Splenic monocytes mediate inflammatory response and exacerbate myocardial ischemia/reperfusion injury in a mitochondrial cell-free DNA-TLR9-NLRP3-dependent fashion.

The spleen contributes importantly to myocardial ischemia/reperfusion (MI/R) injury. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) recruits inflammasomes, initiating inflammatory responses and mediating tissue injury. We hypothesize that myocardial cell-free DNA (cfDNA) activates the splenic NLRP3 inflammasome during early reperfusion, increases systemic inflammatory response, and exacerbates myocardial infarct. Mice were subjected to 40 min of ischemia followed by 0, 1, 5, or 15 min, or 24 h of reperfusion. Splenic leukocyte adoptive transfer was performed by injecting isolated splenocytes to mice with splenectomy performed prior to left coronary artery occlusion. CY-09 (4 mg/kg) was administered 5 min before reperfusion. During post-ischemic reperfusion, splenic protein levels of NLRP3, cleaved caspase-1, and interleukin-1ß (IL-1ß) were significantly elevated and peaked (2.1 ± 0.2-, 3.4 ± 0.4-, and 3.2 ± 0.2-fold increase respectively, p < 0.05) within 5 min of reperfusion. In myocardial tissue, NLRP3 was not upregulated until 24 h after reperfusion. Suppression by CY09, a specific NLRP3 inflammasome inhibitor, or deficiency of NLRP3 significantly reduced myocardial infarct size (17.3% ± 4.2% and 33.2% ± 1.8% decrease respectively, p < 0.01). Adoptive transfer of NLRP3-/- splenocytes to WT mice significantly decreased infarct size compared to transfer of WT splenocytes (19.1% ± 2.8% decrease, p < 0.0001). NLRP3 was mainly activated at 5 min after reperfusion in CD11b+ and LY6G- splenocytes, which significantly increased during reperfusion (24.8% ± 0.7% vs.14.3% ± 0.6%, p < 0.0001). The circulating cfDNA level significantly increased in patients undergoing cardiopulmonary bypass (CPB) (43.3 ± 5.3 ng/mL, compared to pre-CPB 23.8 ± 3.5 ng/mL, p < 0.01). Mitochondrial cfDNA (mt-cfDNA) contributed to NLRP3 activation in macrophages (2.1 ± 0.2-fold increase, p < 0.01), which was inhibited by a Toll-like receptor 9(TLR9) inhibitor. The NLRP3 inflammasome in splenic monocytes is activated and mediates the inflammatory response shortly after reperfusion onset, exacerbating MI/R injury in mt-cfDNA/TLR9-dependent fashion. The schema reveals splenic NLRP3 mediates the inflammatory response in macrophages and exacerbates MI/R in a mitochondrial cfDNA/ TLR9-dependent fashion.

N6-methyladenosine writer METTL3 accelerates the sepsis-induced myocardial injury by regulating m6A-dependent ferroptosis.

Ferroptosis is an iron-dependent and phospholipid peroxidation-mediated cell death, which has been identified to be involved in sepsis-induced injury. However, the in-depth molecular mechanisms of N6-methyladenosine (m6A) and ferroptosis on sepsis-induced myocardial injury are still unclear. Here, in the septic myocardial injury, m6A methyltransferase METTL3 level and methylation level high-expressed in lipopolysaccharide (LPS)-induced cardiomyocytes (H9C2). Functionally, METTL3 silencing repressed the ferroptosis phenotype induced by LPS. Mechanistically, METTL3-mediated m6A methylation on solute carrier family 7 member 11 (SLC7A11) empowered its mRNA with high methylation level. Moreover, YTHDF2 directly bound to the m6A modification sites of SLC7A11 to mediate the mRNA degradation. The m6A modified SLC7A11 mRNA was recognized by YTHDF2, which promoted the decay of SLC7A11 mRNA, consequently up-regulating ferroptosis in sepsis-induced myocardial injury. Together, these findings establish a role of METTL3 in the ferroptosis of LPS-induced cardiomyocytes, and provide potential therapeutic target to treat the sepsis-induced myocardial injury.

RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice.

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the epidermal growth factor receptor (EGFR) signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of methyltransferase like 3 (METTL3), the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3' untranslated regions (3'UTR) accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, thrombospondin-1 (TSP-1), an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

Nrf2 participates in the protective effect of exogenous mitochondria against mitochondrial dysfunction in myocardial ischaemic and hypoxic injury.

OBJECTIVE: Coronary artery disease is one of the leading causes of death worldwide. Treatments including coronary artery intervention can cause complications, such as myocardial ischaemia-reperfusion injury (MIRI). Mitochondrial injury or dysfunction is a key pathology of MIRI. Mitochondrial transplantation is considered a promising therapeutic strategy for cardiac-related diseases, but its mechanism is still unclear. Nrf2 is a prominent player in supporting the structural and functional integrity of mitochondria. In our research, we focused on the effect of Nrf2 in the treatment of MIRI by mitochondrial transplantation. H9C2 cells were subjected to hypoxia/reoxygenation (H/R) and MIRI was induced in wild-type and Nrf2-/- mice by surgical ligation of the left coronary artery to elucidate the mechanism in vitro and in vivo, respectively. Exogenous mitochondria were extracted from healthy H9C2 cells and the pectoralis major and administered to H9C2 cells and mice with MIRI, respectively. Mitochondrial internalization, H9C2 cell injury or apoptosis, cardiac injury/function, mitochondrial function, morphology, mitochondrial dynamics, and the expression of components of the Nrf2 pathway were assessed. We found that exogenous mitochondria were internalized into H9C2 cardiomyocytes. Exogenous mitochondrial transplantation attenuated cardiomyocyte injury, cardiomyocyte apoptosis, and mitochondrial dysfunction. Exogenous mitochondrial transplantation increased the expression of Nrf2 and its downstream targets, attenuated cardiomyocyte injury, cardiac dysfunction, apoptosis, mitochondrial dysfunction, and mitochondrial fusion and fission imbalance, and improved mitophagy after MIRI in wild-type mice but not in Nrf2-/- mice. These results suggested that exogenous mitochondria can be internalized into cardiomyocytes and activate the Nrf2 pathway and that exogenous mitochondria improve cardiac function and ameliorate mitochondrial dysfunction via the Nrf2 pathway.

Pulsed ultrasound attenuates the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury.

OBJECTIVE: Acute hyperglycemia during myocardial infarction worsens outcomes in part by inflammatory mechanisms. Pulsed ultrasound has anti-inflammatory potential in bone healing and neuromodulation. We hypothesized that pulsed ultrasound would attenuate the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury via the cholinergic anti-inflammatory pathway. METHODS: Acute hyperglycemia was induced in wild-type C57BL6 or acetylcholine-receptor knockout (α7nAChR-/-) mice by intraperitoneal injection of glucose. Pulsed ultrasound (frequency 7 MHz, bursting mechanical index 1.2, duration 1 second, repeated every 6 seconds for 2 minutes, 20-second total exposure) was performed at the spleen or neck after glucose injection. Separate mice underwent vagotomy before treatment. The left coronary artery was occluded for 20 minutes, followed by 60 minutes of reperfusion. The primary end point was infarct size in explanted hearts. RESULTS: Splenic pulsed ultrasound significantly decreased infarct size in wild-type C57BL6 mice exposed to acute hyperglycemia and myocardial ischemia-reperfusion injury (5.2% ± 4.4% vs 16.9% ± 12.5% of risk region, P = .013). Knockout of α7nAChR abrogated the beneficial effect of splenic pulsed ultrasound (22.2% ± 12.1%, P = .79 vs control). Neck pulsed ultrasound attenuated the hyperglycemic exacerbation of myocardial infarct size (3.5% ± 4.8%, P = .004 vs control); however, the cardioprotective effect disappeared in mice that underwent vagotomy. Plasma acetylcholine, ß2 adrenergic receptor, and phosphorylated Akt levels were increased after splenic pulsed ultrasound treatment. CONCLUSIONS: Pulsed ultrasound treatment of the spleen or neck attenuated the hyperglycemic exacerbation of myocardial ischemia-reperfusion injury leading to a 3-fold decrease in infarct size. Pulsed ultrasound may provide cardioprotection via the cholinergic anti-inflammatory pathway and could be a promising new nonpharmacologic, noninvasive therapy to reduce infarct size during acute myocardial infarction and improve patient outcomes.

