Neo-peripheral adaptive immune score predicts neoadjuvant chemotherapy for locally advanced breast cancer.

PURPOSE: Whether peripheral immune cell subsets can predict pathological complete response (pCR) in breast cancer patients remains to be elucidated. We aimed to dissect the relationship between peripheral immune cell subsets and pCR. METHODS: Two hundred and twenty-six eligible patients from two prospective clinical trials (SHPD001 and SHPD002) in China were randomly divided into a training cohort and a validation cohort. The breast cancer subtypes in this study included hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative (n = 95), HER2-positive (n = 100), and triple negative (n = 31) breast cancer. We defined the "Neo-Peripheral Adaptive Immune Score" for neoadjuvant chemotherapy (neoPAI Score) based on the percentages of CD4 + T cells, CD8 + T cells, B cells, and the CD4 + /CD8 + ratio in peripheral blood. We also evaluated the ability of the neoPAI Score derived from tumor-infiltrating immune cells (TIICs) to predict survival by employing The Cancer Genome Atlas-Breast Cancer (TCGA-BRCA) database. RESULTS: In the training cohort, multivariate analysis showed that HR status [odds ratio (OR) 0.325; 95% confidence interval (CI) 0.135-0.761; P = 0.010], HER2 status (OR 2.657; 95% CI 1.266-5.730; P = 0.011), Ki67 index (OR 3.191; 95% CI 1.509-6.956; P = 0.003), histological grade (OR 2.297; 95% CI 1.031-5.290; P = 0.045) and neoPAI Score (OR 4.451; 95% CI 1.608-13.068; P = 0.005) were independent predictors of pCR. In the validation cohort, histological grade (OR 3.779; 95% CI 3.793-1.136 × 103; P = 0.008) and neoPAI Score (OR 90.828; 95% CI 3.827-9.843 × 103; P = 0.019) were independent predictors of pCR. The Immune Model that integrated the neoPAI Score was more accurate in predicting pCR than the Clinical Model that exclusively contained clinicopathological parameters in both cohorts. In TCGA-BRCA database, the neoPAI Score constructed from TIICs can predict the progression-free interval (P = 0.048) of breast cancer. CONCLUSION: The neoPAI Score defined by the percentages of peripheral immune cell subsets could be used as a potential biomarker for neoadjuvant chemotherapy efficacy.

Aspirin Blocks the Infarct-Size Limiting Effect of Ischemic Postconditioning in the Rat.

BACKGROUND: Ischemic postconditioning (PostC), repetitive cycles of re-occlusion, and reperfusion of the infarct-related artery immediately after reperfusion have been shown to limit myocardial infarct size in various animal models. Yet, translating the model into the clinical setting was disappointing, several clinical trials showing neutral effect. We hypothesized that aspirin loading could explain the differences between the pre-clinical and clinical studies. METHODS: Male Sprague Dawley rats were subjected to 30-min coronary artery ligation. At 25 min of ischemia, animals received intravenous aspirin (20 mg/kg) or vehicle. Upon reperfusion half of the rats were randomized to PostC (3 cycles of 10-s re-occlusion/10-s reperfusion. After 4-h reperfusion, rats were euthanized. Area at risk was assessed by blue dye and infarct size by 2,3,5-triphenyl-tetrazolium-chloride (TTC). RESULTS: Body weight and the size of the ischemic area at risk were comparable among groups. Infarct size expressed as a percentage of the ischemic area at risk was significantly smaller in the PostC group (13.9 ± 0.4%; p < 0.001) compared to the control group (31.0 ± 2.2%). Aspirin alone had no effect on infarct size (29.0 ± 2.6%). Yet, aspirin completely blocked the protective effect of PostC (33.3 ± 1.1%). CONCLUSIONS: Aspirin, administered before reperfusion, blocks the infarct size limiting effects of PostC in the rat.

Effects of Dapagliflozin on Myocardial Gene Expression in BTBR Mice with Type 2 Diabetes.

BACKGROUND: Dapagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, is approved for the treatment of type 2 diabetes, heart failure, and chronic kidney disease. DAPA-HF and DELIVER trial results demonstrate that the cardiovascular protective effect of dapagliflozin extends to non-diabetic patients. Hence, the mechanism-of-action may extend beyond glucose-lowering and is not completely elucidated. We have previously shown that dapagliflozin reduces cardiac hypertrophy, inflammation, fibrosis, and apoptosis and increases ejection fraction in BTBR mice with type 2 diabetes. METHODS: We conducted a follow-up RNA-sequencing study on the heart tissue of these animals and performed differential expression and Ingenuity Pathway analysis. Selected markers were confirmed by RT-PCR and Western blot. RESULTS: SGLT2 had negligible expression in heart tissue. Dapagliflozin improved cardiac metabolism by decreasing glycolysis and pyruvate utilization enzymes, induced antioxidant enzymes, and decreased expression of hypoxia markers. Expression of inflammation, apoptosis, and hypertrophy pathways was decreased. These observations corresponded to the effects of dapagliflozin in the clinical trials.

