 knockdown. Therefore, we speculated that DOX inhibited mitochondrial fission and fusion simultaneously, yet enabled mitochondrial fusion dominate the mitochondrial dynamics, resulting in mitochondrial elongation as the main manifestation. Notably, blocking mitochondrial elongation by inhibiting Drp1-S637 phosphorylation or OPA1 knockdown aggravated DOX-induced cardiomyocytes death. Based on these results, we propose a novel mechanistic model that DOX-induced mitochondrial elongation is attributed to the equilibrium disturbance of mitochondrial dynamics, which serves as an adaptive response and confers protection against DIC.

FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux.

Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.

Right axillary artery cannulation in acute type A aortic dissection with involvement of the right axillary artery.

OBJECTIVE: The right axillary artery is currently recommended for arterial cannulation in surgery for acute type A aortic dissection. However, the feasibility of cannulation on a dissected right axillary artery remains undetermined. The objective was to examine the feasibility of cannulation on a dissected right axillary artery. METHODS: From 2016 to 2020, 835 patients who underwent acute type A aortic dissection repair were included in this study. Cannulation strategy and perioperative outcomes of patients who did and did not have right axillary artery dissection were compared. Propensity score matching and logistic regression were applied. RESULTS: A total of 124 patients had right axillary artery dissection, and 711 patients did not. Direct right axillary artery cannulation was used for cardiopulmonary bypass in the majority of patients, but with a lower rate in patients with right axillary artery dissection (n = 88 [71.0%] vs n = 579 [81.4%], P = .007). Right axillary artery cannulation failure (n = 3 [2.4%] vs n = 5 [0.7%], P = .102) and related complications (n = 1 [0.8%] vs n = 6 [0.8%], P = 1.000) were rare in both groups. In-hospital mortality (n = 18 [14.5%] vs n = 59 [8.3%], P = .027) and stroke (n = 14 [11.3%] vs n = 42 [5.9%], P = .027) were significantly higher in the right axillary artery dissection group, but after propensity score matching, in-hospital outcomes were comparable. Right axillary artery dissection was not a risk factor for mortality, stroke, right axillary artery cannulation not performed, or right axillary artery cannulation failure. CONCLUSIONS: Direct right axillary artery cannulation is feasible for most patients with acute type A aortic dissection with right axillary artery dissection.

Aortic Balloon Occlusion Technique Does Not Improve Peri-Operative Outcomes for Acute Type A Acute Aortic Dissection Patients With Lower Body Malperfusion.

Background: The management of malperfusion is vital to improve the outcomes of surgery for acute type A acute aortic dissection (ATAAD). Open arch repair under hypothermic circulatory arrest with selective antegrade cerebral perfusion (HCA/sACP) is safe and efficient but associated with inevitable hypothermia and ischemia-reperfusion injury. The aortic balloon occlusion (ABO) technique is shown to be organ protective by allowing higher temperature and shorter circulatory arrest time. In this study, we aimed to evaluate the safety and efficacy of this new technique for ATAAD patients with lower body malperfusion. Methods: Between January 2013 and November 2020, 355 ATAAD patients with lower body malperfusion who underwent arch repair in our institute were enrolled. The patients were divided into 2 groups: ABO group (n = 85) and HCA/sACP group (n = 271). Propensity score matching was performed to correct baseline differences. Results: Using the propensity score matching, 85 pairs were generated. Circulatory arrest time was significantly lower in the ABO group compared with the HCA/sACP group (median, 8 vs. 22 min; p < 0.001). The incidence of in-hospital mortality (10.6 vs. 12.9%; p = 0.812), stroke (7.1 vs. 7.1%; p = 1.000), dialysis (25.9 vs. 32.9%; p = 0.183), hepatic dysfunction (52.9 vs. 57.6%; p = 0.537), tracheostomy (4.7 vs. 2.4%; p = 0.682), paraplegia (1.2 vs. 4.7%; p = 0.368) were comparable between ABO and HCA/sACP groups. Other outcomes and major adverse events were comparable. The multivariable logistic analysis did not recognize ABO technique protective against any major adverse outcomes. Conclusions: For ATAAD patients with lower body malperfusion, the ABO technique allows the performance of arch repair with frozen elephant trunk (FET) under higher temperature and shorter circulatory arrest time. However, ABO technique did not improve perioperative outcomes. Future studies are warranted to evaluate the efficacy of this technique.

