Diabetic Ischemia Research Articles

219-OR: Comparison of the Impact of Autologous Cell Therapy and Conservative Treatment on Chronic Limb-Threatening Ischemia in Patients with Diabetic Foot: A Randomized Controlled Trial

Background: Autologous cell-therapy (ACT) is a new treatment method for patients with diabetes and no-option chronic limb-threatening ischemia (NO-CLTI). We aimed to assess the impact of ACT on CLTI in comparison with standard treatment (ST) in a randomized controlled trial. Methods: Forty diabetic patients with NO-CLTI were randomized to receive either cell therapy (n=20) or standard conservative therapy (n=16); 4 patients dropped out of the study. After 3 months, those in the ST group were switched to ACT (cell therapy crossover). The effect on ischemia and diabetic foot was assessed by changes in transcutaneous oxygen pressure (TcPO2) and the number of healed patients at 3 months. Amputation rates and amputation-free survival (AFS) were assessed at 3 months and then during 12-month follow-up. Results: TcPO2 increased significantly in the ACT group after 1 and 3 months (both p<0.001) whereas TcPO2 in the control group remained unchanged (NS). After the crossover to cell therapy in patients of the ST group, we observed a significant increase in TcPO2 at 1 and 3 months after the injection of ACT (p=0.036 and p<0.001, respectively). We also observed significantly more healed patients at 3 months in the ACT group compared to the ST group (p=0.01). The rates of major amputation and AFS at 3 months were not significantly different between the ACT and ST groups (both NS). In Kaplan-Meier estimate, patients treated initially by ACT showed a trend towards higher AFS rates compared to those treated initially by ST who received ACT after the 3-month delay, but without a significant difference (65 vs. 43.8%). Conclusions: Our study showed that ACT in patients with no-option CLTI and diabetic foot significantly improved limb ischemia and wound healing when compared to standard conservative therapy, without influencing amputation rates and with a trend to higher 1-year AFS in comparison with delayed ACT treatment in ST group. Disclosure M. Dubsky: None. R. Bem: None. A. Nemcova: None. J. Husáková: None. V. Fejfarova: None. A. Jirkovska: None. K. Sutoris: None. Funding Ministry of Health of the Czech Republic (16-27262A, 00023001)

Serum metabolomic profiles reveal the impact of BuZangTongLuo formula on metabolic pathways in diabetic mice with hindlimb ischemia

Ethnopharmacological relevanceBuZangTongLuo Formula (BZTLF) was the decoction of eight traditional Chinese medicines including Astragalus membranaceus, Dioscorea opposita, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng, Fritillariae cirrhosae and Whitmania pigra. This formula has been used as an effective remedy for treatment of diabetic ischemia clinically. Aim of the studyIn previous study, we have reported the therapeutic effect of BZTLF on diabetic vascular dysfunction. However, it remains obscure about the role of metabolic pathways in BZTLF-initiated improvement on hindlimb ischemia. Materials and methodsDiabetic mice with hindlimb ischemia were orally administrated with BZTLF by gavage. The serum samples were prepared for untargeted metabolomic analysis by ultra-performance liquid chromatography-mass spectrometer. The metabolic network was built by integrating metabolite data with the Gene Expression Omnibus (GEO) dataset (GSE3313). Further, quantitative PCR was used to confirm the key target genes. ResultsBZTLF treatment remarkably led to the reversal of changed metabolite levels in serum of diabetic mice with hindlimb ischemia, which mainly derived from bacteria, plant and signaling molecules. Also, BZTLF reshaped the metabolic pathways, especially those responsible for metabolism of lipid, gluthanine and tryptophan. In addition, BZTLF led to the reduction of lysophosphatidic acids (LPAs) and increment of triglycerides (TGs) conjugation with non-saturated fatty acids in serum. BZTLF significantly restored the down-regulation of vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) or the up-regulation of interleukin 4-induced 1 (IL4I1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1) at mRNA level, which were key regulatory genes located in metabolic pathways of glutamate and tryptophan. ConclusionsBZTLF improved hindlimb ischemia in diabetic mice by the positive regulation of metabolome changes in serum.