Recombinant Apyrase (AZD3366) Against Myocardial Reperfusion Injury.

PURPOSE: Recombinant apyrase (AZD3366) increases adenosine production and ticagrelor inhibits adenosine reuptake. We investigated whether intravenous AZD3366 before reperfusion reduces myocardial infarct size (IS) and whether AZD3366 and ticagrelor have additive effects. METHODS: Sprague-Dawley rats underwent 30 min ischemia. At 25 min of ischemia, animals received intravenous AZD3366 or vehicle. Additional animals received intravenous CGS15943 (an adenosine receptor blocker) or intraperitoneal ticagrelor. At 24 h reperfusion, IS was assessed by triphenyltetrazolium chloride. Other rats were subjected to 30 min ischemia followed by 1 h or 24 h reperfusion. Myocardial samples were assessed for adenosine levels, RT-PCR, and immunoblotting. RESULTS: AZD3366 and ticagrelor reduced IS. The protective effect was blocked by CGS15943. The effect of AZD3366 + ticagrelor was significantly greater than AZD3366. One hour after infarction, myocardial adenosine levels significantly increased with AZD3366, but not with ticagrelor. In contrast, 24 h after infarction, adenosine levels were equally increased by AZD3366 and ticagrelor, and levels were higher in the AZD3366 + ticagrelor group. One hour after reperfusion, AZD3366 and ticagrelor equally attenuated the increase in interleukin-15 (an early inflammatory marker after ischemic cell death) levels, and their combined effects were additive. AZD3366, but not ticagrelor, significantly attenuated the increase in RIP1, RIP3, and P-MLKL (markers of necroptosis) 1 h after reperfusion. AZD3366, but not ticagrelor, significantly attenuated the increase in IL-6 and GSDMD-N (markers of pyroptosis) 1 h after reperfusion. At 24 h of reperfusion, both agents equally attenuated the increase in these markers, and their effects were additive. CONCLUSIONS: AZD3366 attenuated inflammation, necrosis, necroptosis, and pyroptosis and limited IS. The effects of AZD3366 and ticagrelor were additive.

SGLT2 Inhibition by Dapagliflozin Attenuates Diabetic Ketoacidosis in Mice with Type-1 Diabetes.

BACKGROUND: SGLT2 inhibitors increase plasma ketone concentrations. It has been suggested that insulinopenia, along with an increase in the counter-regulatory hormones epinephrine, corticosterone, glucagon and growth hormone, can induce ketoacidosis, especially in type-1 diabetes (T1DM). Dehydration precipitates SGLT2 inhibitor-induced ketoacidosis in type-2 diabetes. We studied the effects of dapagliflozin and water deprivation on the development of ketoacidosis and the associated signaling pathways in T1DM mice. METHODS: C57BL/6 mice were fed a high-fat diet. After 7 days, some mice received intraperitoneal injection of streptozocin + alloxan (STZ/ALX). The treatment groups were control + water at lib; control + dapagloflozin + water at lib; control + dapagloflozin + water deprivation; STZ/ALX + water at lib; STZ/ALX + water deprivation; STZ/ALX + dapagloflozin + water at lib; STZ/ALX + dapagloflozin + water deprivation. Dapagliflozin was given for 7 days. In the morning of day 18, food was removed, and water was removed in the water deprivation groups. ELISA, rt-PCR, and immunoblotting were used to assess blood, heart, liver, white and brown adipose tissues. RESULTS: The T1DM mice had ketoacidosis even without water deprivation. Water deprivation increased plasma levels of ß-hydroxybutyrate, acetoacetate, corticosterone, and epinephrine and reduced the levels of adiponectin in T1DM mice. Interleukin (IL) 1ß, IL-6, IL-8, and TNFα were also increased in the T1DM mice with water deprivation. Dapagliflozin attenuated the changes in the T1DM mice without and with water deprivation. Likewise, water deprivation increased the activation of the inflammasome in the heart, liver, and white fat of the T1DM mice and dapagliflozin attenuated these changes. Dapagliflozin reduced the mRNA levels of glucagon receptors in the liver and the increase in GPR109a in white and brown fat. In the liver, dapagliflozin increased AMPK phosphorylation, and attenuated the phosphorylation of TBK1 and the activation of NFκB. CONCLUSIONS: Dapagliflozin reduced ketone body levels and attenuated the activation of NFκB and the activation of the inflammasome in T1DM mice with ketoacidosis.

