This study aims to investigate the effects of ramipril (RPL) on endothelial dysfunction associated with diabetes mellitus using cultured human aortic endothelial cells (HAECs) and a type 2 diabetic animal model. The effect of RPL on vasodilatory function in fat-fed, streptozotocin-treated rats was assessed. RPL treatment of 8 weeks alleviated insulin resistance and inhibited the decrease in endothelium-dependent vasodilation in diabetic rats. RPL treatment also reduced serum advanced glycation end products (AGE) concentration and rat aorta reactive oxygen species formation and increased aorta endothelium heme oxygenase-1 (HO-1) expression. Exposure of HAECs to high concentrations of glucose induced prolonged oxidative stress, apoptosis, and accumulation of AGEs. These effects were abolished by incubation of ramiprilat (RPT), the active metabolite of RPL. However, treatment of HAECs with STO-609, a CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase-β) inhibitor; compound C, an AMPK (AMP-activated protein kinase) inhibitor; and Zn(II)PPIX, a selective HO-1 inhibitor, blocked these beneficial effects of RPT. In addition, RPT increased nuclear factor erythroid 2–related factor-2 (Nrf-2) nuclear translocation and activation in a CaMKKβ/AMPK pathway–dependent manner, leading to increased expression of the Nrf-2–regulated antioxidant enzyme, HO-1. The inhibition of CaMKKβ or AMPK by pharmaceutical approach ablated RPT-induced HO-1 expression. Taken together, RPL ameliorates insulin resistance and endothelial dysfunction in diabetes via reducing oxidative stress. These effects are mediated by RPL activation of CaMKK-β, which in turn activates the AMPK-Nrf-2-HO-1 pathway for enhanced endothelial function.
The vascular endothelium is important in regulating blood flow and tissue homeostasis and protecting us against atherosclerosis and thrombosis (Forstermann and Munzel, 2006). Dysfunction of endothelial cells (ECs) promotes abnormal vascular growth, such as atherosclerosis, and has been considered as an initial trigger of the progression of atherosclerosis in patients with diabetes mellitus (Nakagami et al., 2005). Type 2 diabetes (T2DM) is associated with a two- to fourfold increased risk of both coronary heart disease and stroke as a result of its effects on both the macrovasculature and microvasculature (Fox, 2010). Hyperglycemia is an independent risk factor for the development of cardiovascular disease. Micro- and macrovascular disease can be traced back to hyperglycemia (Stratton et al., 2000; Yu and Lyons, 2005).
Diabetic animals exhibit accelerated disappearance of capillary endothelium (Kohner and Henkind, 1970), morphologic and functional alterations of ECs (Meraji et al., 1987), and weakening of intercellular junctions (Dolgov et al., 1982). In vitro, high glucose (HG) affects endothelial and other vascular cells at the cellular level (Lorenzi and Cagliero, 1991), delays EC replication (Ono et al., 1988), and causes excessive cell death (Lorenzi et al., 1985). Although many signaling intermediates cause HG-induced changes in cardiovascular cellular phenotype, such as proliferation, hypertrophy, and altered function, it is their role as mediators of oxidative stress and apoptosis that has received much attention in recent years. The precise mechanism(s) underlying HG-induced endothelial oxidative stress still needs to be elucidated.
Ramipril (RPL), a potent and competitive angiotensin-converting enzyme inhibitor (ACEI), is used to treat high blood pressure and congestive heart failure. It is metabolized to ramiprilat (RPT) in the liver and kidneys. Recent studies suggested that ACEIs decrease cardiovascular risk and have many beneficial effects on vascular function in patients with advanced atherosclerosis or arterial hypertension (Dagenais et al., 2006). Apart from their antihypertensive effect, ACEIs have anti-inflammatory properties, increase nitric oxide (NO) bioavailability in human vasculature, and decrease cardiovascular risk via interfering directly into the mechanisms of atherogenesis (Watanabe et al., 2005). Despite its increasing clinical use and potential importance, little is known of the underlying mechanism of its endothelium-protecting, anti-inflammatory, and antianthrogenic properties.