MicroRNA-34c Inhibits Osteogenic Differentiation and Valvular Interstitial Cell Calcification via STC1-Mediated JNK Pathway in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD), a common heart valve disease, is increasingly prevalent worldwide and causes high morbidity and mortality. Here, we aimed to investigate a possible role for miR-34c in the development of osteogenic differentiation during CAVD and to find out the underlying mechanisms. Valvular interstitial cells (VICs) were isolated from the clinical aortic valve tissue samples of CAVD patients and patients with acute aortic dissection and collected. Then, RT-qPCR was performed to determine miR-34c expression and western blot analysis was applied to confirm the relevant protein expression in these VICs. Dual luciferase reporter gene assay was applied to confirm the relation between miR-34c and STC1. Alkaline phosphatase (ALP) staining and alizarin red staining was performed to further confirm the degree of calcification in these samples. MiR-34c was lowly expressed and STC1 was highly expressed in the CAVD tissues. Furthermore, STC1 was the target of miR-34c and was negatively regulated by miR-34c. Overexpression of miR-34c in VICs was concomitant with suppression of both STC1 expression and phosphorylation level of c-Jun N-terminal kinase (JNK). In addition, significant decrease of bone morphogenetic protein-2 (BMP2) and osteocalcin, as well as the decrease of calcification degree were also observed in VICs with miR-34c overexpressed. Taken together, miR-34c could inhibit osteogenic differentiation and calcification of VICs by suppressing the STC1/JNK signaling pathway in CAVD, making miR-34c a novel therapeutic target for the treatment of CAVD.

Macrophage K63-Linked Ubiquitination of YAP Promotes Its Nuclear Localization and Exacerbates Atherosclerosis.

The Hippo/Yes-associated protein (YAP) pathway has pivotal roles in innate immune responses against pathogens in macrophages. However, the role of YAP in macrophages during atherosclerosis and its mechanism of YAP activation remain unknown. Here, we find that YAP overexpression in myeloid cells aggravates atherosclerotic lesion size and infiltration of macrophages, whereas YAP deficiency reduces atherosclerotic plaque. Tumor necrosis factor receptor-associated factor 6 (TRAF6), a downstream effector of interleukin-1ß (IL-1ß), triggers YAP ubiquitination at K252, which interrupts the interaction between YAP and angiomotin and results in enhanced YAP nuclear translocation. The recombinant IL-1 receptor antagonist anakinra reduces atherosclerotic lesion formation, which is abrogated by YAP overexpression. YAP level is increased in human and mouse atherosclerotic vessels, and plasma IL-1ß level in patients with STEMI is correlated with YAP protein level in peripheral blood mononuclear cells. These findings elucidate a mechanism of YAP activation, which might be a therapeutic target for atherosclerosis.

Therapeutic Efficacy of Alpha-Lipoic Acid against Acute Myocardial Infarction and Chronic Left Ventricular Remodeling in Mice.

