Chemico-Biological Interactions 213 (2014) 28–36

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Caffeic acid phenethyl ester, a 5-lipoxygenase enzyme inhibitor, alleviates diabetic atherosclerotic manifestations: Effect on vascular reactivity and stiffness Noura Ahmed Hassan a, Hany M. El-Bassossy a,b,⇑, Mona Fouad Mahmoud a, Ahmed Fahmy a a b

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 31 October 2013 Received in revised form 29 December 2013 Accepted 30 January 2014 Available online 6 February 2014 Keywords: Diabetes Atherosclerosis Aorta Vascular reactivity Caffeic acid phenethyl ester

a b s t r a c t Atherosclerosis is a major macrovascular complication of diabetes that increases the risks for myocardial infarction, stroke, and other vascular diseases. The effect of a selective 5-lipoxygenase enzyme inhibitor; caffeic acid phenethyl ester (CAPE) on diabetes-induced atherosclerotic manifestations was investigated. Insulin deficiency or resistance was induced by STZ or fructose respectively. Atherosclerosis developed when rats were left for 8 or 12 weeks subsequent STZ or fructose administration respectively. CAPE (30 mg kg 1 day 1) was given in the last 6 weeks. Afterwards, blood pressure (BP) was recorded. Then, isolated aorta reactivity to KCl and phenylephrine (PE) was studied. Blood glucose level, serum levels of insulin, tumor necrosis factor a (TNF-a) as well as advanced glycation end products (AGEs) were determined. Moreover aortic haem oxygenase-1 (HO-1) protein expression and collagen deposition were also assessed. Insulin deficiency and resistance were accompanied with elevated BP, exaggerated response to KCl and PE, elevated serum TNF-a and AGEs levels. Both models showed marked increase in collagen deposition. However, CAPE alleviated systolic and diastolic BP elevations and the exaggerated vascular contractility to both PE and KCl in both models without affecting AGEs level. CAPE inhibited TNF-a serum level elevation, induced aortic HO-1 expression and reduced collagen deposition. CAPE prevented development of hyperinsulinemia in insulin resistance model without any impact on the developed hyperglycemia in insulin deficiency model. In conclusion, CAPE offsets the atherosclerotic changes associated with diabetes via amelioration of the significant functional and structural derangements in the vessels in addition to its antihyperinsulinemic effect in insulin resistant model. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes mellitus is a chronic disease that affects about 17 million people in the USA [1,2]; there are 170 million cases worldwide, and this figure is expected to be more than double by 2030 [3]. Type II diabetes is the most common form of diabetes, representing Abbreviations: AGEs, advanced glycation end products; ACh, acetyl choline; CAPE, caffeic acid phenethyl ester; BP, blood pressure; PE, phenylephrine; STZ, streptozotocin; TNF-a, tumor necrosis factor-a; HO-1, haem oxygenase-1; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance. ⇑ Corresponding author at: Department of Pharmacology, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260, Jeddah 21589, Saudi Arabia. Tel.: +966 568751075. E-mail addresses: [email protected], [email protected] (H.M. ElBassossy). http://dx.doi.org/10.1016/j.cbi.2014.01.019 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

about 90% of all diabetes worldwide [4]. Diabetic vascular complications affect many tissues, including microvasculature, macrovasculature, nerve and the heart. These complications are the most common causes of mortality, of end-stage renal disease, and of blindness in diabetic patients worldwide [5]. Atherosclerosis is a major macrovascular complication of diabetes that increases risks for myocardial infarction, stroke, and other vascular diseases. In 2001, the American Heart Association defined several factors involved in diabetic atherosclerosis including metabolic factor, oxidation/glucoxidation, and alteration in vascular reactivity [6,7]. The 5-lipoxygenase enzyme, being activated in diabetes caused by either insulin deficiency or resistance [8,9] is known to catalyze the oxygenation of arachidonic acid [10]. The products of arachidonic acid oxygenation were reported to be a function key mediators

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in the pathogenesis of many inflammatory diseases [11,12] including atherosclerosis [12]. Caffeic acid phenethyl ester (CAPE), an active ingredient of honeybee propolis extract, is a flavonoid-like compound that has been used as a traditional medicine in the Far East treating various ailments [13] due to its anti-inflammatory [14] and antioxidant properties [15], CAPE pharmacological effects are thought to be mediated in 5-lipooxygenase inhibition. Previously, it was shown that CAPE inhibits 5-lipooxygenase enzyme in micromolar concentration [10]. CAPE also has immunomodulatory [16], anticarcinogenic [17], neuroprotective [14] and anti-atherosclerotic effects [18]. CAPE has the ability to suppress lipid peroxidation [19] and is also a potent inhibitor of NF-jB activation [20]. Precisely the purpose of the present work is to examine the potentially protective effect of CAPE as a selective 5-lipoxygenase enzyme inhibitor against diabetes-induced atherosclerotic manifestations and illustrate the mechanism of this possible protection.

