The Effect of Garlic and Voluntary Exercise on Cardiac Angiogenesis in Diabetes: The Role of MiR-126 and MiR-210

Roya Naderi Gisou Mohaddes Mustafa Mohammadi Alireza Alihemmati Amirmahdi Khamaneh Rafighe Ghyasi Rana Ghaznavi About the authors

Abstract

Background:

Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment . MiR-126 and miR-210 support angiogenic response in endothelial cells.

Objective:

The present study sought to explore the effect of garlic and voluntary exercise, alone or together, on miR-126 and miR-210 expressions and cardiac angiogenesis in rats with type 1 diabetes.

Methods:

Male Wistar rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Diabetes was induced in the animals by streptozotocin (ip, 50 mg/kg). The rats were then fed raw fresh garlic homogenate (250 mg/kg) or were subjected to voluntary exercise, or to combined garlic and voluntary exercise for 6 weeks. MiR-126 and miR-210 expressions in the myocardium were determined by real time PCR, and the serum lipid profile was measured by enzymatic kits. Angiogenesis was evaluated by immunostaining for PECAM-1/ CD31 in the myocardium.

Results:

Diabetes reduced both cardiac miR-126 expression and angiogenesis (p < 0.05). On the other hand, there was a miR-210 expression increase in the myocardium of diabetic animals (p < 0.001). However, those effects reversed either with garlic or voluntary exercise (p < 0.01). Moreover, treating diabetic rats with garlic and voluntary exercise combined had an additional effect on the expressions of miR-126 and miR-210 (p < 0.001). Furthermore, both voluntary exercise and garlic significantly improved serum lipid profiles (p < 0.001).

Conclusion:

The induction of diabetes decreased angiogenesis in the myocardium, whereas our treatment using long-term voluntary exercise and garlic improved myocardial angiogenesis. These changes were possibly owing to the enhancement of myocardial miR-126 and miR-210 expressions.

Keywords:
Rats; Garlic; Allium Sativum; Exercise; Diabetes Mellitus; microRNAs; Angiogenesis Inducing Agents; Neovascularization; Physologic

Resumo

Fundamento:

O diabetes mellitus (DM) é um dos principais fatores de risco para doenças cardiovasculares, levando à disfunção endotelial e inibição da angiogênese. O miRNA-126 e o miRNA-210 promovem a resposta angiogênica em células endoteliais.

Objetivo:

O presente estudo buscou explorar o efeito do alho e de exercícios físicos voluntários, isoladamente ou em conjunto, nas expressões do miRNA-126 e do miR-210 e na angiogênese cardíaca em ratos com diabetes tipo 1.

Métodos:

Ratos Wistar machos foram divididos em cinco grupos (n = 7): Controle, Diabetes, Diabetes+Alho, Diabetes+Exercícios e Diabetes+Alho+Exercícios. Introduziu-se diabetes nos animais por estreptozotocina (ip, 50 mg/kg). Os ratos foram então alimentados com homogenato de alho fresco cru (250 mg/kg), ou foram submetidos a exercícios voluntários, ou a uma combinação de alho e exercícios voluntários, durante 6 semanas. As expressões do miRNA-126 e do miRNA-210 no miocárdio foram determinadas por PCR em tempo real, e o perfil lipídico sérico foi medido por kits enzimáticos. A angiogênese foi avaliada por imunocoloração por PECAM-1/CD31 no miocárdio

Resultados:

O diabetes reduziu a expressão do miRNA-126 cardíaco e da angiogênese (p < 0,05). Por outro lado, houve um aumento da expressão do miRNA-210 no miocárdio dos animais diabéticos (p < 0,001). No entanto, tais efeitos foram revertidos com alho ou exercícios voluntários (p < 0,01). Além disso, o tratamento de ratos diabéticos conjuntamente com alho e exercícios voluntários teve um efeito adicional sobre as expressões do miRNA-126 e do miRNA-210 (p < 0,001). Além disso, tanto os exercícios voluntários quanto o alho melhoraram significativamente os perfis lipídicos séricos (p < 0,001).

Conclusões:

A indução de diabetes diminuiu a angiogênese no miocárdio, enquanto nosso tratamento com exercícios voluntários de longa duração e alho melhorou a angiogênese miocárdica. Estas alterações devem-se, possivelmente, ao aumento das expressões do miRNA-126 e do miRNA no miocárdio.

Palavras-chave:
Ratos; Alho; Allium Sativum; Exercício; Diabetes Mellitus; microRNAs; Indutores da Angiogênese; Neovascularização Fisiológica

Introduction

Diabetes mellitus (DM) is one of the major risk factors for cardiovascular disease, leading to endothelial dysfunction and angiogenesis impairment.11 Osipova J, Fischer D-C, Dangwal S, Volkmann I, Widera C, Schwarz K, et al. Diabetes-associated microRNAs in pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. J Clin Endocrinol Metab. 2014;99(9):E1661-5. The current trend on research and health care focuses on providing effective therapy with few side effects and low toxicity that can be regularly used to control diabetes complications.22 Tag H, Kalita P, Dwivedi P, Das AK, Namsa ND. Herbal medicines used in the treatment of diabetes mellitus in Arunachal Himalaya, northeast, India. J Ethnopharmacol. 2012;141(3):786-95.