BACKGROUND: We hypothesized that daily administration of a potent antioxidant (α-lipoic acid: ALA) would protect the heart against both acute myocardial infarction (AMI) and left ventricular remodeling (LVR) post-AMI. METHODS AND RESULTS: Two separate studies were conducted. In the AMI study, C57Bl/6 mice were fed ALA daily for 7 d prior to a 45-minute occlusion of the left coronary artery (LCA). Mean infarct size in control mice (fed water) was 60 ± 2%. Mean infarct size in ALA-treated mice was 42 ± 3% in the 15 mg/kg·d group and 39 ± 3% in the 75 mg/kg·d group (both P < 0.05 vs. control). In the LVR study, AMI increased LV end-systolic volume (LVESV) and reduced LV ejection fraction (LVEF) to a similar extent in both groups when assessed by cardiac MRI 1 day after a 2-hour LCA occlusion. Treatment with ALA (75 mg/kg·d) or H2O was initiated 1 day post-AMI and continued until study's end. Both LVESV and LVEF in ALA-treated mice were significantly improved over control when assessed 28 or 56 days post-AMI. Furthermore, the survival rate in ALA-treated mice was 63% better than in control mice by 56 days post-AMI. CONCLUSIONS: Daily oral ingestion of ALA not only protects mice against AMI but also attenuates LVR and preserves contractile function in the months that follow.

Upregulation of microRNA-195 ameliorates calcific aortic valve disease by inhibiting VWF via suppression of the p38-MAPK signaling pathway.

BACKGROUND: Growing evidence has indicated that microRNAs (miRNAs) are involved in the progression of calcific aortic valve disease (CAVD), a progressive pathological condition with no effective pharmacological therapy. This study was set out with the aim to investigate possible roles of miR-195 in CAVD. METHODS AND RESULTS: Initially, the differential expressed genes (DEGs) associated with CAVD were screened out and miRNAs potentially regulating VWF were predicted from microarray analysis. Next, we quantified VWF and miR-195 expression in isolated aortic valve interstitial cells (AVICs) and aortic valve tissues, followed by confirmation of the target relationship between miR-195 and VWF using the dual luciferase reporter assay. Furthermore, we evaluated the biological functions of miR-195 and VWF on ALP activity, cell differentiation, and the levels of miR-195, VWF, Runx2, OCN, ALP, p38 and phosphorylated p38 in AVICs. VWF was highly expressed, while miR-195 was poorly expressed in CAVD. Furthermore, miR-195 targeted VWF and negatively regulated its expression. Upregulation of miR-195 or silencing VWF could reduce ALP activity, calcified deposition, and the mRNA and protein levels of Runx2, OCN, and ALP by inhibiting the p38-MAPK signaling pathway, thereby ameliorating aortic valve calcification in vitro. CONCLUSIONS: On all accounts, miR-195 can potentially inhibit aortic valve calcification by repressing VWF and p38-MAPK signaling pathway, highlighting a theoretical basis for pharmacological treatment of CAVD.

The Effects of Inhibition of MicroRNA-375 in a Mouse Model of Doxorubicin-Induced Cardiac Toxicity.

BACKGROUND Doxorubicin-induced myocardial toxicity is associated with oxidative stress, cardiomyocyte, apoptosis, and loss of contractile function. Previous studies showed that microRNA-375 (miR-375) expression was increased in mouse models of heart failure and clinically, and that inhibition of miR-375 reduced inflammation and increased survival of cardiomyocytes. This study aimed to investigate the effects and mechanisms of inhibition of miR-375 in a mouse model of doxorubicin-induced cardiac toxicity in vivo and in doxorubicin-treated rat and mouse cardiomyocytes in vitro. MATERIAL AND METHODS The mouse model of doxorubicin-induced cardiac toxicity was developed using an intraperitoneal injection of doxorubicin (15 mg/kg diluted in 0.9% saline) for eight days. Treatment was followed by a single subcutaneous injection of miR-375 inhibitor. H9c2 rat cardiac myocytes and adult murine cardiomyocytes (AMCs) were cultured in vitro and treated with doxorubicin, with and without pretreatment with miR-375 inhibitor. RESULTS Doxorubicin significantly upregulated miR-375 expression in vitro and in vivo, and inhibition of miR-375 re-established myocardial redox homeostasis, prevented doxorubicin-induced oxidative stress and cardiomyocyte apoptosis, and activated the PDK1/AKT axis by reducing the direct binding of miR-375 to 3' UTR of the PDK1 gene. Inhibition of PDK1 and AKT abolished the protective role of miR-375 inhibition on doxorubicin-induced oxidative damage. CONCLUSIONS Inhibition of miR-375 prevented oxidative damage in a mouse model of doxorubicin-induced cardiac toxicity in vivo and in doxorubicin-treated rat and mouse cardiomyocytes in vitro through the PDK1/AKT signaling pathway.