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in 10% formalin for immunohistochemistry techniques to measure HO-1 expression and collagen deposition. 2.3. Biochemical analysis Glucose was determined in tail blood by a glucose meter (Bionime GmBH) using noble metal electrode strips. Serum AGEs was measured as previously described [23]; the serum was diluted 1:15 with saline and the fluorescence intensity (kex = 370, kem = 440 nm) was recorded by LS45 fluorescence spectrophotometer (PerkinElmerÒ, Cairo, Egypt). Serum insulin level was measured by enzyme-linked immunosorbent assay (ELISA, Millipore, Cairo, Egypt) that uses plate coated with monoclonal anti-rat insulin antibodies. Serum TNF-a level was determined by ELISA using QuantikineÒ kit (R&D systems, Cairo, Egypt) using rat TNF-a and antibodies raised against the rat TNF-a. 2.4. Blood pressure measurement

2. Materials and methods 2.1. Animals Male Wistar rats weighing 85–105 g for insulin resistance section and 135–155 g for the insulin deficiency section (Faculty of Pharmacy, Zagazig University, Zagazig, Egypt) were housed in clear polypropylene cages (four rats per cage) and kept on equal duration of dark-light cycle, under constant environmental conditions. Rats received normal rodent pellet diet and water ad libitum. Experimental protocol was approved by Zagazig Ethical Committee for Animal Handling. 2.2. Study protocol Rats were randomly divided into four experimental groups (eight animals each) in each model as follows; control, caffeic acid phenethyl ester-treated control (C-CAPE), insulin deficient (ID) or resistant (IR), and caffeic acid phenethyl ester-treated insulin deficient (ID-CAPE) or resistant (IR-CAPE). ID was induced by intraperitoneal injection of streptozotocin (STZ, 50 mg kg 1). ID was confirmed by the development of postprandial hyperglycemia (300–400 mg dl 1) two weeks after STZ injection. IR was induced by adding fructose (10%) to every day drinking water for 12 weeks. We started with rats weighting 85–115 g as they were found to be less resistant to induction of insulin resistance in our previous work [21,22]. Rats with insulin level above 14 lg l 1 were considered IR rats. Insulin deficiency was induced by a single intraperitoneal injection of STZ (50 mg kg 1). Rats with moderate hyperglycemia (postprandial glucose level between 250 and 350 mg dl 1) were used in this study. CAPE (30 mg kg 1 day 1) was daily administered by oral gavage as suspension in distilled water in the last 6 weeks of study. Control group received distilled water as a vehicle instead. At the end of the study (twelve hours after the last CAPE administration), body weight and blood pressure (BP) were recorded. Then, the whole blood was collected (under light ether anesthesia) from the retro-orbital plexus, glucose level was determined in blood and the rest of blood was centrifuged (3000g, 4 °C, 20 min) to separate serum. Serum was divided into aliquots and stored at 20 °C till analyzed for insulin level, TNF-a and AGEs. The descending thoracic aorta was carefully excised through abdominal opening, placed in a Petri dish containing cold Krebs–Henseleit buffer (KHB) composed of (in mM): NaCl 118.1, KH2PO4 1.2, KCl 4.69, NaHCO3 25.0, MgSO4 0.5, glucose 11.7 and CaCl2 2.5. The aorta was cut into 3 rings (of 3 mm length) after cleaning the connective tissue and fat. For each animal, one aortic ring was used for studying vascular reactivity. The two other aortic rings were fixed