Dual Function of a in vivo Albumin-Labeling Tracer for Assessment of Blood Perfusion and Vascular Permeability in Peripheral Arterial Disease by PET.

BACKGROUND: Peripheral arterial disease (PAD) leads to tissue ischemia in the extremities. Enhanced vascular permeability plays a critical role in targeted delivery of drugs for effective therapeutic angiogenesis and resultant blood perfusion recovery. However, optimal tracers for evaluating this process in PAD patients are lacking. At this time, we employed a novel in vivo albumin-labeling tracer of dual function, termed as 18F-NEB, to assess blood perfusion as well as vascular permeability by positron emission tomography (PET). METHODS AND RESULTS: After successful establishment of mouse hindlimb ischemia (HI) model, static PET imaging was performed 15 min and 2 h post injection (p.i.) of 18F-NEB at 1, 3, 5, 7, 10 and 14 days post-surgery respectively. Gradual recovery of blood supply was detected by PET scan 15 min p.i. and collaborated by serial Laser Doppler. In addition, the highest vascular permeability observed by high local uptake of 18F-NEB at 2 h p.i. was consistent with histological examinations. Furthermore, we quantitatively evaluated the effect of vascular endothelial growth factor (VEGF) stimulus on vascular permeability and blood perfusion by PET scan using 18F-NEB probe in HI model, which were also confirmed by immunohistological results. CONCLUSION: The application of 18F-NEB probe alone by PET can successfully achieve dual imaging of blood perfusion as well as vascular permeability at different time points p.i. and monitor their responses to therapy in PAD model. The simple labeling approach and multipurpose feature suggest the great promise of using this imaging probe in theranostic applications for treating ischemic disease.

Noninvasive Evaluation of Angiogenesis and Therapeutic Response after Hindlimb Ischemia with an Integrin-Targeted Tracer by PET.

Background: Peripheral arterial disease (PAD) can severely compromise limb vitality and function. Angiogenesis plays an important role in healing of ischemic lesions. Radiolabeled RGD (Arg-Gly-Asp) peptides specifically targeting α v ß 3 integrin are promising tracers for imaging angiogenesis. In this study, we investigated the application of a one-step labeled RGD in evaluation of angiogenesis and therapy response in a mouse model of hindlimb ischemia (HI) by positron emission tomography (PET). Methods: HI was induced by ablation of the femoral artery in mice. PET imaging using 18F-AlF-NOTA-PRGD2 (18F-PRGD2) tracer was performed at day 0 (pre-surgery) and days 3, 7, 14, and 21 after surgery to evaluate hindlimb angiogenesis longitudinally and noninvasively. The control peptide RAD (Arg-Ala-Asp) labeled with a similar procedure and a block agent were used to confirm the specific binding of 18F-PRGD2 to α v ß 3 integrin. Ex vivo CD31 staining was performed to detect angiogenesis. In addition, the angiogenic therapy response was monitored with 18F-PRGD2 tracer and immunofluorescence staining to confirm the imaging data. Results: The successful establishment of HI model was confirmed by ultrasound imaging and laser doppler perfusion imaging (LDPI). Specific binding of 18F-PRGD2 to α v ß 3 integrin was validated by minimal tracer uptake of the control peptide RAD and significant decrease of tracer accumulation when a block agent was added. Local accumulation of 18F-RRGD2 in ischemic hindlimb was detected as early as 3 days and reached a peak at 7 days after surgery. The temporal change of focal tracer uptake was positively correlated with the pattern of vascular density. Moreover, vascular endothelial growth factor (VEGF) treatment increased the tracer uptake and enhanced angiogenesis, which is consistent with integrin ß 3 expression. Conclusions: PET imaging of a one-step labeled tracer 18F-PRGD2 targeted to α v ß 3 integrin allows longitudinal monitoring of ischemia-induced angiogenesis and noninvasive assessment of VEGF treatment response in a mouse model of hindlimb ischemia. The simple synthesis procedure and in vivo performance of this PET tracer enables the feasibility of future clinical translation in ischemic cardiovascular diseases.