The myocardial infarct-exacerbating effect of cell-free DNA is mediated by the high-mobility group box 1-receptor for advanced glycation end products-Toll-like receptor 9 pathway.

INTRODUCTION: Damage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE). METHODS: C57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer. RESULTS: Free HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40'/60' IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20'/60' IRI, injection of 40'/5' plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40'/0' coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40'/0' coronary perfusate and 40'/reperfusion (min) plasma but not in those with 10' ischemia. In C57BL/6 mice without IRI, 40'/5' plasma significantly increased the interleukin-1ß protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 µg/g) or mitochondrial DNA (0.5 µg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice. CONCLUSIONS: Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9-dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.

Infarct-Sparing Effect of Adenosine A2B Receptor Agonist Is Primarily Due to Its Action on Splenic Leukocytes Via a PI3K/Akt/IL-10 Pathway.

BACKGROUND: Adenosine A2B receptor (A2BAR) agonist reduces myocardial reperfusion injury by acting on inflammatory cells. Recently, a cardiosplenic axis was shown to mediate the myocardial postischemic reperfusion injury. This study aimed to explore whether the infarct-squaring effect of A2BAR agonist was primarily due to its action on splenic leukocytes. METHODS: C57BL6 (wild type [WT]) mice underwent 40 min of left coronary artery occlusion followed by 60 min of reperfusion. A2BAR knockout (KO) and interleukin (IL)-10KO mice served as donors for splenic leukocytes. Acute splenectomy was performed 30 min before ischemia. The acute splenic leukocyte adoptive transfer was performed by injecting 5 × 106 live splenic leukocytes into splenectomized mice. BAY 60-6583, an A2BAR agonist, was injected by i.v. 15 min before ischemia. The infarct size (IS) was determined using 2,3,5-triphenyltetrazolium chloride and Phthalo blue staining. The expression of p-Akt and IL-10 was estimated by Western blotting. Immunofluorescence staining assessed the localization of IL-10 expression. RESULTS: BAY 60-6583 reduced the myocardial IS in intact mice but failed to reduce the same in splenectomized mice, which had a smaller IS than intact mice. BAY 60-6583 reduced the IS in splenectomized mice with the acute transfer of WT splenic leukocytes; however, it did not protect the heart of splenectomized mice with the acute transfer of A2BRKO splenic leukocytes. Furthermore, BAY 60-6583 increased the levels of p-Akt and IL-10 in the WT spleen. Moreover, it did not exert any protective effect in IL-10KO mice. CONCLUSIONS: A2BAR activation before ischemia stimulated the IL-10 production in splenic leukocytes via a PI3K/Akt pathway, thereby exerting anti-inflammatory effects that limited the myocardial reperfusion injury.

Stimulation of the Beta2 Adrenergic Receptor at Reperfusion Limits Myocardial Reperfusion Injury via an Interleukin-10-Dependent Anti-Inflammatory Pathway in the Spleen.