Blood pressure (BP) was indirectly recorded in a slightly restrained conscious rat by tail cuff method as described in our previous work [22]. Rats were trained on the restrainers and the warming chamber for 3 days before measurements for 20 min per day. BP measurements were performed between 7:00 AM and 12:00 AM by the same investigator. After 10 min stabilization in the warming chamber at 35 °C, automated inflation–deflation cycle was repeated 10 times. The mean of 5 readings within a 10 mmHg range was considered as the blood pressure. 2.5. Vascular reactivity Isolated aortic rings were suspended in individual organ chambers (30 ml) containing KHB at 37 °C and aerated with 95% oxygen, 5% carbon dioxide under 11.2 mN resting tension. Isometric force transducers (Biegestab K30, Hugosachs Elektronik, March, Germany) were used to determine rings tension. The force transducers were connected to a four channel Power Lab Data Interface Module connected to a PC running Chart software (v7, ADI Instruments, Cairo, Egypt). Rings were initially equilibrated for 60 min. During this time, the bath solution was changed twice. Before starting vascular reactivity measurements, vessel viability was assessed by exposing arteries to KCl (80 mM) twice to ensure stable responses. For studying the contractile responsiveness of aorta, cumulative concentrations of KCl (10–100 mM) or phenylephrine (PE, 10– 9 M to 10–5 M) were added to the organ bath and the response was recorded. 2.6. Haem oxygenase-1(HO-1) immunohistochemistry Immunohistochemistry of HO-1 protein in rat aortic sections was performed using the method described by Szocs et al. [24] with slight modification. It depends upon the use of a primary antibody to detect the HO-1 in sections followed by fluorophore conjugated antibody. The sections were deparaffinised in xylene and rehydrated. Endogenous peroxidase activity was saturated by incubating slides in 3% H2O2 solution in cold methanol for 30 min. Antigen retrieval solution was performed by incubation with citrate buffer at 90 °C for 30 min followed by rinsing with PBS. After blocking non-specific binding, sections were incubated with 50 ll of the rabbit anti-heme oxygenase-1 primary antibody (dilution 1:200 in blocking solution) overnight in cold room. Then sections were rinsed in 3 changes of 1  PBS (5 min each) then incubated with 50 ll of the Alexa fluor conjugated gout secondary antibody (dilution 1:10,000 in blocking solution) for 2 h in dark then rinsed with 3 changes of 1  PBS (5 min each) then the coverslip was mounted with 20 ll of fluorescence mounting media and left in dark before

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examination by LEICA DM500 fluorescence microscope with excitation at k = 478 and emission at k = 495 nm. Images were acquired with minimum gain and identical acquisition parameters. Quantitative comparisons of fluorescence were carried out by Image J software (National Institutes of Health, Bethesda, MD, USA). The aorta from each rat was divided between all treatment groups. No specific staining was observed in sections treated with the secondary antibody alone.

Aortic tissue sections (5 lm) were fixed by neutral formalin solution (10% at room temperature). Collagen–Masson’s trichrome stain was used to detect collagen in tissue sections and then examined under light microscope.

Including fructose (10%) in the daily drinking water for 12 weeks led to a significant insulin resistance indicated by the elevated serum insulin level (P < 0.001) compared to control group with no significant change in serum glucose level. Oral administration of CAPE (30 mg kg 1 day 1) in the last 6 weeks prevented (P < 0.001) the developed hyperinsulinemia. The IR animals showed a significant elevation in AGE level (P < 0.05). However, CAPE did not affect AGEs level. The IR animals had higher serum level of circulating TNF-a (P < 0.001) compared to control while CAPE administration, normalized the circulating level of serum TNF-a (P < 0.001, Table 2). Oral administration of CAPE (30 mg kg 1 day 1) in the last 6 weeks to normal animals did not affect insulin level, blood glucose level, circulating AGEs or TNF-a levels compared with control group (Tables 1 and 2).

2.8. Drugs and chemicals

3.2. Blood pressure

The following drugs and chemicals were used: STZ, PE (Sigma– Aldrich, Cairo, Egypt) and CAPE (Xi’an App-Chem Bio (Tech), Xi’an, China). STZ was dissolved in pre-chilled distilled water and injected immediately (prepared for every three rats a time). CAPE was prepared fresh every day by dissolving in distilled water. ACh and PE were dissolved in Krebs Henslite buffer. All other chemicals were supplied from El-Nasr. Co, Egypt and they were of analytical grade.