Concomitant surgical atrial fibrillation ablation is safe and efficacious in patients undergoing double valve replacement - A cohort study.

BACKGROUND: Current European Society of Cardiology Guidelines recommend concomitant atrial fibrillation (AF) ablation for all symptomatic patients undergoing other cardiac surgeries, but the safety and potential benefits of concomitant atrial fibrillation (AF) ablation at the time of double valve replacement (DVR: aortic and mitral valve replacement) remains unexamined. MATERIALS AND METHODS: We conducted a retrospective review of 238 patients with AF who underwent DVR with or without concomitant surgical ablation (Ablation group, n = 113; Non-ablation group, n = 125) at a single institute from April 2006 to September 2011. RESULTS: There were no significant group differences in early postoperative mortality and morbidity, late survival, and freedom from major cardiac and cerebrovascular events (MACCEs). However, the Ablation group exhibited higher rates of sinus rhythm restoration at discharge (86.7% vs. 5.6%, P < 0.01) and at last follow-up (71.2% vs. 8.5%, P < 0.01). Follow-up echocardiography demonstrated smaller left atrial dimension and higher ejection fraction in the Ablation group (both P < 0.01). CONCLUSION: Concomitant surgical ablation for AF did not increase perioperative mortality or morbidity in patients undergoing DVR, but significantly increased sinus rhythm restoration, improved heart function, and decreased oral anticoagulation requirements.

ZP2495 Protects against Myocardial Ischemia/Reperfusion Injury in Diabetic Mice through Improvement of Cardiac Metabolism and Mitochondrial Function: The Possible Involvement of AMPK-FoxO3a Signal Pathway.

Coronary heart disease patients with type 2 diabetes were subject to higher vulnerability for cardiac ischemia-reperfusion (I/R) injury. This study was designed to evaluate the impact of ZP2495 (a glucagon-GLP-1 dual-agonist) on cardiac function and energy metabolism after myocardial I/R injury in db/db mice with a focus on mitochondrial function. C57BLKS/J-lepr+/lepr+ (BKS) and db/db mice received 4-week treatment of glucagon, ZP131 (GLP-1 receptor agonist), or ZP2495, followed by cardiac I/R injury. The results showed that cardiac function, cardiac glucose metabolism, cardiomyocyte apoptosis, cardiac mitochondrial morphology, and energetic transition were improved or ameliorated by ZP2495 to a greater extent than that of glucagon and ZP131. In vitro study showed that ZP2495, rather than glucagon, alleviated mitochondrial depolarization, cytochrome C release, and mitochondria ROS generation in neonatal rat ventricular myocytes subjected to high-glucose and simulated I/R injury conditions, the effects of which were weaker in the ZP131 group. Furthermore, the expressions of Akt, FoxO3a, and AMPK phosphorylation were elevated by ZP2495 to a greater extent than that of ZP131. In conclusion, ZP2495 may contribute to the improvement of cardiac function and energy metabolism in db/db mice after myocardial I/R injury by improving mitochondrial function possibly through Akt/FoxO3a and AMPK/FoxO3a signal pathways.

The effect of antegrade pulmonary blood flow following a late bidirectional Glenn procedure.