Exercise is a powerful therapeutic strategy to improve overall cardiovascular health.33 Chipkin SR, Klugh SA, Chasan-Taber L. Exercise and diabetes. Cardiol Clin. 2001;19(3):489-505. However, exhaustive exercise may be problematic as it can cause the production of reactive oxygen species (ROS).44 Huang KC, Wu WT, Yang FL, Chiu YH, Peng TC, Hsu BG, et al. Effects of freshwater clam extract supplementation on time to exhaustion, muscle damage, pro/anti-inflammatory cytokines, and liver injury in rats after exhaustive exercise. Molecules. 2013;18(4):3825-38. Therefore, voluntary exercise, in which the animal has free access to a running wheel, may be a model with more positive effects.55 Wu G, Rana JS, Wykrzykowska J, Du Z, Ke Q, Kang P, et al. Exercise-induced expression of VEGF and salvation of myocardium in the early stage of myocardial infarction. Am J Physiol Heart Circ Physiol. 2009;296(2):H389-H95. There is evidence that aerobic training can promote cardiac angiogenesis,66 Da Silva ND Jr, Fernandes T, Soci UP, Monteiro AW, Phillips MI, DE Oliveira EM. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62.,77 Leosco D, Rengo G, Iaccarino G, Golino L, Marchese M, Fortunato F, et al. Exercise promotes angiogenesis and improves beta-adrenergic receptor signalling in the post-ischaemic failing rat heart. Cardiovasc Res. 2008;78(2):385-94. in which the vascular endothelial growth factor (VEGF) has a critical role.55 Wu G, Rana JS, Wykrzykowska J, Du Z, Ke Q, Kang P, et al. Exercise-induced expression of VEGF and salvation of myocardium in the early stage of myocardial infarction. Am J Physiol Heart Circ Physiol. 2009;296(2):H389-H95. However, the underlying mechanisms of exercise have yet to be fully elucidated.

One of the most traditional plants in herbal medicine is Allium sativum L, which has been reported to have beneficial health effects. It is used as a therapeutic agent in various disorders such as cancer, cardiovascular disease, and diabetes through different mechanisms, including inhibition or stimulation of angiogenesis.22 Tag H, Kalita P, Dwivedi P, Das AK, Namsa ND. Herbal medicines used in the treatment of diabetes mellitus in Arunachal Himalaya, northeast, India. J Ethnopharmacol. 2012;141(3):786-95.,88 Naderi R, Mohaddes G, Mohammadi M, Alihemmati A, Badalzadeh R, Ghaznavi R, et al. Preventive effects of garlic (Allium sativum) on oxidative stress and histopathology of cardiac tissue in streptozotocin-induced diabetic rats. Acta Physiol Hung. 2015;102(4):380-90.,99 Bayan L, Koulivand PH, Gorji A. Garlic: a review of potential therapeutic effects. Avicenna J Phytomed. 2014;4(1):1-14. Considering the effects of garlic in protecting against cardiovascular disease, as well as its effects on angiogenesis in different tissues, it is interesting to examine the effects of garlic on both myocardial angiogenesis and its related mechanisms.

MiRs are small non-coding RNAs that function in RNA silencing and the post-transcriptional regulation of gene expression.1010 Yin K-J, Hamblin M, Chen YE. Angiogenesis-regulating microRNAs and ischemic stroke. Curr Vasc Pharmacol. 2015;13(3):352-65. MiRs are essential intracellular mediators in many processes such as inflammation, mitochondrial metabolism, apoptosis, and angiogenesis, which can be adjusted through exercise.1111 Baggish AL, Hale A, Weiner RB, Lewis GD, Systrom D, Wang F, et al. Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J Physiol. 2011;589(Pt 16):3983-94. Therefore, miRs can be clinically useful in the treatment of several disorders. Moreover, miRs are released in urine and in the bloodstream following tissue injury, which makes them useful biomarkers for early detection, diagnosis, and prognosis of disorders. Recently, these molecules have been found to be involved in cardiovascular diseases.1212 Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell. 2008;15(2):261-71. This includes a high expression of miR-126 in the heart endothelium, as well as its involvement in angiogenesis.1212 Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell. 2008;15(2):261-71.,1313 Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, et al. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell. 2008;15(2):272-84. Circulating levels of miR-126 are reduced in diabetes,1414 de Boer HC, van Solingen C, Prins J, Duijs JM, Huisman MV, Rabelink TJ, et al. Aspirin treatment hampers the use of plasma microRNA-126 as a biomarker for the progression of vascular disease. Eur Heart J. 2013;34(44):3451-7.,1515 Liu Y, Gao G, Yang C, Zhou K, Shen B, Liang H, et al. The role of circulating microRNA-126 (miR-126): a novel biomarker for screening prediabetes and newly diagnosed type 2 diabetes mellitus. Int J Mol Sci. 2014;15(6):10567-77. suggesting that its deficiency may impair vascularisation.1616 Breen EC, Johnson EC, Wagner H, Tseng HM, Sung LA, Wagner PD. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol. 1996;81(1):355-61. Moreover, Fasanaro et al.1717 Fasanaro P, D'Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem. 2008;283(23):15878-83. reported that hypoxia-driven miR-210 supports angiogenic response in endothelial cells and that its blockade by anti-miR transfection inhibits the formation of capillary-like structures.1717 Fasanaro P, D'Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem. 2008;283(23):15878-83.