ID induced by STZ led to significant elevations in systolic, diastolic BP and the pulse BP (all at P < 0.001) compared to control group. CAPE administration alleviated the observed elevation in systolic and diastolic BP (both at P < 0.001) and significantly reduced the pulse BP (at P < 0.01, Fig. 1). Similarly, fructose -induced IR led to significant elevations in both systolic and diastolic BP (both at P < 0.001) and significant elevation in pulse BP (at P < 0.05) compared to control group. CAPE administration alleviated the observed elevation in systolic and diastolic BP (both at P < 0.001, Fig. 2). CAPE administration to normal animals did not significantly affect systolic BP, diastolic BP or pulse BP compared with control group (Figs. 1 and 2).

2.7. Collagen deposition

2.9. Statistical analysis Values are expressed as mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by Newman– Keuls’ post hoc test. For the concentration–response curves, the maximum response (Emax) was calculated by non-linear regression analysis of individual curves using computer based fitting program (Prism 5, Graph pad, CA, USA).

3.3. Vascular reactivity Fig. 3 shows that ID induced by STZ led to aorta hyperresponsiveness to KCl and PE, reflected by the significant increase in Emax (both at P < 0.001). CAPE administration alleviated the exaggerated response of isolated aorta to KCl (P < 0.001) and to PE (P < 0.01, Fig. 3). Similarly, fructose-induced IR increased aorta responsiveness to KCl and PE, reflected by the significant increase Emax (P < 0.001, P < 0.01 respectively). Also, CAPE administration alleviated the exaggerated response of isolated aorta to KCl and PE (P < 0.001 and P < 0.05 respectively, Fig. 4). CAPE administration to normal animals did not significantly affect aorta responsiveness to PE or KCl compared with control group (Figs. 3 and 4).

3. Results 3.1. Biochemical parameters As shown in Table 1, single injection of STZ in a dose of 50 mg/kg led to a sustained elevation in blood glucose level (P < 0.001) for 8 weeks compared to control. Oral administration of CAPE (30 mg kg 1 day 1) in the last 6 weeks to ID animals did not have any significant effect on the developed hyperglycemia. The ID animals had a significantly higher level of serum AGEs (P < 0.05) compared to control. CAPE administration didn’t affect AGEs level as well. On the other hand, ID animals had significantly higher serum level of TNF-a (P < 0.001) compared to control. CAPE administration inhibited the elevations in serum level of circulating TNF-a (P < 0.05).

3.4. Haem oxygenase-1 immunohistochemistry (IHC) Neither ID induced by STZ nor fructose-induced IR show any significant change in HO-1 expression from control group while, CAPE administration resulted in significant increase in HO-1 expression (P < 0.05) in both ID and Ir models (Figs. 5 and 6). CAPE

Table 1 Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on body weight, blood glucose level, serum advanced glycation end products (AGEs) and serum tumor necrosis factor alpha (TNFa) in STZ (50 mg kg 1, 8 weeks)-induced insulin deficient (ID) rats. Treatment

Body weight (g)

Glucose (mg dl

Control C-CAPE ID ID-CAPE

255.9 ± 8.8 259.0 ± 11.3 268.0 ± 16.6 215.8 ± 14.9

121.1 ± 2.7 105.7 ± 12.5 413.8 ± 54.8*** 442.3 ± 48.8

Values are expressed as the mean ± S.E of mean; N = 8 animals. * P < 0.05. *** P < 0.001, compared with the corresponding control group values. # P < 0.05.

1

)

1

AGE (fluorescent unit)

TNFa (lg l

76.1 ± 6.6 69.5 ± 9.9 123.1 ± 11.9⁄ 118.3 ± 12.1

74.0 ± 4.4 75.4 ± 6.5 188.9 ± 16.4*** 151.9 ± 14.0#

)

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Table 2 Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on body weight, insulin level, serum advanced glycation end products (AGEs) and serum tumor necrosis factor alpha (TNFa) in fructose (10% in drinking water, for 12 weeks)-induced insulin resistant (IR) rats. Treatment

Body weight (g)

Insulin (lg l

Control C-CAPE IR IR-CAPE

255.8 ± 7.2 251.7 ± 12.1 379.5 ± 21.3*** 357.7 ± 10.6

10.8 ± 0.4 7.9 ± 1.3 16.3 ± 1.0*** 4.2 ± 0.7###

1

)

1

AGE (fluorescent unit)

TNFa (lg l

69.9 ± 4.5 61.9 ± 8.2 122.0 ± 9.8* 125.8 ± 17.3

71.6 ± 5.0 72.5 ± 5.8 192.0 ± 8.5*** 122.1 ± 9.7###

)

Values are expressed as the mean ± S.E of mean; N = 8 animals. * P < 0.05. *** P < 0.001, compared with the corresponding control group values. ### P < 0.001 compared with the corresponding IR group values; by One Way ANOVA and Newman Keuls post hoc test.