Ticagrelor and Dapagliflozin Have Additive Effects in Ameliorating Diabetic Nephropathy in Mice with Type-2 Diabetes Mellitus.

PURPOSE: Ticagrelor and dapagliflozin can suppress the activation of the NOD-like receptor 3 (NLRP3)-inflammasome and activate AMP-activated protein kinase (AMPK). The anti-inflammatory effects of dapagliflozin has been shown to depend on AMPK activation. Dapagliflozin and ticagrelor have been shown to have additive effects on the progression of diabetic cardiomyopathy in BTBR ob/ob mice with type-2 diabetes. We assessed whether dapagliflozin and ticagrelor have additive effects on the activation of the NLRP3-inflammasome and the progression of diabetic nephropathy in mice with type-2 diabetes. METHODS: Eight-week-old BTBR received either no-drug, dapagliflozin (1.5 mg/kg/d), ticagrelor (100 mg/kg/d), or their combination for 12 weeks. Blood was assessed weekly for glucose and urine for glucose and albumin. After 12 weeks, blood creatinine, cystatin C, inflammasome activation, and insulin were assessed by ELISA. Renal cortex samples were assessed by hematoxylin and eosin and periodic acid-Schiff staining. RT-PCR and immunoblotting were used to evaluate fibrosis and the activation of Akt, AMPK and the inflammasome. RESULTS: Both ticagrelor and dapagliflozin reduced serum creatinine and cystatin C levels and urinary albumin. Both drugs attenuated the increase in glomerular area and mesangial matrix index. Both drugs decreased collagen-1 and collagen-3 expression and the activation of the NLRP3-inflammasome. Both drugs increased P-AMPK levels, but only dapagliflozin increased P-Akt levels. Overall, the protective effects of dapagliflozin and ticagrelor were additive. CONCLUSIONS: Dapagliflozin and ticagrelor attenuated the progression of diabetic nephropathy in BTBR ob/ob mice with additive effects of the combination. This was associated with AMPK activation and reduced activation of the NLRP3 inflammasome, whereas only dapagliflozin increased Akt activation.

Diagnosis of Occlusion Myocardial Infarction in Patients with Left Bundle Branch Block and Paced Rhythms.

PURPOSE OF REVIEW: A number of criteria have been developed to aid with the diagnosis of occlusion myocardial infarction (OMI) in patients with left bundle branch block (LBBB) and ventricular paced rhythms (VPR). The current guidelines do not provide clear preference for any specific ECG criteria in LBBB and paced rhythm patients. RECENT FINDINGS: This review delineates the difficulties of electrocardiographic diagnosis of OMI in both LBBB and VPR patients. We describe the original Sgarbossa and the newer criteria and their diagnostic performances. We highlight the expected changes of newer pacing modalities and how they may interfere with the electrocardiographic diagnosis of OMI. We recommend utilizing the Cai et al. algorithm, which combines clinical assessment with the Smith Modified Sgarbossa ECG criteria, for both LBBB and right ventricular pacing patients with suspected OMI. There is limited data concerning ECG changes of OMI in patients with the newer pacing modalities, such as biventricular, His-bundle, or left bundle branch pacing.

Acupuncture Reduces Hypertrophy and Cardiac Fibrosis, and Improves Heart Function in Mice with Diabetic Cardiomyopathy.

PURPOSE: To assess the effects of electro-acupuncture (EA) on glycemic control, myocardial inflammation, and the progression of diabetic cardiomyopathy in mice with type 2 diabetes. METHODS: Db/Db mice received EA at PC6+ST36 (DM-Acu), non-acupoint simulation (DM-Sham), or no treatment (DM). EA was applied for 30 min per day, 5 days a week for 4 weeks. Heart function was assessed by echocardiography. Myocardium was assessed by RT-PCR, immunoblotting, and histology. Serum TNF-α, IL-1α, IL-1ß, IL-6, and IL-8 were measured. RESULTS: DM-Acu, but not DM-Sham, reduced fasting blood glucose without affecting body weight. DM decreased systolic function. DM-Acu, but not DM-Sham, attenuated the decrease in systolic function. Heart weight was significantly smaller in the DM-Acu than in the DM and DM-Sham groups. Percent fibrosis and apoptosis were reduced in the DM-Acu, but not the DM-Sham, group. Serum levels of IL-1α, IL-1ß, IL-6, IL-8, ICAM-1, MCP-1, and TNF-α were significantly lower in the DM-Acu than in the DM or DM-Sham groups. Protein levels of P-Akt and P-AMPK and mRNA levels of phosphoinositide-3-kinase regulatory subunit 6 (PIK3r6) were significantly higher in the DM-Acu group. Myocardial mRNA and protein levels of insulin-like growth factor 1 receptor (IGF1R) were significantly lower in the DM and DM-Sham groups compared with the DM-Acu group. CONCLUSIONS: EA reduced serum glucose; prevented DM-induced hypertrophy and deterioration of systolic function, inflammation, and fibrosis; and restored IGF1R, P-Akt, and P-AMPK levels in mice with type 2 diabetes mellitus.