Role of Cilostazol in Rats with Diabetic Lower Limb Ischemia and Its Influences on Changes in Matrix Metallopeptidase-9 and Matrix Metalloproteinase-8

Objective: To assess cilostazol’s effect on rats with diabetic lower limb ischemia. Methods: 36 Sprague-Dawley rats were randomly separated into normal group (n = 12), model group (n = 12) and cilostazol group (n = 12). The model of diabetic lower limb ischemia was prepared and normal saline was intraperitoneally injected postoperatively into model group. Besides, cilostazol was administrated through intraperitoneal injection after establishment of model in cilostazol group. After intervention for 6 weeks, doppler blood flow imager was employed to detect blood flow, and the expressions of MMP-8 and MMP-9 were assessed via immunohistochemistry, western blotting or quantitative polymerase chain reaction (qPCR). TUNEL assay was performed to evaluate cell apop-tosis. Results: Doppler blood flow imager showed that compared with normal group, perfusion unit (PU) index was significantly reduced in the other two groups (P < 0.05) with higher value in cilosta-zol group (P < 0.05). The immunohistochemistry results revealed that in comparison with those in normal group, MMP-8 and MMP-9 positive staining was significantly elevated in the other two groups (P < 0.05) with higher level in cilostazol group than model group. In addition, MMP-8 and MMP-9 expressions were significantly elevated in other two groups (P < 0.05), with higher level in cilostazol group (P < 0.05). Moreover, the vascular wall smooth muscle cells in the other two groups had a significantly elevated apoptosis rate (P < 0.05) with higher apoptosis in cilostazol group (P < 0.05). Conclusion: Cilostazol up-regulates MMP-8 and MMP-9 to increase the apoptosis of vascular wall smooth muscle cells, thereby improving the blood flow in rats with diabetic lower limb ischemia.

Nitric Oxide Resistance, Induced in the Myocardium by Diabetes, Is Circumvented by the Nitric Oxide Redox Sibling, Nitroxyl.

Aim: Impairment of tissue responsiveness to exogenous and endogenous nitric oxide (NO•), known as NO• resistance, occurs in many cardiovascular disease states, prominently in diabetes and especially in the presence of marked hyperglycemia. In this study, we sought to determine in moderate and severe diabetes (i) whether NO• resistance also occurs in the myocardium, and (ii) whether the NO• redox sibling nitroxyl (HNO) circumvents this. Results: The spectrum of acute NO• effects (induced by diethylamine-NONOate), including vasodilation, and enhanced myocardial contraction and relaxation were impaired by moderately diabetic rats ([blood glucose] ∼20 mM). In contrast, acute HNO effects (induced by isopropylamine-NONOate) were preserved even in more severe diabetes ([blood glucose] >28 mM). Intriguingly, the positive inotropic effects of HNO were significantly enhanced in diabetic rat hearts. Further, progressive attenuation of soluble guanylyl cyclase (sGC) contribution to myocardial NO• responses occurred with increasing severity of diabetes. Nevertheless, activation of sGC by HNO remained intact in the myocardium. Innovation: Diabetes is associated with marked attenuation of vascular and myocardial effects of NO and NO donors, and this NO• resistance is circumvented by HNO, suggesting potential therapeutic utility for HNO donors in cardiovascular emergencies in diabetics. Conclusion: These results provide the first evidence that NO• resistance occurs in diabetic hearts, and that HNO largely circumvents this problem. Further, the positive inotropic and lusitropic effects of HNO are enhanced in a severely diabetic myocardium, a finding that warrants further mechanistic interrogation. The results support a potential role for therapeutic HNO administration in acute treatment of ischemia and/or heart failure in diabetics.

The Degree of Retinopathy Correlates with the Presence of Silent Myocardial Ischemia in Diabetic Patients

Background: Silent myocardial ischemia is a major component of the total ischemic burden for patients with ischemic heart disease. The disease is more prevalent in diabetic patients than their peers, and early detection of the high-risk group would play an integral role in the prevention of sudden cardiovascular accidents that are common in these patients. Methods: A prospective cohort study including asymptomatic 53 diabetic patients with diabetic retinopathy who suffer no ischemic cardiac symptoms was conducted. The presence and degree of diabetic retinopathy were evidenced using fundus examination and optical coherence tomography. All the enrolled patients underwent stress-resting 99mTc SestaMIBI myocardial perfusion scintigraphy to detect the ischemic burden. The relation between diabetic retinopathy and silent myocardial ischemia was stratified using statistical analysis. Results: A total of 13 diabetic patients (24.5%) have silent myocardial ischemia in the form of regional myocardial perfusion abnormalities. The strongest predictors of abnormal tests were the presence of moderate to severe retinopathy, comorbid hypertension and diabetic duration for more than 10 years. Conclusion: Silent myocardial ischemia affects one in four asymptomatic diabetic patients suffering from diabetic retinopathy. The presence of comorbid risk factors such as high-grade retinopathy, hypertension and/or long diabetic duration surge the incidence and considered additional predictors of the disease.

BuZangTongLuo decoction improved hindlimb ischemia by activating angiogenesis and regulating gut microbiota in diabetic mice.