Many diabetes complications are well-known to be associated with lipid disorders. Indeed, dyslipidemia impairs numerous organs and is recognized as an important factor of many diabetic complications, including vascular abnormalities.1818 de Amorim Miranda PH, Monteiro OM, Rossoni JV Jr., Silva ME, de Lima WG, Costa DC. Vildagliptin induces ß-cell neogenesis and improves the lipid profile in a later phase of type 1 diabetes. Curr Pharm Biotechnol. 2015;16(1):60-5.

Therefore, the aim of this study was to investigate the effect of voluntary exercise and garlic treatment alone or in combination on miR-126 and miR-210 expressions, serum lipid profile, as well as their relationship with cardiac angiogenesis in diabetes.

Methods

Animals and Experimental Design

The Ethics Committee for Animal Experiments approved the study plan, and all experiments were conducted in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Male Wistar rats (200-250 g) were provided by our university’s colony. All animals were housed in a temperature-controlled facility (21-23°C) maintained on a 12:12-h light-dark cycle, with food and water provided ad libitum.

In this study, thirty-five male rats were divided into five groups (n = 7): Control, Diabetes, Diabetes+Garlic, Diabetes+Exercise, and Diabetes+Garlic+Exercise. Control animals received 0.4 mL of sodium citrate buffer, pH 4.5. Diabetes was induced using a single intraperitoneal dose (50 mg/kg) of Streptozotocin (Sigma, St. Louis, Mo, USA). Blood glucose level was measured 72 hours later using a glucometer (Elegance, Model: no: CT-X10 Germany), and induced diabetes was identified if blood glucose level was > 300 mg/dL (16.67 mmol/L).

In this study, sample size was determined based on our similar previous studies.88 Naderi R, Mohaddes G, Mohammadi M, Alihemmati A, Badalzadeh R, Ghaznavi R, et al. Preventive effects of garlic (Allium sativum) on oxidative stress and histopathology of cardiac tissue in streptozotocin-induced diabetic rats. Acta Physiol Hung. 2015;102(4):380-90.,1919 Naderi R, Mohaddes G, Mohammadi M, Ghaznavi R, Ghyasi R, Vatankhah AM. Voluntary exercise protects heart from oxidative stress in diabetic rats. Adv Pharm Bull. 2015;5(2):231-6.

Voluntary exercise

Rats in the voluntary exercise groups were housed individually in cages with stainless-steel running wheels (1.00 m circumference, TajhizGostar) and were allowed free access to the wheel 24 h per day for 6 weeks. Running distance was monitored daily. If the running distance was below 2000 m/day, that animal was excluded from the study. Sedentary rats were housed in standard holding cages without running wheels for the same period.

Preparing Garlic Homogenate

Garlic (Allium sativum) bulbs were purchased from a local market. Cloves were peeled, sliced, ground into a paste and then dissolved in distilled water. The garlic homogenate was freshly prepared each day.

Sampling

At the end of the 6th week, the rats were deeply anesthetized with pentobarbital sodium (35 mg/kg, i.p.), blood samples were collected from the inferior vena cava to measure lipid profile.

Then the heart was quickly removed through midsternal thoracotomy and the left ventricle was excised, frozen in liquid nitrogen, and stored at deep freeze (-70°C) for later measurements. The myocardium was used for miR extraction, real-time PCR study and angiogenesis determination.

MiR Extraction and Real-Time PCR

MiR was extracted from the myocardium using miRCURYTMRNA isolation kit (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol.2020 Biyashev D, Veliceasa D, Topczewski J, Topczewska JM, Mizgirev I, Vinokour E, et al. miR-27b controls venous specification and tip cell fate. Blood. 2012;119(11):2679-87.,2121 Lässer C, Eldh M, Lötvall J. Isolation and characterization of RNA-containing exosomes. J Vis Exp. 2012(59):e3037. The procedure was performed based on the spin column using a proprietary resin as a matrix to separate RNA from other cell components. RNA content and purity were measured using the Nanodrop 1000 spectrophotometer (Thermo scientific, Wilmington, DE 19810 USA). MiR-126 expression profile was obtained for total RNA extracts using universal a cDNA synthesis kit. Briefly, total RNA containing microRNA was polyadenylated and cDNA was synthesized using a poly(T) primer with a 3’ degenerate anchor and a 5’ universal tag (Exiqon, Vedbaek, Denmark). Each cDNA was used as a template for microRNA quantitative real-time PCR by using the SYBR Green master mix (Exiqon, Vedbaek, Denmark). LNA (Locked Nucleic Acid) forward and reverse primer sets (Exiqon, Vedbaek, Denmark) for microRNA are listed in Table 1. Real-time PCR reactions were performed with a Bio-Rad iQ5 detection System (Bio-Rad, Richmond, CA, USA). The amount of PCR products was normalized with housekeeping rno-miR-191 for miR-126 and miR-210.3737 Sorensen CM, Ding J, Zhang Q, Alquier T, Zhao R, Mueller PW, et al. Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem. 2010;43(12):948-56. We used the 2-(ΔΔCt) method to determine the relative quantitative levels of miR-126 and miR-210. Results were expressed as the fold-difference to the relevant controls.