BACKGROUND: In addition to the airway-relaxing effects, ß2 adrenergic receptor (ß2AR) agonists are also found to have broad anti-inflammatory effects. The current study was conducted to define the role of ß2AR agonists in limiting myocardial ischemia/reperfusion injury (IRI). Methods and Results: Adult male wild-type (WT) and interleukin (IL)-10 knockout (KO) mice underwent a 40-min left coronary artery ligation and 60-min reperfusion. A selective ß2AR agonist, Clenbuterol, at doses of 0.1 µg or 1 µg/g weight i.v. 5 min before reperfusion, significantly reduced myocardial infarct size (IS) by 28% and 39% (vs. control, P<0.05) in WT mice respectively, but had no protective effect in IL-10 KO mice. Inhalational therapy with nebulized Clenbuterol, Albuterol, Salmeterol or Arformoterol immediately before ischemia significantly reduced IS (P<0.05) in WT mice. Splenectomy similarly reduced IS as Clenbuterol-treated mice, but intravenous Clenbuterol did not further reduce IS in splenectomized mice. In splenectomized WT mice, acute transfer of isolated splenocytes, not the Clenbuterol-pretreated splenocytes, restored the myocardial IS to the level of intact mice. Intravenous Clenbuterol significantly increased splenic protein levels of ß2AR, phosphorylated Akt and IL-10 and plasma IL-10, and inhibited the expression of pro-inflammatory mRNAs. CONCLUSIONS: Both intravenous and inhalational ß2AR agonists exert a cardioprotective effect against IRI by activating the anti-inflammatory ß2AR-IL-10 pathway.

The spleen contributes importantly to myocardial infarct exacerbation during post-ischemic reperfusion in mice via signaling between cardiac HMGB1 and splenic RAGE.

The spleen plays a critical role in post-infarct myocardial remodeling. However, the role of the spleen in exacerbating myocardial infarction (MI) during acute ischemia/reperfusion (I/R) injury is unknown. The present study tests the hypothesis that splenic leukocytes are activated by substances released from ischemic myocardium to subsequently exacerbate myocardial injury during reperfusion. The left coronary artery in C57BL/6 mice underwent various durations of occlusion followed by 60 min of reperfusion (denoted as min/min of I/R) with or without splenectomy prior to I/R injury. Splenectomy significantly decreased myocardial infarct size (IS) in 40'/60' and 50'/60' groups (p < 0.05); however, it had no effect on IS in 10'/60', 20'/60' and 30'/60' groups (p = NS). In the 20'/60' group, infusion of 40-min ischemic heart homogenate (40-IHH) upon reperfusion increased IS by >threefold versus infusion of 10-IHH (p < 0.05). Splenectomy abolished the infarct-exacerbating effect of 40-IHH, which was restored by splenic leukocyte adoptive transfer (SPAT). Furthermore, depletion of HMGB1 in the 40-IHH group abolished its infarct-exacerbating effect (p < 0.05), and 40-IHH failed to increase IS in both RAGE(-/-) mice and splenectomized wild-type mice with SPAT from RAGE(-/-) mice. The injection of 40-IHH significantly increased formyl peptide receptor 1 (FPR1) expression in sham spleens when compared to 10-IHH-treated sham and control mice. cFLFLF, a specific FPR1 antagonist, reduced myocardial neutrophil infiltration and abrogated the infarct-exacerbating effect of 40-IHH during reperfusion. A cardio (HMGB1)-splenic (RAGE receptor) signaling axis exists and contributes to myocardial infarct exacerbation during reperfusion after prolonged ischemic insults by activating splenic leukocytes. The FPR1 is a potential therapeutic target for inhibiting the cardio-splenic axis that augments infarct size during post-ischemic reperfusion.

Repeatability and variability of myocardial perfusion imaging techniques in mice: Comparison of arterial spin labeling and first-pass contrast-enhanced MRI.