Seven traditional medicinal plants (including Astragalus membranaceus, Dioscorea hemsleyi, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng and Fritillariae cirrhosae) and one insect leech (Whitmania pigra Whitman) were combined into BuZangTongLuo formula (BZTLF) under the guidance of traditional Chinese medicine. BZTLF is potentially effective against diabetic vascular complications. Previous studies failed to clarify the molecular mechanism through which BZTLF suppressed diabetic ischemia. In this study, we aimed to explore whether BZTLF treatment could prevent the occurrence of type 2 diabetic (T2D) hindlimb ischemia in mice. Further, we investigated the regulatory effect of BZTLF on angiogenesis-related VEGF signaling pathway and gut microbiota dysfunction in diabetic ischemia mice. C57BL/6J mice fed with high-fat diet (HFD) received STZ injection and femoral artery ligation to build T2D diabetic hindlimb ischemia model. Mice were gavaged with BZTLF (5 g [raw materials]/kg/d) or with metformin plus atorvastatin for three weeks. Laser doppler imaging system was utilized for the visualization of blood flow. Histochemistry analysis was performed for microvascular vessel staining. Western blot was applied to detect the protein changes of signaling molecules responsible for VEGF pathway. Finally, 16S rDNA gene sequencing was conducted for analysis of gut microbiota structure. BZTLF treatment remarkably restored blood flow and capillary density of diabetic hindlimb ischemia. And the protein changes of VEGF signaling molecules were reversed in BZTLF-treated diabetic ischemia mice, including the decreased VEGF and HIF-1α, and the increased NO, eNOS and p-ERK1/2. The gut microbiota analysis suggests that BZTLF treatment increased the abundances of several beneficial bacteria (Akkermansia, Bifidobacterium and Bacteroides), while decreased the populations of some harmful bacteria(Blautia, Weissella, Escherichia Shigella and Kurthia). By using Spearman's correlation analysis, these changed gut flora were positively/negatively correlated with VEGF signaling pathway or glycometabolic parameters. BZTLF displayed beneficial effects on diabetic hindlimb ischemia by reshaping the gut microbiota structure and stunning the VEGF/HIF-1α pathway.

P712Non-neuronal cholinergic system prevents coronary vascular dysfunction in diabetic heart

Abstract Background Diabetic individuals suffer extensive myocardial damage during ischemia due to impaired ATP production and coronary vascular dysfunction. The cardiomyocytes possess a non-neuronal cholinergic system (NNCS) as it has choline acetyltransferase (ChAT) to synthesize acetylcholine (ACh). ACh released from cardiomyocytes activates hypoxia-inducible factor-1 pathway in an auto/paracrine manner under non-hypoxic condition. Activation of this pathway via NNCS promotes angiogenesis and is a promising mechanism to target ischemia in diabetes. Aim To investigate if activation of NNCS could improve the coronary vasculature in diabetic heart. Methods Type-2 diabetic db/db mice with ventricle-specific ChAT transgene (db/db-ChAT-tg) and control db/db mice of 12- and 24-weeks old were used. Catheterization of the jugular vein and carotid artery was performed in combination with synchrotron radiation microangiography to visualize the in-vivo coronary circulation. Changes of the coronary circulation to ACh (10μg/kg/min) and sodium nitroprusside (SNP, 10μg/kg/min) were assessed. Immunofluorescence analysis was performed to measure the density of arterioles and capillaries ex-vivo. Results In comparison to db/db mice, the number of second and third order vessels was higher in the db/db-ChAT-tg mice of 12- and 24-weeks old under baseline condition. In response to ACh and SNP, number of third order vessels were further increased in the db/db-ChAT-tg mice of both ages. However, the magnitude of the diameter changes in db/db-ChAT-tg mice was comparable to that in db/db mice of both ages. Besides, the db/db-ChAT-tg mice had increased density of arterioles and capillaries compared to the db/db mice of both ages. Conclusion NNCS-induced angiogenesis prevents coronary vascular dysfunction in diabetic heart.

P5400CD34+ cells predict long-term cardiovascular mortality in people with critical limb ischemia: a possible pathogenic role of the microRNA-21/PDCD4 axis