Table 1
Target sequence list for miRs

Immunostaining for PECAM-1/ CD31

To investigate angiogenesis in the myocardium, transversal sections of the ventricles at their midportion were immediately isolated and fixed in 10% buffered-formalin solution, dehydrated in ascending grades of alcohol and embedded in paraffin. Then, serial 3 µm-thick sections were cut from them and floated onto charged glass slides according to standard histological processing. Tissue pieces were deparaffinised in xylene and dehydrated in a graded series of ethanol. Slides were incubated sequentially in proteinase K and 0.3% hydrogen peroxide to block endogenous peroxidase activity. Sections were overlaid by primary antibody CD31 (Santa Cruz, USA) - an angiogenesis marker - and incubated at +4°C overnight. Afterwards, the sections were washed and incubated with standard avidin-biotin complex (ABC; Santa Cruz) according to the protocol. Then the slides were incubated in DAB (Diamino-benzidine, Santa Cruz) as the chromagen, and counterstained with Mayer's hematoxylin. Finally, the sections were cleared in xylene, mounted with Entellan and analyzed with a light microscope.

Assessment of immunostaining

To evaluate immunostaining, 3 to 5 sections of 1 mm22 Tag H, Kalita P, Dwivedi P, Das AK, Namsa ND. Herbal medicines used in the treatment of diabetes mellitus in Arunachal Himalaya, northeast, India. J Ethnopharmacol. 2012;141(3):786-95. were randomly selected at a magnification of 400×, depending on the size of the sample section. Both staining intensity and number of positive cells were evaluated semi-quantitatively. Intensity scoring for CD31 staining was obtained within each area at a 400× magnification. Each endothelial cell cluster of immunoreactivity expressing CD31 and forming lumen or vessels was counted as individual microvessels. Vascular structures positive for CD31 were counted for 5 to 6 slides per animal and 10 fields per slide.

To assess immunostaining, we used the granulation tissue as a positive control, and the intensity of the staining was scored as follows: 0 (<10%); 1 (10% to 25%); 2 (25% to 50%); 3 (50% to 75%) or 4 (75% to 100%).2222 Mirzaei Bavil F, Alipour MR, Keyhanmanesh R, Alihemmati A, Ghiyasi R, Mohaddes G. Ghrelin decreases angiogenesis, HIF-1a and VEGF protein levels in chronic hypoxia in lung tissue of male rats. Adv Pharm Bull. 2015;5(3):315-20.

Lipid profile measurement

Blood samples were obtained from the inferior vena cava, then centrifuged at 3500 rpm for 10 min at 4°C, and serum was collected. Triglycerides serum level was determined by enzymatic kits (ZiestChem Diagnostic kits, Iran) using glycerol as the standard. Additionally, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) levels were determined based on enzymatic methods by diagnostic kits, (ZiestChem, Iran) using cholesterol as the standard.

Statistical analysis

All results are expressed as mean ± SEM for seven animals, and analyses were performed using SPSS statistical software version 16. All parameters were tested for normality using the theone-sample Kolmogorov-Smirnov test. Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test. The significant level was set at p < 0.05.

Results

Effects of garlic and voluntary exercise on miR-126 in the myocardium

As shown in Figure 1, myocardial miR-126 expression level was significantly lower (p < 0.05) in rats with diabetes than in the control group. Treatment with garlic (p < 0.001), voluntary exercise (p < 0.01), or both combined increased significantly (p < 0.001) the myocardial miR-126 expression in diabetic rats compared to the diabetes group. Moreover, the Diabetes+Garlic+Exercise group had significantly higher level of miR-126 expression compared to the garlic treatment group (p < 0.05) and the just voluntary exercise group (p < 0.01) in diabetic animals.

Figure 1
Real-time quantitative PCR analysis of miR-126 in the heart tissue of experimental groups. The values represent means ± S.E.M for 7 animals. *p < 0.05 vs control group, $$p < 0.01 and$$$ p < 0.001 vs diabetes group, && p < 0.01 vs Diabetes+Exercise group, and #p < 0.05 vs Diabetes+Garlic group.