Oxidative stress is a powerful pathogenic mechanism in atherosclerosis (Stocker and Keaney, 2004) by inducing cellular injury, mitochondria dysfunction, and apoptosis (Madamanchi and Runge, 2007). Antioxidant heme oxygenase-1 (HO-1) represents the crucial antioxidant enzyme in vascular endothelium and exerts a protective effect against the presence of coronary artery disease (Holweg et al., 2004; Stocker and Perrella, 2006). Ishikawa et al. (2001a, 2001b) suggested that inducers of HO-1 diminish lesion size in Watanabe heritable hyperlipidemic rabbits and low-density lipoprotein receptor–deficient mice. Juan et al. (2001) showed that selective overexpression of HO-1 using adenovirus-mediated gene transfer of HO-1 reduces lesion size in apolipoprotein E–deficient mice. In addition, previous studies suggest that AMPK prevents oxidative stress associated with diabetes (Xie et al., 2008; Sasaki et al., 2009). RPL was reported to reduce albuminuria in diabetic rats fed a high protein diet by angiotensin-converting enzyme inhibition and may have important implications for the treatment of human diabetic nephropathy (O'Brien et al., 1989; Forbes et al., 2002). Furthermore, RPT protects against free radical injury in isolated working rat hearts (Pi and Chen, 1989). Despite extensive studies, the molecular mechanisms by which RPL exerts its antioxidant defensive and endothelial protecting function are still unclear. Therefore, in the present study, we have examined the protective effects of RPL in human ECs against HG-induced cellular oxidative stress. Our major focus is to determine whether RPL induces the expression of antioxidant enzyme HO-1 that can detoxify toxic free radicals, executes it protective effects through CaMKKβ/AMPK pathway, and alleviates endothelial dysfunction in type 2 diabetic rats. The results of the present study provide further evidence for a functional role of RPL in ECs in relation to the prevention of the cardiovascular complications of T2DM.
Materials and Methods
Human aortic ECs (HAEC) from healthy male adult subject were supplied by Cell Applications (San Diego, CA) and maintained in EBM-2 growth media supplemented with the EGM-2 bullet kit. Passages 2 and 6 were used for all experiments. Cells were cultured in growth media containing 5 mM glucose for the normal glycaemia condition or 25 mM glucose for the hyperglycemia condition (HG).
A rat model of T2DM in a nonobese and outbred rat strain was established, as described previously (Reed et al., 2000). In brief, male Sprague-Dawley rats (n = 40), 7 weeks old, were fed normal chow (12% of calories as fat) or high-fat diet (40% of calories as fat) for 2 weeks and were then injected with streptozotocin (STZ, 50 mg/kg i.v.). Fat-fed/STZ rats were not insulin-deficient compared with normal chow-fed rats, but had hyperglycemia and a somewhat higher insulin response to an oral glucose challenge. This T2DM model replicates the natural history and metabolic characteristics of the human syndrome and is suitable for pharmaceutical research. All animal studies were carried out in accordance with the guidelines for the care and use of laboratory animals established by Wuhan University College of Medicine; on the basis of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health Publication No. 85-23, revised 1996, Public Health Service Approved Animal Welfare Assurance Code A5645-01).
For studies of the effect of RPL on aortic reactive oxygen species (ROS) formation and phosphorylation of CaMKI and AMPK as well as HO-1 protein expression, animals were divided into four groups of eight rats: a control group, untreated diabetes group (T2DM), RPL-treated diabetes group (T2DM + RPL), and diabetes group treated with RPL and compound C (T2DM + RPL + compound C). The T2DM group received RPL (10 mg/kg per day in drinking water) for 8 weeks. Some rats were treated with compound C (0.2 mg/kg b.wt. per day delivered intraperitoneally).
The overnight-fasted rats were anesthetized with sodium pentobarbital (75 mg/kg i.p.). The thoracic aortas (from the diaphragm to the aortic arch) were removed and cleaned of blood and surrounding adipose tissues. The endothelium was denuded with a rubber scraper and immediately processed for HO-1 protein assays as well as CaMKI and AMPK phosphorylation level measurements. The vessels were either snap frozen and stored at −80°C or immediately processed for in vitro vascular reactivity studies.
Vascular Function Study.
The effects of the high-fat diet/STZ and RPT treatment on vascular reactivity were evaluated in vitro using aortic rings in organ chambers (PowerLab; ADInstruments, Colorado Springs, CO), as described previously (Christon et al., 2005; Bourgoin et al., 2013). Aortas isolated from T2DM, T2DM + RPL, T2DM + RPL + compound C, and T2DM + RPL + Zn(II)PPIX (aortas from T2DM + RPL treated with 5 μM Zn(II)PPIX for 2 hours) were mounted in organ bath in 5 ml of Kreb’s solution at 37°C and gassed with 95% O2 plus 5% CO2, under a tension of 2 g, for a 1-hour equilibration period. Contractile response was evoked by U46619. At the plateau of contraction, accumulative acetylcholine or sodium nitroprusside was added into the organ bath to induce the endothelium-dependent or -independent relaxation.