PURPOSE: Preclinical imaging of myocardial blood flow (MBF) can elucidate molecular mechanisms underlying cardiovascular disease. We compared the repeatability and variability of two methods, first-pass MRI and arterial spin labeling (ASL), for imaging MBF in mice. METHODS: Quantitative perfusion MRI in mice was performed using both methods at rest, with a vasodilator, and one day after myocardial infarction. Image quality (score of 1-5; 5 best), between-session coefficient of variability (CVbs ), intra-user coefficient of variability (CVintra-user ), and inter-user coefficient of variability (CVinter-user ) were assessed. Acquisition time was 1-2 min for first-pass MRI and approximately 40 min for ASL. RESULTS: Image quality was higher for ASL (3.94 ± 0.09 versus 2.88 ± 0.10; P < 0.05). Infarct zone CVbs was lower with first-pass (17 ± 3% versus 46 ± 9%; P < 0.05). The stress perfusion CVintra-user was lower for ASL (3 ± 1% versus 14 ± 3%; P < 0.05). The stress perfusion CVinter-user was lower for ASL (4 ± 1% versus 17 ± 4%; P < 0.05). CONCLUSION: For low MBF conditions such as infarct, first-pass MRI is preferred due to better repeatability and variability. At high MBF such as at vasodilation, ASL may be more suitable due to superior image quality and lower user variability. First-pass MRI has a substantial speed advantage. Magn Reson Med 75:2394-2405, 2016. © 2015 Wiley Periodicals, Inc.

Development of target-specific liposomes for delivering small molecule drugs after reperfused myocardial infarction.

Although reperfusion is essential in restoring circulation to ischemic myocardium, it also leads to irreversible events including reperfusion injury, decreased cardiac function and ultimately scar formation. Various cell types are involved in the multi-phase repair process including inflammatory cells, vascular cells and cardiac fibroblasts. Therapies targeting these cell types in the infarct border zone can improve cardiac function but are limited by systemic side effects. The aim of this work was to develop liposomes with surface modifications to include peptides with affinity for cell types present in the post-infarct myocardium. To identify peptides specific for the infarct/border zone, we used in vivo phage display methods and an optical imaging approach: fluorescence molecular tomography (FMT). We identified peptides specific for cardiomyocytes, endothelial cells, myofibroblasts, and c-Kit + cells present in the border zone of the remodeling infarct. These peptides were then conjugated to liposomes and in vivo specificity and pharmacokinetics were determined. As a proof of concept, cardiomyocyte specific (I-1) liposomes were used to deliver a PARP-1 (poly [ADP-ribose] polymerase 1) inhibitor: AZ7379. Using a targeted liposomal approach, we were able to increase AZ7379 availability in the infarct/border zone at 24h post-injection as compared with free AZ7379. We observed ~3-fold higher efficiency of PARP-1 inhibition when all cell types were assessed using I-1 liposomes as compared with negative control peptide liposomes (NCP). When analyzed further, I-1 liposomes had 9-fold and 1.5-fold higher efficiencies in cardiomyocytes and macrophages, respectively, as compared with NCP liposomes. In conclusion, we have developed a modular drug delivery system that can be targeted to cell types of therapeutic interest in the infarct border zone.

Adenosine 2B Receptor Activation Reduces Myocardial Reperfusion Injury by Promoting Anti-Inflammatory Macrophages Differentiation via PI3K/Akt Pathway.

BACKGROUND: Activation of the adenosine A2B receptor (A2BR) can reduce myocardial ischemia/reperfusion (IR) injury. However, the mechanism underlying the A2BR-mediated cardioprotection is less clear. The present study was designed to investigate the potential mechanisms of cardioprotection mediated by A2BR. METHODS AND RESULTS: C57BL/6 mice underwent 40-minute ischemia and 60-minute reperfusion. ATL-801, a potent selective A2BR antagonist, could not block ischemic preconditioning induced protection. BAY 60-6583, a highly selective A2BR agonist, significantly reduced myocardial infarct size, and its protective effect could be blocked by either ATL-801 or wortmannin. BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10 min of reperfusion, and this phosphorylation could also be blocked by ATL-801 or wortmannin. Furthermore, BAY 60-6583 significantly increased M2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts, which also could be blocked by wortmannin. Meanwhile, confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalized to CD206+ cells in both control and BAY 60-6583 pretreated hearts. CONCLUSION: Our results indicated that pretreatment with BAY 60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects, probably by modulating macrophages phenotype switching via a PI3K/Akt pathway.