Effects of garlic and voluntary exercise on miR-210 in the myocardium

As shown in Figure 2, the expression of miR-210 significantly increased (p < 0.001) in animals with diabetes compared with the control group. Treatment with garlic (p < 0.01), voluntary exercise (p < 0.01), or both combined reduced significantly (p < 0.001) the myocardial miR-210 expression in diabetic rats compared to the diabetes group. The combined Garlic+Voluntary Exercise group significantly lowered miR-210 expression compared to the Diabetes+Exercise (p < 0.05) and Diabetes+Garlic (p < 0.01) groups

Figure 2
Real-time quantitative PCR analysis of miR-210 in the heart tissue of experimental groups. The values represent means ± S.E.M for 7 animals. ***p < 0.001 vs control group, $$p < 0.01 and $$$p < 0.001 vs diabetes group, &p < 0.05 vs Diabetes + Exercise group, and ##p < 0.01 vs Diabetes + Garlic group.

Effect of garlic and voluntary exercise on angiogenesis in the myocardium

Immunostaining with CD31 marker was performed for the assessment of angiogenesis in the transversal section of the ventricles at their midportion. Brown stained tissues show CD-31 immunostained endothelial cells. Figure 4 shows the scores for staining intensity, which are as follows: 0 (<10%); 1 (10% to 25%); 2 (25% to 50%); 3 (50% to 75%) or 4 (75% to 100%). As shown in Figures 3 and 4, statistical analysis of our immunohistochemical study revealed that angiogenesis decreased significantly (p < 0.01) in the diabetes group compared to the control group. Six weeks of garlic treatment, voluntary exercise, or a combination thereof in the diabetes groups increased significantly (p < 0.001) the angiogenesis in their left ventricle compared to the diabetes group (Figure 3 and 4). Combined garlic consumption and exercise in diabetic animals induced more angiogenesis compared to garlic alone and exercise alone, though the difference was not significant.

Figure 3
Immunohistochemical detection of CD31 in myocardial vessels of different groups. Brown stained tissues show CD-31 immunostained endothelial cells in: (A) Control; (B) Diabetes; (C) Diabetes+Garlic; (D) Diabetes+Exercise; and (E) Diabetes+Garlic+Exercise. The intensity of immunostaining for CD31 (arrow head) decreased in the diabetes group compared to the control group. Garlic treatment and exercise alone or combined increased angiogenesis in diabetes compared to the diabetes group (Magnification was 400x).

Figure 4
Effects of garlic treatment and voluntary exercise on angiogenesis in different experimental groups. The intensity of the staining was scored as: 0 (<10%); 1 (10-25%); 2 (25-50%); 3 (50-75%); and 4 (75-100%). The values represent means ± S.E.M for 7 animals. **p < 0.01 vs control group and $$$p < 0.001 vs diabetes group.

Effect of garlic and voluntary exercise on serum lipid profile

Lipid profile alterations in different groups are shown in Table 2. The induction of diabetes in the animals increased significantly (p < 0.001) the serum TGs and LDL levels while lowering serum HDL and HDL/LDL compared to the control animals. Voluntary exercise reduced significantly (p < 0.05) the serum triglycerides levels in the diabetes group compared with the control group. Six weeks of garlic treatment alone or with voluntary exercise decreased significantly (p < 0.01) the triglycerides levels in the animals with diabetes. In these, serum LDL levels decreased significantly (p < 0.001) after garlic alone and exercise alone or a combination thereof. However, serum HDL level was significantly increased (p < 0.001) by garlic treatment, voluntary exercise, or a combination thereof in diabetic rats. Furthermore, the HDL:LDL ratio was significantly higher (p < 0.001) in the Diabetes+Garlic, Diabetes+Exercise and Diabetes+Garlic+Exercise groups compared with diabetes group.

Table 2
Serum lipid profile in different groups after 6 weeks (Mean ± SEM, n = 7)

Discussion

The present study has shown that the induction of diabetes impaired serum lipid profile, decreased myocardial angiogenesis and miR-126 expression, and increased myocardial expression of miR-210. However, the treatment with garlic alone, voluntary exercise alone or both combined ameliorated these effects in the myocardium of diabetic animals. Interestingly, treating diabetic rats simultaneously with garlic and voluntary exercise had an additional effect on the cardiac expression of miR-126 and miR-210. In line with our study, research has shown that diabetes leads to an impaired function of early endothelial progenitor cells, which results in a reduced capacity of neovascularisation and angiogenesis in the myocardium of diabetic rats.2323 Khazaei M, Fallahzadeh AR, Sharifi MR, Afsharmoghaddam N, Javanmard SH, Salehi E. Effects of diabetes on myocardial capillary density and serum angiogenesis biomarkers in male rats. Clinics. 2011;66(8):1419-24. VEGF, as an inducer of angiogenesis, is a highly specific mitogen for endothelial cells.2424 Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev. 2004;56(4):549-80. It is well-known that the expression of VEGF-A and its receptors decreases in the myocardium of diabetic rats and humans.2525 Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, et al. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic states: a possible explanation for impaired collateral formation in cardiac tissue. Circulation. 2002;105(3):373-9. However, the actual process of VEGF and angiogenesis reduction in the diabetic heart has not been fully elucidated.