Measurement of ROS.
The generation of intracellular ROS was determined by analyzing the oxidation of the nonfluorescent 2′,7′-dihydrodichlorofluorescein diacetate (DCFH2-DA; Molecular Probes) to the fluorescent dichlorofluorescein (DCF), as described previously (Wang et al., 2008). DCF emission was recorded using a microplate reader Wallac 1420 VICTOR2 (PerkinElmer Life and Analytical Sciences, Waltham, MA). The confluent HAECs in 96-well plates were preincubated with 2′,7′-dihydrodichlorofluorescein diacetate (10 µM) for 30 minutes. Cells were washed three times in phosphate-buffered saline, followed by measurement of fluorescence intensity at 485-nm excitation and 538-nm emission spectra. The ROS formation in rat aorta was determined, as described previously (Korystov et al., 2009; Merry et al., 2010). In brief, the aorta was removed and rinsed with cold (4°C) 10 mM Hanks’-HEPES solution (pH 7.4). Residuary fat was carefully cleaned. The aorta from the aorta arc to the point of branching of kidney arteries was cut into seven 5-mm sections. Aortic sections were cut lengthwise, turned inside out with the endothelium outside, and attached to the tip of the plastic pipette. Aorta segments then were placed in glass flasks in 2.5 ml of Hanks’-HEPES solution, pH 7.4, and incubated for 30 minutes at 37°C with shaking for adaptation before the addition of DCFH2-DA. DCFH2-DA (20 μM) was added, and aorta segments were incubated for 20 minutes at 37°C with shaking. The DCFH2-DA containing solutions were removed after the completion of incubation, and aorta sections were rinsed twice with cold Hanks’-HEPES solution, followed by fluorescence measurement. These procedures were performed in dark conditions to avoid photo-oxidation. Data are presented as the fold increase in DCF fluorescence compared with that in unstimulated cells or aorta tissues.
Glutathione (GSH) and the disulfide dimer, GSH disulfide, were measured with the Glutathione Assay Kit II (Merck, NJ). Cell lysate was harvested, and a deproteination step was carried out using metaphosphoric acid and triethanolamine, according to the kit instructions.
Cells were cultured in 24-well plates to 90% confluence and incubated with according treatments. Cells were lysed and centrifuged at 200g for 10 minutes. Apoptosis was determined by using a Cell Death Detection ELISA Kit (Roche, Indianapolis, IN). This method is based on a quantitative sandwich enzyme immunoassay principle, using mouse monoclonal antibodies against DNA and histones released into the cytoplasm of apoptotic cells.
Advanced Glycation End Product Measurement.
Cells were cultured in 12-well plates. Advanced glycation end product (AGE) was measured by an enzyme-linked immunosorbent assay method using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO), as described by Araki et al. (1992). Purified human AGE was used to generate the standard curve.
HAEC lysates were incubated with SAMS (synthetic peptide substrate HMRSAMSGLHLVKRR) peptide and [γ-32P]ATP, and the catalytic activity of AMPK was determined by the incorporation of 32P into SAMS peptide, as described previously (Sun et al., 2006).
Immunoblotting was performed in thoracic aorta to detect HO-1, p-CaMKI, and p-AMPK, as previously described (Bourgoin et al., 2008). In brief, the aorta of each rat were individually homogenized in 5 volumes of homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM dithiothreitol, 1% Triton X-100, 0.1 mg/ml phenylmethylsulfonyl fluoride, 50 mM NaF, and a protease inhibitor cocktail (Sigma-Aldrich). Homogenates were then centrifuged (10,000g) for 15 minutes at 4°C. Protein concentrations were measured with the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL), using bovine serum albumin as the standard. Protein samples (50 μg) from aorta or whole-cell lysates of HAECs were separated on 6–12% Tris-glycine gels and transferred to nitrocellulose membranes. Membranes were then probed with antibodies, as indicated, followed by incubation with horseradish peroxidase–associated secondary antibodies before signals were visualized with the enhanced chemiluminescence detection system (Amersham Bioscience).
Immunocytochemical analysis of nuclear factor erythroid 2–related factor-2 (Nrf-2) was performed, as previously described (Kim et al., 2010). Briefly, human umbilical vein ECs (1 × 105 cells/600 ml in a four-well chamber slide) were fixed with 3% formaldehyde for 20 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and preblocked with phosphate-buffered saline containing 3% bovine serum albumin for at least 1 hour. The cells were incubated with anti–Nrf-2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight, followed by incubation with the secondary antibody conjugated with Alexa Fluor 568 (Invitrogen) for 2 hours. After incubation, the cells were mounted with mounting solution containing 1 μg/ml 4′,6′-diamidino-2-phenylindole. Images were obtained with a Nikon E-800 fluorescence microscope.
