Introduction: An early manifestation during the development of diabetes is cardiovascular damage. Studies illustrated the potential effects of peroxisome proliferator-activated receptor (PPAR) agonists on reduction of cardiovascular disease. In this study, we tried to delineate a possible effect of fenofibrate, a PPARa agonist, on coronary angiogenesis in diabetic rats.
Methods: Eighteen male Wistar rats were randomly divided into three groups of control, diabetic and diabetic + fenofibrate (100 mg/kg/day) (n = 6 for all groups). Diabetes was induced by a single dose of intraperitoneal streptozotocin (55 mg/kg). After 21 days, capillary density in the myocardial tissue was evaluated by immunohistochemical staining. The results were reported as capillaries per mm2. Blood samples were taken before and after the induction of diabetes.
Results: Diabetes was associated with reduced myocardial capillary density compared to the control group (121.71 ± 13.32 vs. 153.78 ± 11.08/mm2; p < 0.05). Administration of fenofibrate significantly restored angiogenesis in myocardial tissue of diabetic animals (199.98 ± 20.54 vs. 121.71 ± 13.32 /mm2).
Conclusion: Our results supported the hypothesis regarding a possible beneficial effect of fenofibrate on coronary artery diseases in diabetic subjects.
Keywords: Diabetes, Fenofibrate, Capillary Density (JPMA 62: S-9; 2012).
Diabetes mellitus is a complex metabolic disease with adverse multiple clinical effects such as glucose intolerance, insulin resistance, cardiovascular pathologies and endothelial dysfunction.1 Diabetes global incidence has been estimated to reach 366 million by 2030.2 There has been increasing evidences that many of clinical effects of diabetes may relate to abnormalities of angiogenesis as compared with euglycemic individuals.3,4 On one hand, increased angiogenesis has been seen in the retina or kidneys of diabetic subjects, and on the other hand, impaired coronary collateral vessel development has been implicated.3
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of steroid receptor superfamily. Three isotypes of PPARs have been identified as PPARa, PPARg and PPARb/d. PPARa is highly expressed in liver, heart, kidney, vascular endothelial cells and skeletal muscles.5-7 PPARa agonists are known to ameliorate hyperlipidemia, hyperglycemia and vascular endothelial dysfunction in diabetic patients,9 and improve cardiac function in subjects with diabetes in long term.10,11 Recently, in vivo and in vitro studies have examined the angiogenic properties of PPARa.12,13 This study attempted to investigate the role of fenofibrate, a known PPARa agonist, on coronary angiogenesis in a diabetic animal model.
Materials and Methods
Eighteen male Wistar rats, weighing 180 ± 50 g, were purchased from Pasteur Institute of Iran. They were housed in an environmentally controlled room in a 12 h/12 h light/dark cycle. The animals were fed a standard rat chow and had free access to water ad libitum. All experimental procedures were approved by the Ethics Committee of Isfahan University of Medical Sciences, Isfahan, Iran.
The animals were randomly divided into three groups of control, diabetic and diabetic + fenofibrate. Diabetes was induced by single intraperitoneal injection of streptozotocin (Sigma Co.) at a dose of 55 mg/kg.14 After 48 hours, blood glucose levels were measured. The animals with blood glucose concentrations higher than 16.7 mmol/l were considered as diabetic.15 The control diabetic groups received vehicle while diabetic + fenofibrate received fenofibrate (100 mg/kg/day) by gavage every day. The treatments lasted for 21days.16
Blood samples were taken before and after the experiment. Blood samples were centrifuged at 3000 rpm for 20 minutes and serums were poured in separate eppendorf tubes. Blood glucose levels were measured by a glucometer. Serum total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) levels were determined using calorimetric assay. Low-density lipoprotein cholesterol (LDL-C) was calculated with the Friedewald equation. Serum nitric oxide (NO) concentration was measured by Griess reagent method.
Measurement of capillary density:
After 21 days, the animals were sacrified by cervical dislocation and the left ventricular muscles were immediately dissected and put in 10% formalin overnight. After preparation of 5 µm histological sections, they were deparaffinized and incubated with a rat monoclonal antibody against murine CD31 (Abcam, Cambridge, UK). Then, the counted in ten random microscopic fields (×400) from each tissue preparation were reported as the number of capillaries per square millimeter.17
Data was represented as mean ± SE. Statistical comparisons between groups were evaluated by one way analysis of variance (ANOVA). Paired t-test was used for data analysis before and after drug therapy. Bivariate correlations were calculated using Pearson\'s correlation coefficient. P values less than 0.05 were considered as statistically significant.