There is a variety of miRs in the heart tissue, and these tiny regulators are recognized as novel targets/drugs in numerous fields, including cardiology.1212 Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell. 2008;15(2):261-71. MiR-126 is known as an endothelial-specific miR that modulates angiogenesis in vivo. Several studies have shown miR-126 to support endothelial homeostasis and angiogenesis,1212 Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA, et al. The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell. 2008;15(2):261-71.,1313 Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, et al. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell. 2008;15(2):272-84.,1515 Liu Y, Gao G, Yang C, Zhou K, Shen B, Liang H, et al. The role of circulating microRNA-126 (miR-126): a novel biomarker for screening prediabetes and newly diagnosed type 2 diabetes mellitus. Int J Mol Sci. 2014;15(6):10567-77. which is mediated by SPRED1 and PIK3R2 to promote VEGF signaling.1515 Liu Y, Gao G, Yang C, Zhou K, Shen B, Liang H, et al. The role of circulating microRNA-126 (miR-126): a novel biomarker for screening prediabetes and newly diagnosed type 2 diabetes mellitus. Int J Mol Sci. 2014;15(6):10567-77. In addition, miR-126 activates survival kinases such as ERK and Akt by downregulating its targets and promoting the action of VEGF.2626 Ueki K, Fruman DA, Yballe CM, Fasshauer M, Klein J, Asano T, et al. Positive and negative roles of p85a and p85ß regulatory subunits of phosphoinositide 3-kinase in insulin signaling. J Biol Chem. 2003;278(48):48453-66. Osipova et al reported in their study that urinary miR-126 levels were reduced in the patients with diabetes; however, circulating miR-126 levels in plasma showed no significant difference.11 Osipova J, Fischer D-C, Dangwal S, Volkmann I, Widera C, Schwarz K, et al. Diabetes-associated microRNAs in pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. J Clin Endocrinol Metab. 2014;99(9):E1661-5.

Little information is available about the expression of miR in the myocardium of diabetic rats in response to voluntary exercise. Interestingly, in the present study, we observed that garlic, voluntary exercise and a combination thereof increased the levels of miR-126 expression and angiogenesis in the myocardium. Cardioprotective effects of garlic have been reported in some studies related to improvement of antioxidant activities,88 Naderi R, Mohaddes G, Mohammadi M, Alihemmati A, Badalzadeh R, Ghaznavi R, et al. Preventive effects of garlic (Allium sativum) on oxidative stress and histopathology of cardiac tissue in streptozotocin-induced diabetic rats. Acta Physiol Hung. 2015;102(4):380-90. AMPK-mediated AKT/GSK-3β/HIF-1α activation,2727 Yu L, Di W, Dong X, Li Z, Xue X, Zhang J, et al. Diallyl trisulfide exerts cardioprotection against myocardial ischemia-reperfusion injury in diabetic state, role of AMPK-mediated AKT/GSK-3ß/HIF-1a activation. Oncotarget. 2017;8(43):74791-74805. and Akt-eNOS signaling pathways.2828 Hayashida R, Kondo K, Morita S, Unno K, Shintani S, Shimizu Y, et al. Diallyl trisulfide augments ischemia-Induced angiogenesis via an endothelial nitric oxide synthase-dependent mechanism. Circ J. 2017;81(6):870-8. Moreover, in line with our results, da Silva et al.66 Da Silva ND Jr, Fernandes T, Soci UP, Monteiro AW, Phillips MI, DE Oliveira EM. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62. showed that aerobic training in healthy rats increased cardiac miR-126 expression, which was possibly related to exercise-induced cardiac angiogenesis.66 Da Silva ND Jr, Fernandes T, Soci UP, Monteiro AW, Phillips MI, DE Oliveira EM. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62. Furthermore, studies have demonstrated that exercise enhances angiogenesis in the heart both under healthy2929 Hakkila J. Studies on the myocardial capillary concentration in cardiac hypertrophy due to training; an experimental study with guinea pigs. Ann Med Exp Biol Fenn. 1955;33(Suppl 10):1-82. and pathological conditions,55 Wu G, Rana JS, Wykrzykowska J, Du Z, Ke Q, Kang P, et al. Exercise-induced expression of VEGF and salvation of myocardium in the early stage of myocardial infarction. Am J Physiol Heart Circ Physiol. 2009;296(2):H389-H95.,77 Leosco D, Rengo G, Iaccarino G, Golino L, Marchese M, Fortunato F, et al. Exercise promotes angiogenesis and improves beta-adrenergic receptor signalling in the post-ischaemic failing rat heart. Cardiovasc Res. 2008;78(2):385-94. which highlights the positive effect of physical activity as a non-pharmacological tool in the treatment of cardiovascular disorders. Considering the increased expression of miR-126 following voluntary exercise, cardiac angiogenesis is possibly related to exercise-induced miR-126 expression and VEGF modulation, which upregulates angiogenic pathways such as MAPK and PI3K/Akt/eNOS.66 Da Silva ND Jr, Fernandes T, Soci UP, Monteiro AW, Phillips MI, DE Oliveira EM. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62.