Fig. 1. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on systolic (a), diastolic (b) and pulse (c) BP in STZ (50 mg kg 1, 8 weeks)induced insulin deficient (ID) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group values; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the corresponding ID group values; by One Way ANOVA and Newman Keuls post hoc test.

administration to normal animals did not affect HO-1 expression compared with control group (Figs. 5 and 6).

Fig. 2. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on systolic (a), diastolic (b) and pulse (c) BP in fructose (10% in drinking water, for 12 weeks)-induced insulin resistant (IR) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group values; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the corresponding IR group values; by One Way ANOVA and Newman Keuls post hoc test.

4. Discussion 3.5. Collagen deposition Both ID induced by STZ and fructose-induced IR was associated with a marked increase in collagen deposition as stained by the blue color. While CAPE administration resulted in clear reduction in collagen synthesis in both models. CAPE administration to normal animals did not show major change in collagen content compared with control animals (Figs. 7 and 8).

The present study is the first to report on the protective effect of caffeic acid phenethyl ester (CAPE) on atherosclerotic deleterious vascular effects of insulin deficiency and resistance in diabetes. CAPE administration virtually abolished the diabetes-induced atherosclerotic manifestations without affecting the developed hyperglycemia. These findings suggest CAPE as a possible protective natural ingredient against the toxic effect of diabetes on the vasculature.

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Fig. 3. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on the isolated aorta responsiveness to KCl (a) and PE (b) in STZ (50 mg kg 1, 8 weeks)-induced insulin deficient (ID) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group Emax values; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the corresponding ID group values; by One Way ANOVA and Newman Keuls post hoc test.

Six findings; each demonstrates CAPE counteraction of diabetes-induced atherosclerosis and will be discussed in the same sequence lately; (i) CAPE abrogated hypertension progression associated with diabetes, (ii) CAPE abolished the exaggerated contractile responses of aorta accompanied diabetes, (iii) CAPE ameliorated the elevation of TNF-a linked with diabetes, (iv) CAPE induced the expression of haem-oxygenase-1 enzyme in aorta sections and (v) CAPE reduced collagen synthesis compared with insulin deficient or resistant groups. Briefly, these findings established convincing evidence that CAPE offsets the atherosclerotic manifestations of diabetes via ameliorating significant vascular functional and structural derangements caused by diabetes in both insulin deficiency and insulin resistance models. Being a disorder of insulin deficiency and/or resistance with well distinguished characters, diabetes had to be well investigated into the two etiology patterns to see the effectiveness of CAPE. A single dose of STZ (50 mg kg 1, ip) established insulin deficiency through destruction of pancreatic b-cells [25] with persistent hyperglycemia within 2 weeks. On the other hand, insulin resistance model was confirmed by the developed hyperinsulinemia after 6 weeks of including fructose (10%) in the daily drinking water [26]. Remaining as one of the most important risk factors for atherosclerosis, hypertension per se was demonstrated to be associated with diabetes mellitus [27]. This study found that diabetes was associated with elevation in systolic blood pressure, diastolic blood pressure and pulse pressure in both ID and IR animals. This finding is in harmony with a previous study showing increase in both systolic and diastolic blood diabetes pressure in insulin deficiency and insulin resistance diabetes [22]. It was asserted previously by our lab that elevated systolic (afterload) BP in diabetic animals was

Fig. 4. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on the isolated aorta responsiveness to KCl (a) and PE (b) in fructose (10% in drinking water, for 12 weeks)-induced insulin resistant (IR) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group Emax values; #P < 0.05, ##P < 0.01, ### P < 0.001 compared with the corresponding IR group Emax values; by One Way ANOVA and Newman Keuls post hoc test.