Abstract Background/Introduction The annual rate of restenosis, amputation, and death of subjects with type 2 diabetes (T2DM) and critical limb ischemia (CLI) is very high even after successful revascularization. We have recently demonstrated that the in-vitro migratory ability of proangiogenic bone marrow (BM)-derived CD45dimKDR+CXCR4+ CD34+ cells predicts cardiovascular mortality at 18 months after percutaneous angioplasty (PTA) in people with T2DM and CLI. Purpose To verify the predictor at a longer follow-up time and to investigate the underpinning mechanism. Methods We analyzed the rate of cardiovascular mortality in the previously studied cohort of 119 subjects at a six-year follow-up. In addition, we compared apoptosis and angiogenic capability of MAC-sorted BM-CD34+ cells in a newly-recruited series of patients with T2DM and CLI and control nondiabetic subjects (CTRL). Following the screening of a spectrum of microRNA (miR) using PCR-based Exiqon technology, we validated the modulated miRs in BM-CD34+ cells from 6 new donors per group. Finally, we focused on miR-21 and its inhibitory targets, by assessing the effect of miR-21 modulation of functional outcomes, including apoptosis (caspase assay and Annexin V positive staining) and the induction of in-vitro endothelial networking on Matrigel. Results Multivariable regression model analysis confirmed that CD34+ cell migration forecasts cardiovascular mortality after revascularization. BM-CD34+ cells isolated from T2DM-CLI donors undergo apoptosis in a higher percentage and are less proangiogenic in-vitro than cells from CTRL. Of the 56 microRNAs commonly expressed in CD34+ cells from the two studied groups, 6 miRs were differentially expressed. Biological validation confirmed a significant down-modulation of miR-21 in CD34+ cells from T2DM-CLI patients. The expression of the miR-21 inhibitory target, PDCD4, a regulator of apoptosis, was increased in CD34+ cells from T2DM-CLI patients compared to CTRL (1.6±0.6 vs. 74.4±14.9 average±SE 2ddCt via PCR). Silencing miR-21 in CD34+ cells from CTRL phenocopied the T2DM-CLI behavior, resulting in significantly increased apoptosis and inhibition of EC networking. Likewise, exposure of endothelial cells to patient-derived CD34+ CCM induced apoptosis and reduced the network formation ability on Matrigel, with these functional liabilities being associated with miR-21 inhibition and PDCD4 upregulation. Conclusions Migratory activity of CD34+ cells predicts cardiovascular mortality at 6 years follow-up after PTA in subjects with T2DM and CLI. Complicated T2DM is associated with a specific miR signature in BM CD34+ cells which could negatively impact on vascular cell survival and angiogenic capacity, thereby possibly contributing to adverse cardiovascular outcomes. The miR-21/ PDCD4 duo may represent a therapeutic target to rescue diabetes-associated complications. Acknowledgement/Funding British Heart Foundation grant RG/13/17/30545, Cariplo Foundation (code: 2016-0922)

Abstract 149: Endothelial-specific Overexpression of Metallothionein Prevents Diabetes Mellitus-induced Impairment in Ischemia Angiogenesis via Preservation of Hif-1á/sdf-1 in Endothelial Progenitor Cells

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Whether oxidative protection of EPCs by an antioxidant, such as the protein metallothionein (MT) can have positive angiogenic effects and by what mechanisms remain unclear. Endothelial-specific MT overexpression (JTMT) mice were made diabetic by high fat diet followed by streptozocin administration (HFD/STZ). Diabetic hind limb ischemia (HLI) was established by femoral artery ligation. Bone marrow mononuclear cells (MNCs) collected from JTMT mice were transplanted into db/db mice with HLI. Blood reperfusion was monitored. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia. Compared with wild-type (WT) littermates, JTMT mice were resistant to diabetes-induced impairment in ischemia angiogenesis and blood reperfusion in HFD/STZ diabetes. Similarly, transplantation of MT overexpressing MNCs showed better therapeutic effect on db/db mice with HLI than transplantation of WT MNCs. These changes were accompanied by increased mobilization and infiltration of EPCs in JTMT mice, and enhanced incorporation of EPCs into capillaries in JTMT MNCs transplanted db/db mice. Furthermore, EPCs from JTMT mice exhibited augmented cell survival, tube formation, and migration capacities under diabetic ischemia-like conditions. Mechanistically, the expression of hypoxia-inducible factor 1α (HIF-1α) and the secretion of stromal cell-derived factor (SDF-1) in blood and expression in ischemic tissues were upregulated in JTMT and JTMT-MNC transplanted db/db mice, and in cultured JTMT EPCs, which were accompanied by marked amelioration of oxidative stress. However, MT-mediated elevation of SDF-1 and improvements of function in EPCs were all abrogated by siRNA knockdown of HIF-1α expression without effect on the anti-oxidative capacity of MT. Endothelial-specific MT elevation is sufficient to protect against diabetes mellitus-induced impairment of ischemia angiogenesis by promoting EPC function. The benefits of MT are predominantly mediated by upregulation of the HIF-1α/SDF-1 pathway.

Two-year analysis of changes in the optic nerve and retina following anti-VEGF treatments in diabetic macular edema patients.