Dapagliflozin and Ticagrelor Have Additive Effects on the Attenuation of the Activation of the NLRP3 Inflammasome and the Progression of Diabetic Cardiomyopathy: an AMPK-mTOR Interplay.

PURPOSE: Ticagrelor, a P2Y12 receptor antagonist, and dapagliflozin, a sodium-glucose-cotransporter-2 inhibitor, suppress the activation of the NLRP3 inflammasome. The anti-inflammatory effects of dapagliflozin depend on AMPK activation. Also, ticagrelor can activate AMPK. We assessed whether dapagliflozin and ticagrelor have additive effects in attenuating the progression of diabetic cardiomyopathy in T2DM mice. METHODS: Eight-week-old BTBR and wild-type mice received no drug, dapagliflozin (1.5 mg/kg/day), ticagrelor (100 mg/kg/day), or their combination for 12 weeks. Heart function was evaluated by echocardiography and heart tissue samples were assessed for fibrosis, apoptosis, qRT-PCR, and immunoblotting. RESULTS: Both drugs attenuated the progression of diabetic cardiomyopathy as evident by improvements in left ventricular end-systolic and end-diastolic volumes and left ventricular ejection fraction, which were further improved by the combination. Both drugs attenuated the activation of the NOD-like receptor 3 (NLRP3) inflammasome and fibrosis. The effect of the combination was significantly greater than each drug alone on myocardial tissue necrotic factorα (TNFα) and interleukin-6 (IL-6) levels, suggesting additive effects. The combination had also a greater effect on ASC, collagen-1, and collagen-3 mRNA levels than each drug alone. While both drugs activated adenosine mono-phosphate kinase (AMPK), only dapagliflozin activated mTOR and increased RICTOR levels. Moreover, only dapagliflozin decreased myocardial BNP and Caspase-1 mRNA levels, and the effects of dapagliflozin on NLRP3 and collagen-3 mRNA levels were significantly greater than those of ticagrelor. CONCLUSIONS: Both dapagliflozin and ticagrelor attenuated the progression of diabetic cardiomyopathy, the activation of the NLRP3 inflammasome, and fibrosis in BTBR mice with additive effects of the combination. While both dapagliflozin and ticagrelor activated AMPK, only dapagliflozin activated mTOR complex 2 (mTORC2) in hearts of BTBR mice.

Ticagrelor Improves Remodeling, Reduces Apoptosis, Inflammation and Fibrosis and Increases the Number of Progenitor Stem Cells After Myocardial Infarction in a Rat Model of Ischemia Reperfusion.

BACKGROUND/AIMS: We assessed the effects of ticagrelor, aspirin and prasugrel, started 7days after myocardial ischemia-reperfusion injury on remodeling, inflammation and fibrosis in the rat. We examined whether ticagrelor can affect the number of progenitor cells in the border zone. Ticagrelor, started 24h after myocardial ischemia-reperfusion injury, attenuates the decrease in heart function and adverse remodeling, an effect which is blocked by aspirin. METHODS: Rats underwent 40min ischemia followed by reperfusion. Oral dosing with vehicle, ticagrelor (300mg/kg/d), aspirin (20mg/kg/d), their combination or prasugrel (15mg/kg/d) started 7days after infarction. Echocardiography was used to assess systolic function. Heart tissue were analyzed by rt-PCR, immunoblotting, ELISA and immunohistochemistry 2weeks after infarction. RESULTS: Both ticagrelor and aspirin attenuated the decrease in systolic function and remodeling, an effect that was blocked by their combination. Ticagrelor and aspirin attenuated the increase in ANP, BNP, collagen-I and collagen-III. Again, the effect was blocked by their combination. Ticagrelor increased c-Kit, Sca-1, Ki-67, CD34, attenuated the decrease in CD105 mRNA levels, and attenuated the increase in CD31, whereas aspirin increased Ki-67, suppressed the increase in CD31 and attenuated the decrease in CD105 mRNA levels. Prasugrel did not display any effects. CONCLUSION: Ticagrelor attenuated adverse remodeling and deterioration of left ventricular systolic function despite starting treatment after the myocardial ischemia-reperfusion injury is completed. Aspirin had similar effects; however, when combined with ticagrelor, the protective effects were significantly attenuated. Ticagrelor increased the levels of several markers of stem cells and regeneration, suggesting cardiac healing by recruiting regenerative cells into the infarct.