OBJECTIVES: The effect of antegrade pulmonary blood flow (APBF) has never been studied in the bidirectional Glenn (BDG) procedure performed late. METHODS: Records of 112 consecutive patients who had a BDG procedure during a 10-year period were reviewed retrospectively. The patients were divided into 2 groups based on whether APBF occurred following the BDG procedure (APBF group, n = 81) or not (non-APBF group, n = 31). The median age at the BDG procedure was 6.16 ± 3.93 years in the APBF group and 6.12 ± 4.40 years in the non-APBF group. RESULTS: Demographics and pre- and intraoperative variables were comparable for both groups. Follow-up data were obtained for patients at the BDG stage and for those who had undergone the Fontan completion. Both oxygen saturation levels (81.72 ± 1.976% vs 78.32 ± 2.344%, P < 0.01) and pulmonary pressure (13.59 ± 1.376 mmHg vs 12.90 ± 0.978 mmHg, P = 0.012) were higher in the APBF group immediately after the BDG procedure. Both the duration of chest tube drainage and the total length of stay were longer in the APBF group. The pre-Glenn measurements showed a mean McGoon ratio of 1.68 ± 0.114 in the APBF group and 1.67 ± 0.098 in the non-APBF group (P = 0.474). The McGoon ratios measured before the Fontan procedure were also comparable (1.669 ± 0.726 vs 1.685 ± 0.669, P = 0.576). At the pre-Fontan measurement, there was no significant difference in mean pulmonary artery pressures between the groups (13.72 ± 1.368 vs 13.50 ± 1.265, P = 0.653). Fifty-nine patients underwent the Fontan completion (43 from the APBF group and 16 from the non-APBF group) procedure with a median of 1.2 (APBF group) and 1.4 (non-APBF group) years after the BDG procedure. No significant differences between groups were observed in arterial oxygen saturation levels, incidence of systemic atrioventricular valve regurgitation or ventricular dysfunction in survivors at the last follow-up visit. CONCLUSIONS: The BDG procedure can be safely performed at a relatively older age (∼6 years). APBF increases oxygen saturation but also prolongs pleural effusion and hospital stay. Medium-term outcomes and the Fontan completion rate in the APBF and the non-APBF groups are comparable. Further large studies and long-term follow-up are needed to clarify the effect of APBF in patients who have the late BDG.

Bilateral versus unilateral antegrade cerebral perfusion in total arch replacement for type A aortic dissection.

BACKGROUND: Antegrade cerebral perfusion (ACP) is the most widely used cerebral protection strategy for complex aortic repair and includes unilateral (u-ACP) and bilateral (b-ACP) techniques. The superiority of b-ACP over u-ACP has been the subject of much debate. Focusing on type A aortic dissection requiring total arch replacement, we investigated the clinical effects of b-ACP versus u-ACP. METHODS: Between September 2006 and August 2014, 203 patients presenting with type A aortic dissection (median age, 51.0 ± 13 years; range, 17-72 years; 128 males) underwent total aortic arch replacement with hypothermic circulatory arrest. ACP was used in all patients, including u-ACP in 82 (40.3%) and b-ACP in 121 (59.7%). RESULTS: There was no significant difference between the u-ACP and b-ACP groups in terms of cardiopulmonary bypass (CPB) time, cross-clamp time, or circulatory arrest time. Overall 30-day mortality was comparable in the 2 groups (11.6% for b-ACP vs 20.7% for u-ACP; P = .075). The prevalence of postoperative permanent neurologic dysfunction (PND) was comparable as well (8.4% vs 16.9%; P = .091). Mean ventilation time was lower in the b-ACP group (95.5 ± 45.25 hours vs 147.0 ± 82 hours; P < .001). Mean lengths of stay in the intensive care unit and the hospital overall were comparable in the 2 groups (intensive care unit: 16 ± 17.75 days vs 17 ± 11.5 days, P = .454; hospital: 26.5 ± 20.6 days vs 24.8 ± 10.3 days, P = .434). The P values from logistic regression models indicated that in the 2 groups combined, CPB time and circulatory arrest time were independent risk factors for both mortality and PND. CONCLUSIONS: In this, the first published study focusing on the efficacy of u-ACP and b-ACP in total arch replacement for type A aortic dissection, the b-ACP group did not demonstrate significantly lower 30-day mortality or PND rate compared with the u-ACP group. Future large-sample studies are warranted to thoroughly examine this critical issue.

Kappa-Opioid Agonist U50,488H-Mediated Protection Against Heart Failure Following Myocardial Ischemia/Reperfusion: Dual Roles of Heme Oxygenase-1.