An important hypoxia-induced miR, miR-210 is stimulated following hypoxia and HIF activation.3030 Chan YC, Banerjee J, Choi SY, Sen CK. miR 210: The master hypoxamir. Microcirculation. 2012;19(3):215-23. The elevation of miR-210 gene expression is evidence of hypoxic conditions in the cardiac muscle, in which hypoxia stimulates a number of physiological responses such as angiogenesis through HIF-1α-induced miR-210 expression.3131 XU L, WANG F, Wei W, DAI W-q, HE S-s, WANG X-p, et al. Effects of hypoxia on the expressions of hypoxia-inducible factor-1 alpha and miR-210 in hepatocellular carcinoma HepG2 cells. Tumor. 2011;31(6):502-7. MiR-210 upregulation is a major element of endothelial cell response to hypoxia, which leads to angiogenesis via its target gene Ephrin-A3.1717 Fasanaro P, D'Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem. 2008;283(23):15878-83. The upregulation of miR-210 and VEGF has been shown to enhance myocardium angiogenesis in acute myocardial infarction in response to Huoxue Anxin Recipe.3232 Wang J, Zhang Y, Liu YM, Guo LL, Wu P, Dong Y, et al. Huoxue Anxin Recipe () promotes myocardium angiogenesis of acute myocardial infarction rats by up-regulating miR-210 and vascular endothelial growth factor. Chin J Integr Med. 2016;22(9):685-90. Greco et al.3333 Greco S, Fasanaro P, Castelvecchio S, D'Alessandra Y, Arcelli D, Di Donato M, et al. MicroRNA dysregulation in diabetic ischemic heart failure patients. Diabetes. 2012;61(6):1633-41. described that, in addition to hypoxia, hyperglycemia is another stimulator that upregulates miR-210 expression, which is observed in diabetes.3333 Greco S, Fasanaro P, Castelvecchio S, D'Alessandra Y, Arcelli D, Di Donato M, et al. MicroRNA dysregulation in diabetic ischemic heart failure patients. Diabetes. 2012;61(6):1633-41. Osipova et al.11 Osipova J, Fischer D-C, Dangwal S, Volkmann I, Widera C, Schwarz K, et al. Diabetes-associated microRNAs in pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. J Clin Endocrinol Metab. 2014;99(9):E1661-5. showed that miR-210 level was upregulated in plasma and urine of type 1 diabetic children,11 Osipova J, Fischer D-C, Dangwal S, Volkmann I, Widera C, Schwarz K, et al. Diabetes-associated microRNAs in pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. J Clin Endocrinol Metab. 2014;99(9):E1661-5. as well as in cardiomyocytes and endothelial cells in diabetic patients.3333 Greco S, Fasanaro P, Castelvecchio S, D'Alessandra Y, Arcelli D, Di Donato M, et al. MicroRNA dysregulation in diabetic ischemic heart failure patients. Diabetes. 2012;61(6):1633-41. In line with these studies, we showed that the induction of diabetes increased myocardial miR-210 level, which was reduced by both garlic, voluntary exercise and a combination of both. Similarly, a recent study demonstrated that plasma miR-210 levels decreased in chronic kidney disease after acute exercise.3434 Van Craenenbroeck AH, Ledeganck KJ, Van Ackeren K, Jürgens A, Hoymans VY, Fransen E, et al. Plasma levels of microRNA in chronic kidney disease: patterns in acute and chronic exercise. Am J Physiol Heart Circ Physiol. 2015;309(12):H2008-16. On the contrary, some studies have shown that miR-210 was not responsive during acute, exhaustive exercise, sustained aerobic exercise1111 Baggish AL, Hale A, Weiner RB, Lewis GD, Systrom D, Wang F, et al. Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J Physiol. 2011;589(Pt 16):3983-94. and swimming3535 Fernandes T, Baraúna VG, Negrão CE, Phillips MI, Oliveira EM. Aerobic exercise training promotes physiological cardiac remodeling involving a set of microRNAs. Am J Physiol Heart Circ Physiol. 2015;309(4):H543-52. in the heart tissue. Furthermore, both garlic and exercise have been shown to be involved in providing good glycemic control and prevention against long-term diabetic complications.33 Chipkin SR, Klugh SA, Chasan-Taber L. Exercise and diabetes. Cardiol Clin. 2001;19(3):489-505.,88 Naderi R, Mohaddes G, Mohammadi M, Alihemmati A, Badalzadeh R, Ghaznavi R, et al. Preventive effects of garlic (Allium sativum) on oxidative stress and histopathology of cardiac tissue in streptozotocin-induced diabetic rats. Acta Physiol Hung. 2015;102(4):380-90.,1919 Naderi R, Mohaddes G, Mohammadi M, Ghaznavi R, Ghyasi R, Vatankhah AM. Voluntary exercise protects heart from oxidative stress in diabetic rats. Adv Pharm Bull. 2015;5(2):231-6. Therefore, in the present study, the decrease of miR-210 expression back to normal levels seems to stem from glycemic control. Additionally, garlic extract-mediated angiogenesis probably occurs through the upregulation of the neovasculogenic c-kit protein expression and the activation of the PI3-K/Akt/NF-κB signaling pathways,3636 Chiang EP, Chiu SC, Pai MH, Wang YC, Wang FY, Kuo YH, et al. Organosulfur garlic compounds induce neovasculogenesis in human endothelial progenitor cells through a modulation of MicroRNA 221 and the PI3-K/Akt signaling pathways. J Agric Food Chem. 2013;61(20):4839-49. which regulates e-NOS activation and NO production.1111 Baggish AL, Hale A, Weiner RB, Lewis GD, Systrom D, Wang F, et al. Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J Physiol. 2011;589(Pt 16):3983-94.