Body weight and blood samples:
Table illustrates serum lipid profile, blood glucose and body weight of the experimental groups. Body weight significantly decreased in diabetic rats over time (p < 0.05) while the reduction in body weight among diabetic rats that received fenofibrate was insignificant. On the other hand, body weight increased in the control group. Fasting blood glucose level in the diabetic rats was higher than the control group. In addition, administration of fenofibrate failed to reduce blood glucose. While plasma levels of TG significantly decreased, HDL-C levels significantly increased in the diabetic group treated with fenofibrate compared to the untreated group (p < 0.05). Serum NO level in the fenofibrate-treated diabetic group was significantly higher than the non-treated group (p < 0.05).
Myocardial capillary density:
Myocardial capillary density in the diabetic group was lower than the control group (121.71 ± 13.32 vs. 153.78 ± 11.08 /mm2; p < 0.05). Administration of fenofibrate significantly improved myocardial capillary density in diabetic rats (Figure-1).
Images of some histological sections of ventricular muscles in all experimental groups are presented in Figure-2.
In the correlation analysis, there was a positive correlation between capillary density in myocardial tissue and serum NO concentrations (r = 0.63) (Figure-3).
The present study aimed to determine the impact of diabetes on heart capillary density. It also assessed the efficacy of fenofibrate, a PPARa agonist, administration on glycemic control and myocardial capillary density improvement. It therefore evaluated the glycemic status and capillary density in ventricular muscles of diabetic rats. We found diabetes to be associated with impaired formation of heart collateral vessels. In addition, fenofibrate administration restored heart capillary density without a glucose lowering effect.
Cardiovascular diseases are the main cause of morbidity and mortality in diabetic subjects. One of the suggested mechanisms is defective neovascularization in myocardial tissue under hypoxic condition resulting in reduced myocardial blood flow and increased morbidity.21 Therapeutic angiogenesis is a novel physiological approach for improving tissue perfusion and clinical consequences in diabetes. Imbalanced growth factors and cytokines and defected signal transduction of vascular endothelial growth factor (VEGF) may be two important mechanisms explaining inadequate angiogenesis.3 For first time, Abaci et al. declared diabetic subjects to have reduced coronary artery collateral formation compared to non-diabetics.18 Other studies also suggested the expression of some angiogenic factors such as VEGF and its receptors to decrease in the myocardium of diabetic individuals while production of anti-angiogenic factor (angiostatin) increased.19,20
In this study, we also found that fenofibrate could improve collateral vessel formation in heart muscles of a rat model of type 1 diabetes. In recent years, the angiogenic potentials of PPARa agonists have been studied.1,16,22 Activation of PPARa by fenofibrate and WY14643 (a synthetic PPARa agonist) has been reported to inhibit endothelial cell migration mediating by VEGF via targeting Akt phosphorylation.23 Furthermore, fenofibrate decreases plasma VEGF in patients with hyperlipidemia and atherosclerosis.24 In contrast to these observations and in agreement with our results, Biscetti et al. found PPARa agonists to induce angiogenesis indirectly via upregulation of the angiogenic factor VEGF. However, they found that inhibition of VEGF did not completely inhibit the induced angiogenesis.12 Thus, other angiogenic factors may involve in PPARa stimulated angiogenesis. Similarly, we found increased serum NO concentrations after fenofibrate treatment which suggests a possible mechanism for increased neovascularization. In another study, we found that fenofibrate resulted in ischemia-induced angiogenesis in hindlimb ischemia of diabetic rats via increasing serum NO concentrations (data not published, yet).
In conclusion, our data showed that diabetes is associated with impaired coronary artery collateral formation. We also found the administration of fenofibrate to be able to restore coronary angiogenesis. It therefore can be considered as an bneficial in treatment of diabetic patients with cardiovascular diseases.
This study was supported by a grant (#188138) from Isfahan University of Medical Sciences, Isfahan, Iran.
1. Grundy SM, Benjamin Ij, Burke Gl et al. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American heart association. Circulation 1999; 100: 1134-46.
2. Wild S,Rolglic G,Green A et al, Global prevalence of diabetes: estimates for the year 2000and projections for 2030. Diabetes Care 2004; 27: 1047-53.
3. Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev 2003; 23: 117-45.
4. Soares Raquel, Angiogenesis in diabetes .unraveling the angiogenic paradox The Open Circulation J 2010; 3: 3-9.
5. Moraes L, Bishop-Bailey D, peroxisome proliferator-activated receptors and inflammation. Pharmacol Ther 2006; 110: 371-85.
6. Dreyer C, Krey G, Keller H, et al. Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors Cell. 1992; 68: 879-87.
7. Pozzi A, Capdevila JH. PPARalpha Ligands as Antitumorigenic and Antiangiogenic Agents. PPAR Res 2008; 2008:906542..
8. Evans M, Anderson RA, Graham J, et al. Ciprofibrate therapy improves endothelial function and reduces postprandial lipemia and oxidative stress in type 2 diabetes mellitus. Circulation 2000; 101: 1773-9.
9. Play Ford D, Watts G, Best J, et al. Effect of fenofibrate on brachial artery flow-mediated dilation in type 2 diabetes mellitus . Am J Cardiol 2002; 90: 1254-7.
10. Aasum E, Belke DD, Severson D, et al. Cardiac function and metabolism in type 2 diabetic mice after treatment with BM17.0744, a novel PPAR-alpha activator. Am J Physiol Heart Circ Physiol 2002; 283: H949-57.
11. Ogata T, Miyauchi T, Sakai S, et al. Myocardial fibrosis and diastolic dysfunction in deoxycorticosterone acetate - salt hypertensive rats is ameliorated by the peroxisome proliferator - activated receptor alpha activator fenofibrate, partly by suppressing inflammatory responses with the nuclear factor kappa-B pathway. J Am Coll Cardiol 2004; 43: 1481-8.
12. Biscetti F, Gaetani E, Flex A, et al. Selective activation of peroxisome proliferator-activated receptor (PPAR) alpha and PPAR gamma induces neoangiogenesis through a vascular endothelial growth factor-dependent mechanism. Diabetes 2008; 57: 1394-404.
13. Varet J, Vincent L, Mirshahi P, et al. Fenofibrate inhibits angiogenesis in vitro and in vivo. Cell Mol Life Sci 2003; 60: 810-9.
14. Rivard A, Silver M, Chen D, Annex B, et al. Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF. Am J Pathol 1999; 154: 355-63.
15. Taniyama Y, Morishita R, Hiraoka K, et al. Therapeutic angiogenesis induced by human hepatocyte growthfactor gene in rat diabetic hind limb ischemia model: molecularmechanisms of delayed angiogenesis in diabetes. Circulation 2001; 104: 2344-50.
16. Katayama A, Yamamoto Y, Tanaka K, et al. Fenofibrate enhances neovascularization in a murine ischemic hindlimb model. J Cardiovasc Pharmacol 2009; 54: 399-404.
17. Li P, Kondo T, Numaguchi Y, et al. Role of bradykinin ,nitric oxide and angiotensin II type 2 receptor in imidapril -induced angiogenesis. Hypertention 2008; 51: 252-8.
18. Abaci A, Oguzhan A, Kahraman S, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation 1999; 99: 2239-42.
19. Chou E, Suzuma I, Way KJ, 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: 373-9.
20. Weihhrauch D, Lohr NL, Mraovic B, et al. Chronic hyperglycemia attenuates coronary collateral development of myocardial interstitial fluid by production of angiostatin. Circulation 2004; 109: 2343-8.
21. Waltenberger J. New Horizons in Diabetes Therapy: The Angiogenesis Paradox in Diabetes :Description of the Problem and Presentation of a Unifying Hypothesis Immun, Endoc & Metab Agents in Med Chem 2007; 7: 87-93.
22. Ping Li, Rei Shibata, Sonomi Maruyama, et al. Fenofibrate promotes ischemia-induced revascularization through the adiponectin-dependent pathway, Am J Physiol Endocrinol Metab 2010; DR.
23. Goetze S, Eilers F, Bungenstock, et al. PPAR activators inhibit endothelial cell migration by targeting Akt. Biochem Biophys Res Commun 2002; 293: 1431-7.
24. Blann A, Belgore F, Constans J, et al. Plasma vascular endothelial growth factor and its receptor Flt-1 in patients with hyperlipidemia and atherosclerosis and the effects of fluvastatin or fenofibrate,The American journal of cardiology 2001; 87: 1160-3.