BACKGROUNDS/AIMS: The selective κ-opioid agonist U50,488H protects heart from myocardial ischemia-reperfusion (MI/R) injury. We examined whether U50,488H is also beneficial for MI/R induced heart failure. METHODS: Anesthetized male Sprague-Dawley rats were subjected to 30 min of myocardial ischemia via left anterior descending coronary artery (LAD) occlusion, followed by 4 weeks of reperfusion. Infarct size was examined by Evans blue/triphenyl tetrazolium chloride (TTC) staining. Cardiac function and remodeling were examined by echocardiography and histology. HO-1 gene transcription and expression were measured by RT-PCR and western blot. RESULTS: Compared to vehicle-treated MI/R rats, rats administered a single dose of U50,488H at the beginning of reperfusion exhibited reduced myocardial infarct size, oxidative stress, hypertrophy, and fibrosis, improved mechanical function, and greater neovascularization. U50,488H also increased myocardial heme oxygenase (HO)-1 gene transcription and expression, while pharmacological HO-1 inhibition reversed all protective effects of U50,488H. Furthermore, U50,488H protected control cultured cardiomyoctes against simulated I/R-induced apoptosis but not cultures subjected to shRNA-mediated HO-1 knockdown. Inhibition of HO-1 in the subacute phase of reperfusion reversed the U50,488H-induced increase in neovascularization and suppression of oxidative stress. Finally, U50,488H increased Akt phosphorylation and nuclear translocation of Nrf2, a key HO-1 transcription activator, while inhibition of PI3K-Akt signaling abolished U50,488H-induced Nrf2 nuclear translocation, HO-1 upregulation, and cardioprotection. CONCLUSION: Activation of HO-1 expression through the PI3K-Akt-Nrf2 pathway may mediate the acute and long-term protective effects of U50,488H against heart failure by enhancing cardiomyocyte survival and neoangiogenesis and by reducing oxidative stress.

PEGylated exendin-4, a modified GLP-1 analog exhibits more potent cardioprotection than its unmodified parent molecule on a dose to dose basis in a murine model of myocardial infarction.

A Site-specifically PEGylated exendin-4 (denoted as PEG-Ex4) is an exendin-4 (denoted as Ex4) analog we developed by site-specific PEGylation of exendin-4 with a high molecular weight trimeric poly(ethylene glycol) (tPEG). It has been shown to possess prolonged half-life in vivo with similar receptor binding affinity compared to unmodified exendin-4 by our previous work. This study is sought to test whether PEG-Ex4 is suitable for treating myocardial infarction (MI). In the MI model, PEG-Ex4 was administered every 3 days while equivalent amount of Ex4 was administered every 3 days or twice daily. Animal survival rate, heart function, remodeling and neoangiogenesis were evaluated and compared. Tube formation was examined in endothelial cells. In addition, Western blotting and histology were performed to determine the markers of cardiac hypertrophy and angiogenesis and to explore the possible molecular mechanism involved. PEG-Ex4 and Ex4 showed comparable binding affinity to GLP-1 receptor. In MI mice, PEG-Ex4 given at 3 days interval achieved similar extent of protection as Ex4 given twice daily, while Ex4 given at 3 days interval failed to produce protection. PEG-Ex4 elevated endothelial tube formation in vitro and capillary density in the border area of MI. PEG-Ex4 increased Akt activity and VEGF production in a GLP-1R dependent manner in endothelial cells and antagonism of GLP-1R, Akt or VEGF abolished the protection of PEG-Ex4 in the MI model. PEG-Ex4 is a potent long-acting GLP-1 receptor agonist for the treatment of chronic heart disease. Its protection might be attributed to enhanced angiogenesis mediated by the activation of Akt and VEGF.

In vivo multimodality imaging of miRNA-16 iron nanoparticle reversing drug resistance to chemotherapy in a mouse gastric cancer model.