Hyperglycemia is currently considered to be primarily responsible for the alteration of lipid profile. In general, dyslipidemia is well confirmed in diabetes mellitus; it is known as a criterion for the diagnosis of type I diabetes and potential beta-cell lipotoxin.3737 Sorensen CM, Ding J, Zhang Q, Alquier T, Zhao R, Mueller PW, et al. Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem. 2010;43(12):948-56. It is worth noting that dyslipidemia is related to atherosclerosis and a risk of heart disease.3737 Sorensen CM, Ding J, Zhang Q, Alquier T, Zhao R, Mueller PW, et al. Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem. 2010;43(12):948-56. Dyslipidemia is possibly mediated by the alteration of LXRα expression in the liver and intestine, the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways, and the consequent inhibition of eNOS activity, causing impaired angiogenesis.1515 Liu Y, Gao G, Yang C, Zhou K, Shen B, Liang H, et al. The role of circulating microRNA-126 (miR-126): a novel biomarker for screening prediabetes and newly diagnosed type 2 diabetes mellitus. Int J Mol Sci. 2014;15(6):10567-77.,3838 Mohammadi A, Oshaghi EA. Effect of garlic on lipid profile and expression of LXR alpha in intestine and liver of hypercholesterolemic mice. J Diabetes Metab Disord. 2014;13(1):20. In addition, dyslipidemia is related to decreased levels of circulating miR-126.1313 Fish JE, Santoro MM, Morton SU, Yu S, Yeh RF, Wythe JD, et al. miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell. 2008;15(2):272-84. Riedel et al.3939 Riedel S, Radzanowski S, Bowen TS, Werner S, Erbs S, Schuler G, et al. Exercise training improves high-density lipoprotein-mediated transcription of proangiogenic microRNA in endothelial cells. Eur J Prev Cardiol. 2015;22(7):899-903. showed that exercise in patients with chronic heart failure significantly improved HDL-induced miR-126 expression.3939 Riedel S, Radzanowski S, Bowen TS, Werner S, Erbs S, Schuler G, et al. Exercise training improves high-density lipoprotein-mediated transcription of proangiogenic microRNA in endothelial cells. Eur J Prev Cardiol. 2015;22(7):899-903. In this study, treatment with garlic and voluntary exercise alone and together ameliorated lipid profile in the serum of diabetic rats, which is in agreement with previous studies.66 Da Silva ND Jr, Fernandes T, Soci UP, Monteiro AW, Phillips MI, DE Oliveira EM. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis. Med Sci Sports Exerc. 2012;44(8):1453-62.,99 Bayan L, Koulivand PH, Gorji A. Garlic: a review of potential therapeutic effects. Avicenna J Phytomed. 2014;4(1):1-14.,4040 Kostrzewa-Nowak D, Nowak R, Jastrzebski Z, Zarebska A, Bichowska M, Drobnik-Kozakiewicz I, et al. Effect of 12-week-long aerobic training programme on body composition, aerobic capacity, complete blood count and blood lipid profile among young women. Biochem Med (Zagreb). 2015;25(1):103-13. Therefore, garlic and exercise have possibly modulated angiogenesis in the myocardium of the diabetic animals by modulating serum lipid profile and the expression of pro-angiogenic miRs. With regard to the limitations of this study, we did not measure other factors involved in angiogenesis. Further studies are necessary to clarify the pathophysiological mechanisms of garlic and voluntary exercise in the treatment of diabetic complications.

Conclusion

This study showed that garlic and voluntary exercise modulated serum lipid profile and the expression of miR-126, miR-210, thus increasing angiogenesis in myocardium of diabetic rats. These findings suggest that garlic and voluntary exercise alone and combined may hold benefits in the treatment of diabetes.

  • Sources of Funding
    This study was funded by a grant from Drug Applied Research Center, Tabriz University of Medical Sciences.
  • Study Association
    This article is part of the thesis of Doctoral submitted by Roya Naderi, from Drug Applied Research Center, Tabriz University of Medical Sciences.
  • Ethics approval and consent to participate
    This study was approved by the Ethics Committee on Animal Experiments of the Tabriz University of Medical Sciences under the protocol number 91.4-2.4.

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    Sorensen CM, Ding J, Zhang Q, Alquier T, Zhao R, Mueller PW, et al. Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem. 2010;43(12):948-56.
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Publication Dates

  • Publication in this collection
    Feb 2019

History

  • Received
    05 Feb 2018
  • Reviewed
    02 Aug 2018
  • Accepted
    02 Aug 2018
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