Fig. 5. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on heme oxygenase-1 (HO-1) expression in STZ (50 mg kg 1, 8 weeks)induced insulin deficient (ID) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group values; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the corresponding ID group values; by One Way ANOVA and Newman Keuls post hoc test. Photos at the top are representative fluorescence images of aorta cross sections immunofluorescence stained by HO-1 antibody followed by Alexa FlourÒ conjugated secondary antibody.

related to cardiac complication and/or aortic stiffness while elevated diastolic (preload) BP may be imputed to the alteration in vascular reactivity [21]. Aortic stiffness was assigned to be responsible for augmented pressure in late systole [28]. Also as pulse

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Fig. 6. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on heme oxygenase-1 (HO-1) expression in fructose (10% in drinking water, for 12 weeks)-induced insulin resistant (IR) rats. Symbols indicate mean ± SEM for N = 6–8 animals; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001, compared with the corresponding control group values; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the corresponding IR group values; by One Way ANOVA and Newman Keuls post hoc test. Photos at the top are representative fluorescence images of aorta cross sections immunofluorescence stained by HO-1 antibody followed by Alexa FlourÒ conjugated secondary antibody.

pressure is associated with fibrinolysis as well as atherosclerotic progression in diabetic patients, it was more prominent in patients with diabetes than in non-diabetic people [29]. CAPE administration in a dose of 30 mg kg 1 day 1 abrogated the elevations in systolic and diastolic blood pressure in both insulin deficiency and insulin resistance models and caused a significant reduction of the pulse pressure in insulin deficiency model. Its vascular protective effect seems to be due to a direct effect as

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it did not affect glucose level at the used dose and duration. In addition, the inhibition of hyperinsulinemia development in IR model shares in this effect still without ignoring its direct protective effect. This finding is further supported by previous studies, wherein CAPE has been reported to have a protective effect on hypertension mediated cardiac impairment in cadmium treated rats [30]. In addition, in vitro incubation with caffeic acid phenethyl amide increased coronary flow rate in both normal and diabetic isolated rat hearts [31]. Owing to its important role in the development of atherosclerosis [32] and in particular on diabetes-evoked atherosclerosis [33], vascular reactivity impairment is focused on in this work. However, this does not rule out the endogenous substance(s) involved in blood pressure regulation. Both ID and IR animals’ isolated aorta showed increased responsiveness into KCl and PE. This observation is similar to the observation of other investigators that diabetes was associated with enhanced vasoconstriction to different vasoconstrictors [22,34]. On the other hand, CAPE inhibited the exaggerated contraction of aorta to KCl and PE in both ID and IR models. To investigate the mechanism of the protective effect of CAPE on aorta, we had to measure the expression of heme oxygenase1 (HO-1). We found that CAPE administration was associated with increased aortic expression of HO-1. This is in harmony with previous studies which reported the ability of CAPE to induce HO-1 enzyme expression [35,36] in human umbilical vein endothelial cells. The HO-1 induction also has been shown to be beneficial as it rapidly decrease the undesired pro-oxidant heme [37]. In addition, bilirubin which is a product of HO-1 is able to prevent oxidative injury in vascular smooth muscle cells [38]. Furthermore, carbon monoxide another product of HO-1) inhibits superoxide generation and thus the generation of various vasoconstrictive substances, ameliorating the development of atherosclerosis [39]. Expression of HO-1 prevents the development of atherosclerotic lesions [40], lowers blood pressure [41] and prevents vascular dysfunction

Fig. 7. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on aortic glycogen deposition in STZ (50 mg kg 1, 8 weeks)-induced insulin deficient (ID) rats. Micrographs are representative of cross sections from 6 rats stained with masson’s trichrome (X 1200). Collagen is blue; noncollagen proteins are pink and red. Sections are shown with the lumen on the bottom left side of the frames. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Effect of caffeic acid phenethyl ester (CAPE, 30 mg kg 1 day 1 for the last 6 weeks) on aortic glycogen deposition in fructose (10% in drinking water, for 12 weeks)induced insulin resistant (IR) rats. Micrographs are representative of cross sections from 6 rats stained with masson’s trichrome (X 1200). Collagen is blue; noncollagen proteins are pink and red. Sections are shown with the lumen on the bottom left side of the frames. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