miRNA-16 (miR16) plays an important role in modulating the drug resistance of SGC7901 cell lines to adriamycin (ADR). A variety of viral carriers have been designed for miRNA delivery. However, the safety concerns are currently perceived as hampering the clinical application of viral vector-based therapy. Herein a type of magnetic nanoparticles (MNPs) was designed and synthesized using poly(ethylene glycol) (PEG)-coated Fe3O4 nanoparticles as a miRNA delivery system for the purpose of reducing drug resistance of gastric cancer cells by enforcing miR16 expression in SGC7901/ADR cells. The MNPs with good biocompatibility were synthesized by thermal decomposition, and then conjugated with miRNA via electrostatic interaction producing miR16/MNPs. After co-culture with miR16/MNPs, ADR-induced apoptosis of SGC7901/ADR was examined by MTT and TUNEL. miR16/MNPs treatment significantly increased cell apoptosis in vitro. SGC7901/ADR(fluc) tumor-bearing nude mice under ADR therapy were treated with miR16/MNPs by tail vein injection for in vivo study. After intraperitoneal injection of ADR, tumor volume measurement and fluorescence imaging were performed to for the death of SGC7901/ADR cells in vivo. Results showed that miR16/MNPs were able to significantly suppress SGC7901/ADR tumor growth, probably through increasing SGC7901/ADR cells' sensitivity to ADR. Our results suggest the efficient delivery of miR16 by MNPs as a novel therapeutic strategy for drug resistant tumor treatment.

In Vivo Labeling of Serum Albumin for PET.

UNLABELLED: The purpose of this study was to develop a novel in vivo albumin-labeling method to allow PET of cardiac function after myocardial infarction and vascular leakage and increased permeability in inflammatory diseases and malignant tumors. METHODS: To label albumin in vivo, we synthesized a NOTA (1,4,7-triazacyclononane-N,N',N″-triacetic acid)-conjugated truncated form of Evans blue (NEB). (18)F labeling was achieved by the formation of an (18)F-aluminum fluoride ((18)F-AlF) complex, and (64)Cu labeling was obtained by a standard chelation method. Sixty-minute dynamic PET imaging was performed on normal mice to evaluate the distribution of (18)F-AlF-NEB, which was compared with in vitro-labeled mouse serum albumin ((18)F-fluorobenzyl-MSA). Electrocardiography-gated PET imaging was performed in a mouse model of myocardial infarction. Both dynamic and static PET scans were obtained in a mouse inflammation model induced by local injection of turpentine to evaluate vascular leakage. Tumor permeability was studied by dynamic and late-point static PET using (64)Cu-NEB in a UM-22B xenograft model. RESULTS: NEB was successfully synthesized, and (18)F labeling including work-up took about 20-30 min, with a radiochemical purity greater than 95% without the need for high-performance liquid chromatography purification. Most of the radioactivity was retained in the circulation system at 60 min after injection (26.35 ± 1.52 percentage injected dose per gram [%ID/g]). With electrocardiography-gated PET, ventricles of the heart and major arteries were clearly visualized. The myocardial infarction mice showed much lower left ventricular ejection fraction than the control mice. Inflammatory muscles showed significantly higher tracer accumulation than the contralateral healthy ones. UM-22B tumor uptake of (64)Cu-NEB gradually increased with time (5.73 ± 1.11 %ID/g at 1 h and 8.03 ± 0.77 %ID/g at 2 h after injection). CONCLUSION: The distribution and local accumulation of serum albumin can be noninvasively visualized and quantified by (18)F-AlF-NEB and (64)Cu-NEB PET. The simple labeling and broad applications make these imaging probes attractive for clinical translation.

Comparison of three dimeric 18F-AlF-NOTA-RGD tracers.

PURPOSE: RGD peptide-based radiotracers are well established as integrin αvß3 imaging probes to evaluate tumor angiogenesis or tissue remodeling after ischemia or infarction. In order to optimize the labeling process and pharmacokinetics of the imaging probes, we synthesized three dimeric RGD peptides with or without PEGylation and performed in vivo screening. PROCEDURES: Radiolabeling was achieved through the reaction of F-18 aluminum-fluoride complex with the cyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Three imaging probes were synthesized as (18)F-AlF-NOTA-E[c(RGDfK)]2, (18)F-AlF-NOTA-PEG4-E[c(RGDfK)]2, and (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2. The receptor binding affinity was determined by competitive cell binding assay, and the stability was evaluated by mouse serum incubation. Tumor uptake and whole body distribution of the three tracers were compared through direct tissue sampling and PET quantification of U87MG tumor-bearing mice. RESULTS: All three compounds remained intact after 120 min incubation with mouse serum. They all had a rapid and relatively high tracer uptake in U87MG tumors with good target-to-background ratios. Compared with the other two tracers, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 had the highest tumor uptake and the lowest accumulation in the liver. The integrin receptor specificity was confirmed by co-injection of unlabeled dimeric RGD peptide. CONCLUSION: The rapid one-step radiolabeling strategy by the complexation of (18)F-aluminum fluoride with NOTA-peptide conjugates was successfully applied to synthesize three dimeric RGD peptides. Among the three probes developed, (18)F-AlF-NOTA-E[PEG4-c(RGDfk)]2 with relatively low liver uptake and high tumor accumulation appears to be a promising candidate for further translational research.