Purpose: To evaluate long-term structural and functional changes that happen to the optic nerve and retina following ranibizumab (Lucentis) injections in diabetic macular edema (DME) patients.Methods: Patients with clinically significant DME requiring anti-VEGF injections underwent pre-injection baseline, 6, 12, and 24 month follow-up tests. The tests performed were optical coherence tomography (OCT), best-corrected visual acuity (BCVA), and visual field (VF). Wide-field fluorescein angiogram (IVFA) was performed to monitor the progression of diabetic ischemia.Results: A total of 30 patients requiring anti-VEGF injections and 21 control patients not requiring anti-VEGF injections were enrolled in the study. From baseline, the average macular thickness significantly decreased (p<0.0002) over the 24-month time period. Mean perfused ratio significantly increased (p<0.0005) at 6, 12, and 24 months. Cup volume and vertical cup-to-disk ratio significantly increased (p<0.0014) over the study period. This was verified by masked independent grading of patient optic nerve stereo-photographs by glaucoma specialists. BCVA significantly (p<0.0006) improved over the study period. VFs showed a non-significant trend of deteriorating peripheral vision at 12 and 24 months.Conclusion: Clinically, anti-VEGF therapy appears to affect the optic nerve by increasing cup volume and increasing vertical cup/disk ratio over time. The results provide a cautionary note to monitor both the retina and optic nerve status in patients undergoing frequent injections.

625-P: Metallothionein Improves Angiogenic Function of Endothelial Progenitor Cells via HIF-1α/SDF-1/Akt Pathway in Diabetic Limb Ischemia

Diabetes mellitus associated dysfunction of endothelial progenitor cells (EPCs) may contribute to dysregulation of endothelial regeneration. Metallothionein (MT) acts as an antioxidant in various pathophysiological processes. Whether MT in EPCs has positive angiogenic effects and the mechanisms involved remain unclear. Endothelial MT overexpression (JTMT) mice were made diabetic by high fat diet followed by STZ administration (HFD/STZ). Diabetic hind limb ischemia model was established by femoral artery ligation. Bone marrow derived mononuclear leucocytes (MNCs) collected from JTMT mice were transplanted into db/db mice with hind limb ischemia. Blood reperfusion was monitored and oxidative stress was measured. High glucose and hypoxia conditions in culture were adopted to mimic diabetic ischemia in vitro. MT improved blood perfusion of ischemic hind limb and promoted angiogenesis in HFD/STZ diabetes. Oxidative stress of ischemic muscles was aggravated under diabetic condition, which was reduced in JTMT mice. Compared with WT mice, the expression of HIF-1α, SDF-1, VEGF and p-Akt were all increased in JTMT mice, along with elevation of plasma SDF-1α and VEGF. Moreover, transplantation of MT overexpressed MNCs showed better therapeutic effect on db/db diabetic mice with hind limb ischemia than control or transplantation of normal MNCs. Transplantation was accompanied by elevated HIF-1α, SDF-1 and Akt signaling. Vitro studies demonstrated that MT overexpression reduced high glucose and hypoxia induced EPC dysfunction and preserved high glucose and hypoxia impaired HIF-1α, SDF-1, VEGF and Akt signaling, all of which were abolished by siRNA knockdown of HIF-1α. In conclusion, elevated MT enhances angiogenic function and therapeutic efficacy of EPCs in diabetic limb ischemia. The benefits of MT are predominantly mediated by a HIF-1α/SDF-1/Akt pathway under diabetic ischemia conditions. Disclosure K. Wang: None. X. Dai: None. J. He: None. C. Yang: None. J. Chen: None. K.A. Wintergerst: None. P.N. Epstein: None. L. Cai: None. Y. Tan: None. Funding American Diabetes Association (1-15-BS-018, 1-18-IBS-082 to L.C.); 1-13-JF-53 (to Y.T.)

PEGylated substance P augments therapeutic angiogenesis in diabetic critical limb ischemia

Substance P (SP) is a therapeutic peptide that has been widely used to induce angiogenesis and tissue regeneration. However, its therapeutic efficacy is often limited due to rapid degradation in vivo and a short half-life (˜1min) after systemic administration. In the present study, we chemically modified SP with polyethylene glycol (PEG) to generate long-lasting formulations with increased stability and extended retention time in vivo and evaluated their ability to enhance therapeutic angiogenesis. Compared to the unmodified SP, PEGylated SP (PEG-SP) exhibited significantly increased half-life in vivo (˜360-fold increase in normal mouse and ˜120-fold increase in diabetic mouse). Systemic injection of PEG-SP led to a marked increase in therapeutic efficacy and angiogenesis in diabetic hindlimb ischemia, as evident from the remarkable improvement in salvage of ischemic limb and recovery of blood perfusion in diabetic mice with limb ischemia. These formulations increased endogenous stem cell mobilization to the injured site and modulated the immune system to increase microvessel formation and reduce systemic inflammation. Importantly, PEGylation efficiently reduced the injection doses of SP while still maintaining its therapeutic efficacy. To conclude, our study demonstrated that PEG-SP formulations exhibited an improved in vivo pharmacokinetic profile with enhanced efficacy and clinically acceptable treatment regimens, thus acting as a potent therapeutic for diabetic ischemia and inflammation.