DPP-4 inhibition by linagliptin prevents cardiac dysfunction and inflammation by targeting the Nlrp3/ASC inflammasome.

We compared the effects of linagliptin (Lina, a DPP4 inhibitor) and GLP-1 receptor activation by exenatide followed by exendin-4 in an infusion pump (EX) on infarct size (IS), post-infarction activation of the inflammasome and remodeling in wild-type (WT) and db/db diabetic mice. Mice underwent 30 min ischemia followed by 24 h reperfusion. IS was assessed by TTC. Additional mice underwent permanent coronary artery occlusion. Echocardiography was performed 2w after infarction. Activation of the inflammasome in the border zone of the infarction was assessed by rt-PCR and ELISA 2w after reperfusion. Further in vitro experiments were done using primary human cardiofibroblasts and cardiomyocytes exposed to simulated ischemia-reoxygenation. Lina and EX limited IS in both the WT and the db/db mice. Lina and EX equally improved ejection fraction in both the WT and the db/db mice. mRNA levels of ASC, NALP3, IL-1ß, IL-6, Collagen-1, and Collagen-3 were higher in the db/db mice than in the WT mice. Infarction increased these levels in the WT and db/db mice. Lina more than EX attenuated the increase in ASC, NALP3, IL-1ß, IL-6, Collagen-1 and Collagen-3, TNFα and IL-1ß, and decreased apoptosis, especially in the db/db mice. In vitro experiments showed that Lina, but not EX, attenuated the increase in TLR4 expression, an effect that was dependent on p38 activation with downstream upregulation of Let-7i and miR-146b levels. Lina and EX had similar effects on IS and post-infarction function, but Lina attenuated the activation of the inflammasome and the upregulation of collagen-1 and collagen-3 more than direct GLP-1 receptor activation. This effect depends on p38 activation with downstream upregulation of miR-146b levels that suppresses TLR4 expression.

The Role of Non-coding RNAs in Ischemic Myocardial Reperfusion Injury.

MicroRNAs (miRNA) are non-coding RNAs that regulate gene expression in up to 90% of the human genome through interactions with messenger RNA (mRNA). The expression of miRNAs varies and changes in diseased and healthy states, including all stages of myocardial ischemia-reperfusion and subsequent ischemia-reperfusion injury (IRI). These changes in expression make miRNAs an attractive potential therapeutic target. Herein, we review the differences in miRNA expression prior to ischemia (including remote ischemic conditioning and ischemic pre-conditioning), the changes during ischemia-reperfusion, and the changes in miRNA expression after IRI, with an emphasis on inflammatory and fibrotic pathways. Additionally, we review the effects of manipulating the levels of certain miRNAs on changes in infarct size, inflammation, remodeling, angiogenesis, and cardiac function after either ischemia-reperfusion or permanent coronary ligation. Levels of target miRNA can be increased using molecular mimics ("agomirs"), or can be decreased by using "antagomirs" which are antisense molecules that act to bind and thus inactivate the target miRNA sequence. Other non-coding RNAs, including long non-coding RNAs and circular RNAs, also regulate gene expression and have a role in the regulation of IRI pathways. We review the mechanisms and downstream effects of the miRNAs that have been studied as therapy in both permanent coronary ligation and ischemia-reperfusion models.

Circulating miRNA Expression Profiling and Target Prediction in Patients Receiving Dexmedetomidine.

BACKGROUND/AIMS: Circulating miRNAs could serve as biomarkers for diagnosis or prognosis of heart diseases and cerebrovascular diseases. Dexmedetomidine has protective effects in various organs. The effects of dexmedetomidine on circulating miRNAs remain unknown. Here, we investigated differentially expressed miRNA and to predict the target genes of the miRNA in patients receiving dexmedetomidine. METHODS: The expression levels of circulating miRNAs of 3 patients were determined through high through-put miRNA sequencing technology. Target genes of the identified differentially expressed miRNAs were predicted using TargetScan 7.1 and miRDB v.5. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to conduct functional annotation and pathway enrichment analysis of target genes respectively. RESULTS: Twelve differentially expressed miRNAs were identified. Five miRNAs were upregulated (hsa-miR-4508, hsa-miR-novel-chr8_87373, hsa-miR-30a-3p, hsa-miR-novel-chr16_26099, hsa-miR-4306) and seven miRNAs (hsa-miR-744-5p, hsa-miR-320a, hsa-miR-novel-chr9_90035, hsa-miR-101-3p, hsa-miR-150-5p, hsa-miR-342-3p, and hsa-miR-140-3p) were downregulated after administration of dexmedetomidine in the subjects. The target genes and pathways related to the differentially expressed miRNAs were predicted and analyzed. CONCLUSION: The differentially expressed miRNAs may be involved in the mechanisms of action of dexmedetomidine. Specific miRNAs, such as hsa-miR-101-3p, hsa-miR-150-5p and hsa-miR-140-3p, are new potential targets for further functional studies of dexmedetomidine.