VEGF-loaded graphene oxide as theranostics for multi-modality imaging-monitored targeting therapeutic angiogenesis of ischemic muscle.

Herein we report the design and synthesis of multifunctional VEGF-loaded IR800-conjugated graphene oxide (GO-IR800-VEGF) for multi-modality imaging-monitored therapeutic angiogenesis of ischemic muscle. The as-prepared GO-IR800-VEGF positively targets VEGF receptors, maintains an elevated level of VEGF in ischemic tissues for a prolonged time, and finally leads to remarkable therapeutic angiogenesis of ischemic muscle. Although more efforts are required to further understand the in vivo behaviors and the long-term toxicology of GO, our work demonstrates the success of using GO for efficient VEGF delivery in vivo by intravenous administration and suggests the great promise of using graphene oxide in theranostic applications for treating ischemic disease.

Histone deacetylase inhibitors are neuroprotective and preserve NGF-mediated cell survival following traumatic brain injury.

Acute traumatic brain injury (TBI) is associated with long-term cognitive and behavioral dysfunction. In vivo studies have shown histone deacetylase inhibitors (HDACis) to be neuroprotective following TBI in rodent models. HDACis are intriguing candidates because they are capable of provoking widespread genetic changes and modulation of protein function. By using known HDACis and a unique small-molecule pan-HDACi (LB-205), we investigated the effects and mechanisms associated with HDACi-induced neuroprotection following CNS injury in an astrocyte scratch assay in vitro and a rat TBI model in vivo. We demonstrate the preservation of sufficient expression of nerve growth factor (NGF) and activation of the neurotrophic tyrosine kinase receptor type 1 (TrkA) pathway following HDACi treatment to be crucial in stimulating the survival of CNS cells after TBI. HDACi treatment up-regulated the expression of NGF, phospho-TrkA, phospho-protein kinase B (p-AKT), NF-κB, and B-cell lymphoma 2 (Bcl-2) cell survival factors while down-regulating the expression of p75 neurotrophin receptor (NTR), phospho-JNK, and Bcl-2-associated X protein apoptosis factors. HDACi treatment also increased the expression of the stem cell biomarker nestin, and decreased the expression of reactive astrocyte biomarker GFAP within damaged tissue following TBI. These findings provide further insight into the mechanisms by which HDACi treatment after TBI is neuroprotective and support the continued study of HDACis following acute TBI.

Development of a new thiol site-specific prosthetic group and its conjugation with [Cys(40)]-exendin-4 for in vivo targeting of insulinomas.

A new tracer, N-5-[(18)F]fluoropentylmaleimide ([(18)F]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps ((18)F displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine, and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11-17% radiochemical yield (uncorrected), and specific activity of 20-49 GBq/µmol. [(18)F]FPenM showed comparable labeling efficiency with N-[2-(4-[(18)F]fluorobenzamido)ethyl]maleimide ([(18)F]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys(40)]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and metabolic stability of the radiolabeled [(18)F]FPenM-[cys(40)]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [(18)F]FPenM-[cys(40)]-exendin-4, had high tumor uptake (20.32 ± 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was comparable to the imaging results with [(18)F]FBEM-[cys(40)]-exendin-4 reported by our group.

Comparison of (18)F-labeled CXCR4 antagonist peptides for PET imaging of CXCR4 expression.