Protective effect of insulin on myocardial ischemia-reperfusion injury in diabetic rats based on PI3K/Akt signaling pathway

Objective To study the effect of insulin on phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway in diabetic rats with myocardial ischemia reperfusion injury. Methods Diabetic rats were induced by high-sugar and high-fat diet plus intraperitoneal injection of streptozotocin (40 mg/kg). They were randomly divided into diabetic sham group (group A), diabetes ischemic reperfusion group (group B), diabetes ischemic reperfusion insulin treatment group (group C) and diabetic ischemia reperfusion+ insulin Wortmaninn (PI3K inhibitors) group (group D), 10 in each group. Myocardial ischemia reperfusion model in diabetic rats: heart was exposed between the third and fourth ribs of the left chest, 2 mm from the lower edge of the left atrial ear, and 5-0 sterile suture was used to ligate the anteri-or descending coronary artery (LAD) and the great cardiac vein for 30 min, and then the perfusion was resumed for 120 min.Wortmannin (15 μg/kg) was given through femoral vein 20 min before ligation in group D, and the same amount of normal saline was given in the other 3 groups.Insulin (2 U/kg) was injected subcutaneously in group C and D 10 min before ligation, and the same amount of normal saline was injected subcutaneously in group A and group B. Plasma creatine kinase MB (CK-MB)and troponin I (cTnI) levels were measured in arterial blood after 120 min of reperfusion, and PI3K and Akt expression in myocardial tissue were detected by Western blot. Results Compared with the group A, the plasma levels of cTnI and CK-MB increased and the expressions of PI3K and phosphorylated protein kinase B (p-Akt) in myocardium decreased in the group B (P<0.05). After insulin treatment, the plasma cTnI [(0.89±0.26)μg/L], CK-MB [(9.24±3.16)μg/L] in the myocardial tissue of the group C decreased, while the expression of PI3K (0.341 8±0.03 1) and p-Akt (0.673 1±0.028) in the myocardial tissue increased (P<0.05). After insulin+ Wortmaninn administration, the plasma cTnI [(1.16±0.29)μg/L] and CK-MB [(12.57±3.01)μg/L] in the group D increased , while the expression of PI3K (0.292 7±0.036) and p-Akt (0.531 4±0.030) in the myocardial tissue decreased, with statistically significant difference (P<0.05). Conclusions Insulin can reduce serum CK-MB and cTnI levels in diabetic rats with myocardial ischemia reperfusion injury, possibly by activating PI3K/Akt signal transduction pathway, inhibiting myocardial enzyme release, and improving myocardial ischemia reperfusion injury in diabetic rats to play a protective role in myocardial cells. Key words: 1-phosphatidylinositol 3-kinase; Protein kinases; Insulin; Diabetes mellitus; Reperfusion injury; Rats

Screening of silent myocardial ischemia in asymptomatic diabetic patients: ROle of coronary artery calcium and stress myocardial perfusion scintigraphy

Coronary artery diseases and heart failure are the main causes of morbimortality in diabetes. Screening of silent myocardial ischemia in asymptomatic diabetic patients is very important and is still controversial. Coronary artery calcium (CAC) belongs to innovative technology in non-invasive imagery technique based on tomography without injection and permits cardiovascular risk assessment; its threshold usually is 400. Principal objective was to diagnose silent myocardial ischemia in asymptomatic diabetics based on CAC measurement. A secondary objective was to determine patients’ characteristics when CAC was < or ≥ 400. CAC imaging was performed in every asymptomatic diabetic patient and all patients with CAC ≥ 400 underwent myocardial perfusion scintigraphy. Two hundred and sixty-three patients were included between 2014 and 2017. Eighty patients had a CAC score ≥ 400 and sixty-two of them underwent myocardial perfusion scintigraphy. Silent myocardial ischemia was detected in 14 patients (5%). The predictors of elevated CAC were: age, male gender, hypertension, duration of diabetes, dyslipidemia and microalbuminuria. In this study, evaluating silent ischemia screening, CAC followed by myocardial scintigraphy have an important role in cardiovascular risk assessment. Thirty percent of patients required an imaging test such as perfusion scintigraphy and 5% had silent myocardial ischemia. CAC and myocardial perfusion scintigraphy can improve screening and risk stratification in this population. CAC score can help to guide medical treatment.