Expression Profiling of Circular RNAs and Micrornas in Heart Tissue of Mice with Alcoholic Cardiomyopathy.

BACKGROUND/AIMS: Chronic heavy alcohol consumption may result in alcoholic cardiomyopathy. This study was designed to screen differentially expressed microRNAs and circular RNAs in heart tissue of mice with alcoholic cardiomyopathy to reveal the underlying molecular mechanism. METHODS: Having established a murine alcoholic cardiomyopathy model, we screened differentially expressed microRNAs and circular RNAs in three heart samples from the alcohol-treated and control groups by high-throughput microarray analysis. We analyzed the function and biological signaling pathways of differentially expressed non-coding RNAs closely related to alcoholic cardiomyopathy using bioinformatics software to identify some mRNAs and their biological signaling pathways closely related to alcoholic cardiomyopathy. RESULTS: Nineteen microRNAs and 265 circular RNAs were differentially expressed in the alcohol-treated group compared with the control group. After analyzing gene function and signaling pathways by bioinformatics software, we found that the differentially expressed mRNAs were associated with carbohydrate metabolism. CONCLUSIONS: Chronic alcohol consumption can change the non-coding RNA profile of heart tissue, which is closely related to the pathological mechanisms of alcoholic cardiomyopathy.

SGLT2 Inhibitors and Cardiovascular Outcomes: Current Perspectives and Future Potentials.

PURPOSE OF REVIEW: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been shown to exert benefit on cardiac outcomes. In this review, we provide updates on available clinical data, studies on potential mechanisms for the CV effects, as well as discuss potential clinical implications of these new findings. RECENT FINDINGS: Since the publications of the EMPA-REG and CANVAS trials, large multi-national cohort studies have further shown the cardioprotective effects of SGLT2i. Moreover, new studies examining SGLT2i action on sodium-hydrogen exchanger proteins in both the heart and the kidney, on myocardial energetics and impact on inflammation and atherosclerosis continue to shed light on the multitude of pleotropic effects of these agents. Though more data is needed to substantiate the safety and efficacy, SGLT2i should be considered as a valuable therapy to help reduce CV risk in patients with diabetes. Ultimately, SGLT2i may have utility in preventing progression to diabetes or providing CV protection in patients who do not have diabetes.

Dapagliflozin Attenuates Na+/H+ Exchanger-1 in Cardiofibroblasts via AMPK Activation.

PURPOSE: We assessed whether the SGLT-2 inhibitor dapagliflozin (Dapa) attenuates the upregulation of the cardiac Na+/H+ exchanger (NHE-1) in vitro in mouse cardiofibroblasts stimulated with lipopolysaccharides (LPS) and whether this effect is dependent on adenosine monophosphate kinase (AMPK) activation. METHODS: Mouse cardiofibroblasts were exposed for 16 h to Dapa (0.4 µM), AMPK activator (A769662 (10 µM)), AMPK inhibitor (compound C (CC) (10 µM)), an SGLT-1 and SGLT-2 inhibitor (phlorizin (PZ) (100 µM)), Dapa+CC, or Dapa+PZ, and then stimulated with LPS (10 ng/ml) for 3 h. NHE-1 mRNA levels were assessed by rt-PCR and total AMPK, phosphorylated-AMPK (P-AMPK), NHE-1, and heat shock protein-70 (Hsp70) protein levels in the whole cell lysate by immunoblotting. In addition, NHE-1 protein levels attached to Hsp70 were assessed by immunoprecipitation. RESULTS: Exposure to LPS significantly reduced P-AMPK levels in the cardiofibroblasts. A769662 and Dapa equally increased P-AMPK. The effect was blocked by CC. Phlorizin had no effect on P-AMPK. LPS exposure significantly increased NHE-1 mRNA levels. Both Dapa and A769662 equally attenuated this increase. The effect of Dapa was blocked with CC. Interestingly, none of the compounds significantly affected NHE-1 and Hsp70 protein levels in the whole cell lysate. However, LPS significantly increased the concentration of NHE-1 attached to Hsp70. Both Dapa and A69662 attenuated this association and CC blocked the effect of Dapa. Again, phlorizin had no effect and did not alter the effect of Dapa. CONCLUSIONS: Dapa increases P-AMPK in cardiofibroblasts exposed to LPS. Dapa attenuated the increase in NHE-1 mRNA and the association between NHE-1 and Hsp70. This effect was dependent on AMPK.