PURPOSE: CXCR4 is overexpressed on tumor cells from many types of human cancers. A high level of CXCR4 expression often correlates with poor prognosis, chemotherapy resistance, and metastasis. The development of CXCR4-specific radiotracers for positron emission tomography (PET) imaging will allow in vivo evaluation of receptor expression level for diagnosis or therapeutic evaluation. PROCEDURES: Two new (18)F-labeled radiotracers based on an Ac-TC14012 peptide, [(18)F]FP-Ac-TC14012 and [(18)F]FB-Ac-TC14012, were synthesized and characterized. The affinities of the 2-fluoropropionate (FP)-conjugated or 4-fluorobenzoate (FB)-conjugated peptides to CXCR4-transfected Chinese hamster ovarian (CHO) cells were evaluated in a competitive binding assay with [(125)I]CXCL12 radioligand. The cell uptake and retention of [(18)F]FP-labeled and [(18)F]FB-labeled peptides were measured. The tumor targetability and pharmacokinetics of these two tracers were also evaluated by microPET imaging and biodistribution studies. RESULTS: The labeled peptides retained high binding affinity to CXCR4 and showed much higher uptake in CXCR4-positive CHO cells than in CXCR4-negative cells in vitro. The smaller and more hydrophilic [(18)F]FP prosthetic group resulted in higher affinity and lower nonspecific cell uptake compared to the [(18)F]FB-labeled peptide. Both radiotracers showed much higher accumulation in CXCR4-positive than CXCR4-negative tumor xenografts in mice and allowed clear visualization of CXCR4 expression by PET. Among the two, [(18)F]FP-Ac-TC14012 showed higher tumor uptake and better tumor-to-background contrast. Unlike their N-terminal 4-F-benzoate analogs, these two tracers had minimal blood retention, likely due to reduced red blood cell binding. Metabolic organs, such as the liver and kidney, also showed high uptake. When blocked with low-dose cold peptide (10 µg), the tumor uptake was significantly increased, most likely due to the increased concentration in blood circulation, as evidenced by decreased liver uptake. CONCLUSION: These results demonstrate that the [(18)F]FP-labeled Ac-TC14012 peptide with high tumor uptake, low nonspecific binding, and good tumor-to-background contrast promises [(18)F]FP-Ac-TC14012 as a PET tracer for in vivo PET imaging of CXCR4 expression.

α-Lipoic acid reduces infarct size and preserves cardiac function in rat myocardial ischemia/reperfusion injury through activation of PI3K/Akt/Nrf2 pathway.

BACKGROUND: The present study investigates the effects and mechanisms of α-Lipoic acid (LA) on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in rat hearts subjected to in vivo myocardial ischemia/reperfusion (MI/R) injury. METHODOLOGY/PRINCIPAL FINDINGS: Male adult rats underwent 30 minutes of ischemia followed by 3, 24, or 72 h of reperfusion. Animals were pretreated with LA or vehicle before coronary artery ligation. The level of MI/R- induced LDH and CK release, infarct size, cardiomyocyte apoptosis and cardiac functional impairment were examined and compared. Western blot analysis was performed to elucidate the mechanism of LA pretreatment. The level of inflammatory cytokine TNF-α released to serum and accumulated in injured myocardium as well as neutrophil accumulation in injured myocardium were also examined after MI/R injury. Our results reveal that LA administration significantly reduced LDH and CK release, attenuated myocardial infarct size, decreased cardiomyocytes apoptosis, and partially preserved heart function. Western blot analysis showed that LA pretreatment up-regulated Akt phosphorylation and Nrf2 nuclear translocation while producing no impact on p38MAPK activation or nitric oxide (NO) production. LA pretreatment also increased expression of HO-1, a major target of Nrf2. LA treatment inhibited neutrophil accumulation and release of TNF-α. Moreover, PI3K inhibition abolished the beneficial effects of LA. CONCLUSIONS/SIGNIFICANCE: This study indicates that LA attenuates cardiac dysfunction by reducing cardiomyoctyes necrosis, apoptosis and inflammation after MI/R. LA exerts its action by activating the PI3K/Akt pathway as well as subsequent Nrf2 nuclear translocation and induction of cytoprotective genes such as HO-1.