Splenic leukocytes mediate the hyperglycemic exacerbation of myocardial infarct size in mice.

Acute hyperglycemia during acute myocardial infarction is associated with worse myocardial injury and increased mortality. Using a mouse model of myocardial ischemia/reperfusion injury, we tested the hypothesis that acute hyperglycemia activates splenic leukocytes and subsequently exacerbates myocardial infarct size. We then examined whether the adverse effects of hyperglycemia could be attenuated by a potent anti-inflammatory agent (an agonist of the adenosine A2A receptor) administered immediately prior to reperfusion. C57BL6 (WT) mice underwent 30-min LAD occlusion and 60-min reperfusion with or without prior splenectomy. Acute hyperglycemia before ischemia increased myocardial infarct size (IS) by 43% (p < 0.05). Splenectomy before ischemia did not change IS (vs. control, p = NS) but did serve to prevent the exacerbation of IS by hyperglycemia. Acute hyperglycemia activated splenic leukocytes by increasing formyl peptide receptor expression and reactive oxygen species production before ischemia, and enhanced splenic neutrophil release with resultant peripheral neutrophilia and increased myocardial neutrophil infiltration during reperfusion. Acute adoptive transfer of splenic leukocytes to splenectomized mice before ischemia restored the hyperglycemic exacerbation of infarct size. ATL146e, an adenosine 2A receptor (A2AR) agonist, abolished neutrophilia during reperfusion and reduced IS in hyperglycemic mice. ATL146e also reduced IS in splenectomized hyperglycemic mice with transfer of WT splenic leukocytes, but not with transfer of splenic leukocytes from A2AR knockout mice. Acute hyperglycemia prior to myocardial ischemia and reperfusion exacerbates IS by activating splenic leukocytes. ATL146e administered at reperfusion suffices to abrogate the hyperglycemic exacerbation of IS by acting on A2ARs on splenic leukocytes.

The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors.

Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 µg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.

Atorvastatin at reperfusion reduces myocardial infarct size in mice by activating eNOS in bone marrow-derived cells.

BACKGROUND: The current study was designed to test our hypothesis that atorvastatin could reduce infarct size in intact mice by activating eNOS, specifically the eNOS in bone marrow-derived cells. C57BL/6J mice (B6) and congenic eNOS knockout (KO) mice underwent 45 min LAD occlusion and 60 min reperfusion. Chimeric mice, created by bone marrow transplantation between B6 and eNOS KO mice, underwent 40 min LAD occlusion and 60 min reperfusion. Mice were treated either with vehicle or atorvastatin in 5% ethanol at a dose of 10 mg/kg IV 5 min before initiating reperfusion. Infarct size was evaluated by TTC and Phthalo blue staining. RESULTS: Atorvastatin treatment reduced infarct size in B6 mice by 19% (p<0.05). In eNOS KO vehicle-control mice, infarct size was comparable to that of B6 vehicle-control mice (p = NS). Atorvastatin treatment had no effect on infarct size in eNOS KO mice (p = NS). In chimeras, atorvastatin significantly reduced infarct size in B6/B6 (donor/recipient) mice and B6/KO mice (p<0.05), but not in KO/KO mice or KO/B6 mice (p = NS). CONCLUSIONS: The results demonstrate that acute administration of atorvastatin significantly reduces myocardial ischemia/reperfusion injury in an eNOS-dependent manner, probably through the post-transcriptional activation of eNOS in bone marrow-derived cells.