GLP-1 Receptor Agonists and Cardiovascular Disease: a Meta-Analysis of Recent Cardiac Outcome Trials.

PURPOSE: The aim of this study is to examine the cardioprotective properties of Glucagon-like peptide-1 receptor agonist, a class of antihyperglycemic therapy, via meta-analysis of four recently published cardiovascular outcomes trials. METHODS: Meta-analysis was performed pooling data from the ELIXA, LEADER, SUSTAIN-6 and EXSCEL trials. A random effects model was used to generate risk ratio with 95% confidence interval for cardiovascular and safety outcomes. RESULTS: A total of 33,457 patients were included in the meta-analysis. Based on the study, GLP-1R agonists significantly reduced all-cause mortality (RR 0.89; 95% CI 0.82 to 0.96) and cardiovascular mortality (RR 0.88; 95% CI 0.80 to 0.97) when compared to placebo. When long-acting agents were analyzed alone, reduction in major adverse cardiac events (RR 0.88; 95% CI 0.81 to 0.97) and non-fatal strokes (RR 0.87; 95% CI 0.76 to 0.99) also showed significance. CONCLUSION: Overall, GLP-1R agonists appear to have cardioprotective properties likely via modification of metabolic parameters such as glycemic control, weight loss, and improvement in blood pressure. Additional studies are warranted to compare cardiovascular outcomes among the different agents.

Combined SGLT2 and DPP4 Inhibition Reduces the Activation of the Nlrp3/ASC Inflammasome and Attenuates the Development of Diabetic Nephropathy in Mice with Type 2 Diabetes.

BACGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors and dipeptidyl peptidase-4 inhibitors (DPP4I) are used to treat type 2 diabetes (T2DM). DPP4 inhibitors (DPP4) attenuate Nlrp3 inflammasome activation in the kidney. SGLT2 inhibition reduces inflammation and attenuates the progression of diabetic nephropathy (DN). The effects of dapagliflozin (Dapa) on the activation of the Nlrp3 inflammasome and the combined effect of SGLT2 and DPP4 on T2DM-induced inflammasome activation and progression of DN have not been previously studied. We assessed whether Dapa attenuates the inflammasome activation and progression of DN in T2DM mice and whether these effects can be augmented by adding DPP4I saxagliptin (Saxa). METHODS AND RESULTS: Male BTBR ob/ob and wild-type (WT) mice received vehicle, Dapa, or Dapa+Saxa for 8 weeks. Serum BUN in the WT mice was 16.9 ± 0.8 mg/dl. It increased to 55.7 ± 2.8 mg/dl in the BTBR mice. Dapa alone reduced BUN to 31.4 ± 1.2 mg/dl. A greater effect was seen in the Dapa+Saxa combination (24.8 ± 0.8 mg/dl). Serum creatinine was 0.16 ± 0.02 and 1.01 ± 0.04 mg/dl in the WT and BTBR mice, respectively. Dapa and Dapa+Saxa attenuated the increase of creatinine to 0.65 ± 0.02 and 0.40 ± 0.03 mg/dl, respectively. Serum cystatin C was elevated in the BTBR mice (3.9 ± 0.1 vs. 0.6 ± 0.2 ng/ml) as compared to WT mice. Dapa (2.4 ± 0.1) and Dapa+Saxa (1.4 ± 0.1) attenuated this increase. Kidney weight was higher in the BTBR than that of WT mice. Dapa reduced the kidney/body weight ratio in the BTBR mice. Dapa+Saxa tended to have greater effect, but the difference was not significant. mRNA levels of NALP3, ASC, IL-1ß, IL-6, caspase-1, TNF-α, collagen-1, and collagen-3 significantly increased in the kidneys of the BTBR compared to the WT mice. Dapa alone and to a greater extent, Dapa+Saxa, attenuated the activation of the inflammasome. Yet, the combination did not result in greater attenuation of the collagen-1 and collagen-3 mRNA levels. The P-AMPK/total AMPK ratio was lower in the BTBR mice than in the WT mice. Dapa and Dapa+ Saxa equally increased the ratio. CONCLUSIONS: Dapa attenuates T2DM-induced activation of the inflammasome and progression of DN in BTBR ob/ob mice. Adding Saxa to Dapa augmented attenuation of the inflammasome, but had no significant effect on kidney weight or collagen-1 and collagen-3 mRNA levels. Future clinical trials are necessary to study the effect of combined SGLT2 inhibitor and incretin therapy on renal outcomes in patients with T2DM.

Unraveling the Interaction of Aspirin, Ticagrelor, and Rosuvastatin on the Progression of Atherosclerosis and Inflammation in Diabetic Mice.