13 - Use of adeno-associated viruses to inhibit Glutaredoxin-1 in mouse skeletal muscle

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that reverses glutathione (GSH) adducts of protein thiols generated through oxidative post-translational modifications. GSH-adducts regulate cellular signaling and transcription factors. We previously found that after femoral artery ligation Glrx knockout mice improved blood flow recovery in relation to activation of hypoxia-inducible factor (HIF)-1α via GSH adducts, indicating Glrx as an anti-angiogenic enzyme. Objective We hypothesized that local Glrx gene inhibition might improve poor blood flow recovery after ischemia in aging or diabetes. To inhibit Glrx gene expression in vitro and in vivo, we developed an improved protocol to produce adeno-associated viruses (AAVs) with a bicistronic expression system coding for Glrx shRNA (shGlrx) and mVenus, a yellow fluorescent protein. Methods Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation, aqueous two-phase partitioning, and an iodixanol gradient, which resulted in AAVs with adequate titer and purity for in vivo use. We tested the AAVs on C2C12 skeletal muscle cells and mouse gastrocnemius muscles. Results Quiescent confluent C2C12 cells transduced with a shGlrx-containing AAV exhibited a decrease of ~75% in the Glrx mRNA and protein levels after 4 days. Intramuscular injection (4.4 x 1012 viral genome/50 μl) of AAV-shGlrx transduced mVenus expression in skeletal muscle efficiently, but non-injected muscle, heart, or liver showed no mVenus expression. However, 4 weeks after intramuscular injection, Glrx protein levels remained unchanged compared to control-AAV injected or non-injected muscle, and inflammatory markers were increased. Six weeks after virus injection, Glrx expression in the muscle decreased 80% at the mRNA and 50% at the protein level. Conclusion Intramuscular injection of AAV-shGlrx resulted in muscle-specific attenuation of Glrx expression after at least 6 weeks. AAV intramuscular injection caused local inflammation in the early stage which may counteract the effects of shGlrx. (R01HL133013, R03AG051857, R01DK103750)

Abstract 536: Increased O-linked Glycosylation in Diabetic Myocardium of Mice and Human

Objectives: The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Our focus was to establish the difference of O-linked glycosylation in diabetic (DM) and Non-diabetic (ND) heart of mouse and human in the presence or absence of cardioplegic ischemia and cardiopulmonary bypass (CP/CPB). Methods and results: Right atrial tissue was harvested pre- and post-CP/CPB from the ND patients and patients with type-2 DM undergoing cardiac surgery (n = 4/group).The post-CP/CPB tissue samples were from a atrial tissue segment exposed to cold, hyperkalemic blood cardioplegia (ischemia) and a brief period (10 minutes) of reperfusion. Ventricular tissue samples were also obtained from mice with and without type-2 DM (n =3/group). Thin Layer Chromatography (TLC) was used to measure the contents of acetyl coenzyme A (acetyl-CoA) in the tissue lysates of mouse and human myocardium. Western blotting and immunohistochemistry analysis were employed to determine the expression/localization of O-Linked β-N-acetylglucosamine (O-GlcNAc) in the tissue lysates and paraffin-embedded tissue sections. Diabetes tends to increase the amounts of acetyl-CoA and O-GlcNAc (20% increase vs. ND, P&lt;0.05) in the myocardium compared with ND. Post-CP/CPB further induced more acetyl-CoA and this effect was more pronounced in the diabetic atrial myocardium than that of ND. Immunofluorescence experiments with O-GlcNAc-specific antibody and paraffin-embedded tissue sections from human atria and mouse heart show a significant number of cells stain positively with the antibody in the diabetes sections (3 folds increases in optical density vs. ND, P &lt;0.01). The protein oxidation and ratio of NADH/NAD in the DM myocardium were significantly increased compared with ND (P&lt;0.05). Conclusion: Diabetes and cardioplegic ischemia are associated with an increase in O-linked glycosylation in the mouse and human myocardium, suggesting that diabetes and cardioplegic ischemia affect myocardial metabolism via hexosamine biosynthetic pathway.

CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells in Diabetic Limb Ischemia

Stromal cell-derived factor 1 (SDF-1)-chemokine receptors CXCR4/7 axis plays a vital role in diabetic limb ischemia. Our previous studies demonstrated that elevated CXCR7 improves therapeutic efficacy of endothelial progenitor cells (EPCs) in diabetic limb ischemia. The effects of a specific CXCR7 agonist TC14012 on diabetic hind limb ischemia remains untested. We hypothesize that CXCR7 expression is reduced in diabetic EPCs and TC14012 improve angiogenic function of EPCs in diabetic limb ischemia. Early WT-EPCs were transfected with siRNA against CXCR7, and early db/db-EPCs were infected with CXCR7 lentivirus. Tube formation and capillary density of ischemic gastrocnemius muscle (GS) and soleus muscle (SS) at 4 weeks post-ischemic surgery were adopt for indication of angiogenesis. Diabetic hind limb ischemia (HLI) models were established by femoral artery ligation, followed by administration of TC14012 subcutaneously. Total and cell surface expression of CXCR7 were significantly decreased in EPCs from db/db mice compared with those from WT mice, whereas no significant differences of CXCR4 expression were observed. Tube formation by EPCs from db/db mice was also impaired compared with WT-EPCs. siRNA knockdown of CXCR7 was accompanied by impaired tube formation. Conversely, increasing CXCR7 levels in db/db EPCs completely reversed impaired tube formation function. High glucose (HG) dose dependently decreased the expression of CXCR7 in vitro but not that of CXCR4. HG also impaired tube formation of EPC from WT mice. Tube formation function of HUVEC was impaired after treated with HG (25mM) for 24 hours, which could be prevented by TC14012. Most importantly, TC14012 significantly improved blood flow restoration of ischemic hind limb starting from 2 weeks after ligation and promoted angiogenesis of ischemic GS and SS. Diabetes attenuates CXCR7 expression and impairs angiogenesis of EPCs. TC14012 improves blood reperfusion of diabetic hind limb ischemia. Disclosure K. Wang: None. X. Dai: None. J. Chen: None. P.N. Epstein: None. L. Cai: None. K.A. Wintergerst: None. Y. Qian: None. Y. Tan: None.

High-density lipoprotein-mimicking nanodiscs carrying peptide for enhanced therapeutic angiogenesis in diabetic hindlimb ischemia

Therapeutic strategies using endogenous stem cell mobilizer can provide effective cell-free therapy for addressing various ischemic diseases. In particular, substance P (SP) exhibited therapeutic regeneration by facilitating mobilization of endogenous stem cells from bone marrow to the injured sites. However, its therapeutic effect has been limited due to short half-life and rapid degradation of administered SP peptides in vivo. Here we sought to develop high-density lipoprotein (HDL)-mimicking nanodiscs conjugated with SP (HDL-SP) in order to increase the in vivo half-life, bone marrow targeting, and therapeutic efficacy of SP for the treatment of diabetic peripheral ischemia. Conjugation of SP onto HDL nanodisc led to remarkable ∼3215- and ∼1060-fold increase in the ex vivo and in vivo half-lives of SP, respectively. Accordingly, HDL-SP nanodiscs improved retention of SP in bone marrow after systemic administration, leading to efficient mobilization of stem cells from bone marrow into blood circulation and reduction of systemic inflammation. Consequently, nanodisc based SP peptide delivery promoted blood vessel formation, blood perfusion recovery and markedly improved limb salvage in diabetic hindlimb ischemia model relative to administration of free SP without nanodisc modification. Therefore, HDL-SP nanodisc can provide a novel strategy for the treatment of diabetic ischemia and HDL nanodisc modification could be potentially useful for the extension of plasma circulation of other labile peptides.

Liraglutide Activates the Nrf2/HO-1 Antioxidant Pathway and Protects Brain Nerve Cells against Cerebral Ischemia in Diabetic Rats.

This study aimed to determine the effect of liraglutide pretreatment and to elucidate the mechanism of nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase-1 (HO-1) signaling after focal cerebral ischemia injury in diabetic rats model. Adult male Sprague-Dawley rats were randomly divided into the sham-operated (S) group, diabetes mellitus ischemia (DM + MCAO) group, liraglutide pretreatment normal blood glucose ischemia (NDM+MCAO+L) group, and liraglutide pretreatment diabetes ischemia (DM + MCAO + L) group. At 48 h after middle cerebral artery occlusion (MCAO), neurological deficits and infarct volume of brain were measured. Oxidative stress brain tissue was determined by superoxide dismutase (SOD) and myeloperoxidase (MPO) activities. The expression levels of Nrf2 and HO-1 of brain tissue were analyzed by western blotting. In the DM + MCAO + L group, neurological deficits scores and cerebral infarct volume seemed to decrease at 48 h after MCAO cerebral ischemia compared with those in DM + MCAO group (P < 0.05). In addition, the expression of Nrf2 and HO-1 increased in 48 h at liraglutide pretreatment groups after MCAO cerebral ischemia if compared with those in the DM + MCAO group (P < 0.05). Furthermore, the DM + MCAO + L group has no significant difference compared with the NDM + MCAO + L group (P > 0.05). To sum up, alleviating effects of liraglutide on diabetes complicated with cerebral ischemia injury rats would be related to Nrf2/HO-1 signaling pathway.

Sitagliptin-mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes.

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.