[37] in a variety of experimental conditions. In brief, HO-1 enzyme was found to participate in the defense mechanisms against inflammatory molecules [42,43]. Therefore, it appears that the anti-hypertensive effect of CAPE may be mediated, at least in part, through up regulation of HO-1 enzyme. In diabetes, the relative deficit in the secretion and/or action of insulin increases AGEs formation [44]. AGEs and their intermediates have been found to contribute to vascular complications associated with diabetes [45] and are linked to the pathogenesis of atherosclerosis [46]. In this study both ID and IR models were associated with elevation in AGE. This is in accordance with what shown previously as an association there between elevations in AGEs and both ID [21] and IR models [47,48] of diabetes. Furthermore, AGEs was known to induce the expression of endothelin-1, a potent vasoconstrictor [49]. Treatment with CAPE didn’t reduce the level of the elevated AGEs in both insulin deficiency and insulin resistance models which can be explained by the persistent hyperglycemia observed in ID-CAPE in this study. Chronic hyperglycemia can lead to the formation of AGEs as well via non-enzymatic glycation reactions in patients with diabetic mellitus [50]. Recent studies, demonstrated that the interaction of AGE modified proteins in diabetes with different AGE–receptor complexes induces the synthesis and release of cytokines like TNF-a [49,51]. There is an accumulating evidence which indicates the key role of TNF-a in the progression of atherogenesis [52] as it was found to facilitate inflammation, a fundamental process in atherogenesis [27,53]. TNFa stimulation can result in the activation of a caspase cascade leading to apoptosis [54] and activation of nuclear factor kappa B [55,56] which is mediated through TNFa receptor activation [57]. Interestingly, TNF-a was found to contribute to the enhanced vascular constrictor response to PE in both in vitro [58] and in vivo [59]. In this study we observed that serum level of TNF-a was increased significantly in both ID and IR animals. This is in harmony with previous studies which found associations

between both IR [58] and ID [60] and increased TNF-a level. On the other hand, CAPE administration resulted in significant reduction of serum TNF-a level. This result therefore points to a possible role of TNF-a inhibition in the vasculoprotective effect of CAPE and it is in consistence with what previously reported that CAPE can reduce the circulating level of TNF-a [19,61]. In addition, CAPE administration decreased serum level of interleukin 1 beta, the important inflammatory cytokine, in diabetic mice [62]. Moreover, CAPE inhibited TNF-a induced endothelial Tissue Factor protein expression in human aortic endothelial cells [63]. The catechols in caffeic acid phenethyl ester seem to be responsible for TNF-a inhibition [64]. Collagen provides the basic functional features of the most vulnerable tissues including the vascular system. Collagen fibers are inextensible to provide mechanical strength allowing flexibility between various organs of the body [65]. In the vasculature, the collagen network determines the degree of stiffness of the vessel wall, which plays a vital role in mechanical properties of blood vessels [66]. The vascular collagen undergoes significant nonenzymatic glycosylation with time, which favors the atherosclerotic process, especially the accelerated diabetic atherogenesis [65,67,68]. Herein, the collagen deposition was clearly increased in the diabetic aortas compared to controls in both ID and IR models. This is harmonious with previous reports showing elevated levels of collagen in the diabetic aorta sections [69]. Mechanical stretch was found to activate collagen synthesis by whole artery segments and this may be the link between local hemodynamic forces and plaque collagen formation [70]. This process increases vessel stiffness [71]. On the other hand, treatment with CAPE resulted in marked reduction in collagen synthesis in aorta sections of both ID and IR animals. This result is in consistent with DeFronzo study in which CAPE administration prevented collagen deposition in esophageal wall following caustic esophageal burn [72]. In spite of lack of positive effect of CAPE on AGEs level in this study, CAPE revealed a beneficial ef-

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fect on collagen deposition in both ID and IR models. This beneficial effect of CAPE on collagen deposition in ID model can be clarified by its direct hypertensive effect thus reducing mechanical stretch which as previously mentioned stimulates collagen synthesis. While in case of IR model besides the antihypertensive effect of CAPE, the antihyperinsulinemic effect of CAPE may also contribute to preventing collagen deposition as insulin per sue was found to augment collagen synthesis in the vascular wall [73]. In conclusion, CAPE recoups the atherosclerotic manifestations of diabetes via ameliorating significant vascular functional and structural derangements in addition to its antihyperinsulinemic effect in the IR model.

[16]

[17]

[18]

[19]

[20]

Conflict of interest [21]

The authors have no conflict of interest to disclose. Acknowledgements This work is funded by a research Grant ID 1024 provided by the Science and Technology Development Fund, Egypt.

[22]

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