PURPOSE: We explored the effects of rosuvastatin, aspirin, ticagrelor, and clopidogrel, alone or in combinations on the progression of atherosclerosis and inflammasome activation in diabetic mice. Statins and ticagrelor increase the production of 15-epi-lipoxin A4 via cyclooxygenase-2. Aspirin alone increases 15-epi-lipoxin A4, but when combined with statins, cyclooxygenase-2 is completely blocked. METHODS: ApoE-/-/db+/db+ double-knockout mice received rosuvastatin (5 mg/kg/day), aspirin (25 mg/kg/day), ticagrelor (300 mg/kg/day), clopidogrel (75 mg/kg/day), or their combination for 14 weeks. Serum 15-epi-lipoxin A4 levels and aortic wall cholesterol content, IL-1ß, IL-6, and TNF-α levels, and plaque area were assessed. RESULTS: Aspirin, ticagrelor, and rosuvastatin increased 15-epi-lipoxin A4 levels. The combination of rosuvastatin + ticagrelor provided an additive effect. Aspirin attenuated the effect of both ticagrelor and rosuvastatin. Aspirin, ticagrelor, and rosuvastatin reduced the area of the atherosclerotic plaque. The combination of ticagrelor + rosuvastatin provided additive effects. There was a negative interaction when aspirin was combined with ticagrelor or rosuvastatin. Aspirin, ticagrelor, and rosuvastatin decreased serum IL-1ß and IL-6 levels. There was no interaction between aspirin and ticagrelor or aspirin and rosuvastatin, whereas combining rosuvastatin and ticagrelor provided an additive effect. Aspirin, ticagrelor, and rosuvastatin all decreased TNF-α levels. Aspirin attenuated the effect of both ticagrelor and rosuvastatin, and there was no additive effect of combining ticagrelor + rosuvastatin. CONCLUSIONS: We found an intricate interaction between aspirin, ticagrelor, and rosuvastatin, as aspirin reduced both ticagrelor and rosuvastatin ability to ameliorate inflammation and atherosclerosis. In contrast, we found additive effects when ticagrelor and rosuvastatin were combined.

SGLT-2 Inhibition with Dapagliflozin Reduces the Activation of the Nlrp3/ASC Inflammasome and Attenuates the Development of Diabetic Cardiomyopathy in Mice with Type 2 Diabetes. Further Augmentation of the Effects with Saxagliptin, a DPP4 Inhibitor.

PURPOSE: We assessed whether (1) dapagliflozin (Dapa, an SGLT2-inhibitor) attenuates the deterioration of heart function Nlrp3 and inflammasome activation in diabetic mice. (2) The effects can be augmented with saxagliptin (Saxa), a DDP4-inhibitor. (3) Dapa effect is possibly SGLT2-independent on cardiofibroblasts in vitro. METHODS: Type 2 diabetic (BTBR ob/ob) and wild-type (WT) mice received vehicle, Dapa, or Dapa+Saxa for 8 weeks. Glucose tolerance test and echocardiogram were performed. Cardiofibroblasts from WT and BTBR hearts were incubated with Dapa and exposed to LPS. RESULTS: Left ventricular ejection fraction (LVEF) was 81 ± 1% in the WT and 53 ± 1% in the T2D-cont mice. Dapa and Dapa+Saxa improved LVEF to 68 ± 1 and 74.6 ± 1% in the BTBR mice (p < 0.001). The mRNA levels of NALP3, ASC, IL-1ß, IL-6, caspase-1, and TNFα were significantly higher in the BTBR compared to the WT hearts; and Dapa and Dapa+Saxa significantly attenuated these levels. Likewise, protein levels of NLRP3, TNFα, and caspase-1 were higher in the BTBR compared to the WT hearts and Dapa, and to a greater extent Dapa+Saxa, attenuated the increase in the BTBR mice. Collagen-1 and collagen-3 mRNA levels significantly increased in the BTBR mice and these increases were attenuated by Dapa and Dapa+Saxa. P-AMPK/total-AMPK ratio was significantly lower in the BTBR mice than in the WT mice. Dapa and Dapa+Saxa equally increased the ratio in the BTBR mice. This in vitro study showed that NALP3, ASC, IL-1ß, and caspase-1 mRNA levels were higher in the BTBR cardiofibroblasts and attenuated with Dapa. The effect was AMPK-dependent and SGLT1-independent. CONCLUSIONS: Dapa attenuated the activation of the inflammasome, fibrosis, and deterioration of LVEF in BTBR mice. The anti-inflammatory, anti-fibrotic effects are likely SGLT2- and glucose-lowering-independent, as they were replicated in the in vitro model. The effects on remodeling were augmented when Saxa was added to Dapa. Yet, adding Saxa to Dapa did not result in a greater effect on myocardial fibrosis and collagen levels.