Hyperinsulinemic-Euglycemic Clamp Experiment.
The hyperinsulinemic-euglycemic clamp experiment for the determination of insulin sensitivity was performed, as described previously (Ai et al., 2005). In brief, food was withdrawn 12 hours before the experiment. After anesthesia with amobarbital sodium (25 mg/kg i.p.), the rats were cannulated in the jugular vein for infusion of glucose and insulin (dual cannula) and in the carotid artery for sampling. All cannulae were tunneled subcutaneously and encased in silastic tubing (0.08 cm) sutured to the skin. After infusion of glucose (10%) and insulin (1 IU/ml) through dual cannula with constant velocity, the blood glucose levels were measured. To keep the blood glucose in a relatively steady state, the rate of glucose infusion was continuously adjusted. Glucose injection rate (GIR) was measured under homeostasis six times during the experiment.
Short Insulin Tolerance Test.
Insulin sensitivity assay by short insulin tolerance test using capillary blood glucose was performed, as described previously (Ai et al., 2005). Rats were weighed and put into cages after fasting overnight. Blood glucose was detected six times after insulin (0.05 U/kg i.p.) using a blood glucose detector. Abscissa indicates time, and ordinate expresses nature logarithm of blood glucose. Regression coefficient (r) or slope was determined by linear regression, and KITT was calculated by multiplying r by 100. K value indicates insulin sensibility, with smaller K values for lower sensibilities.
Glucose and Insulin Measurements.
Plasma glucose concentrations were detected with the glucose oxidase method (Richterich et al., 1974), using a Beckman glucose analyzer (Beckman Instruments, Palo Alto, CA). Insulin levels were measured by radioimmunoassay with porcine insulin standards and polyethylene glycol for separation (Desbuquois and Aurbach, 1971).
All data are given as the mean ± S.D.; n is the number of rats. Western analysis data are expressed relative to the control, assigning a value of 1 to the control group baseline mean. Data were analyzed using Student's t test or two-way analysis of variance, as appropriate. A P value of <0.05 was taken as significant difference between data sets.
RPT Reduces ROS Generation, Oxidative Stress, and Apoptosis in Human ECs Exposed to HG.
Elevated glucose concentrations have profound effects on cell function. In this study, we demonstrated that, compared with 5 mM glucose, incubation of HAECs with 25 mM glucose (HG) for up to 3 days increased ROS formation in a time-dependent manner, as indicated by DCF fluorescence. RPT (0.3 and 0.4 μM) reduced ROS liberation in HAECs exposed to HG after 1 day of treatment and continued to reduce ROS over a period of 3 days (Fig. 1A). The ability of RPT to reduce ROS formation was concentration-dependent. The effect of 0.3 μM RPT on ROS generation was similar to 0.4 μM RPT. Thus, 0.3 μM RPT treatment of 24 hours was chosen for further study. RPT also reduced ROS production in human umbilical vein ECs exposed to HG for 1–3 days (data not shown). HAECs exposed to HG exhibited increased molar ratio of GSSG/GSH, which indicated increased oxidative stress in HAEC, and RPT treatment reduced GSSG/GSH ratio (GSSG = the oxidized form of GSH; Fig. 1B). The ability of 0.3 μM RPT to decrease the GSSG/GSH ratio in HAECs exposed to HG was similar to 500 U/ml polyethylene glycol-catalase, a positive control. Moreover, apoptosis in HAECs exposed to HG for 24 hours was inhibited by RPT (Fig. 1C). The AGE accumulation in vascular endothelium is an irreversible complication of HG and is associated with metabolic memory in diabetic patients (Ihnat et al., 2007). In the present study, we showed that RPT suppressed AGE accumulation in HAECs exposed to HG after 1 day of treatment (Fig. 1D).
RPT Increases AMPK Phosphorylation and Activity in ECs through CaMKKβ Activation.
Phosphorylation of residue Thr172 is important in inducing AMPK enzyme activity (Hardie, 2007, 2011). RPT could increase AMPK phosphorylation at Thr172 in HAECs exposed to normal glucose and HG conditions (Fig. 2A). In addition, STO-609, a CaMKKβ inhibitor, inhibited the effect of RPT on AMPK phosphorylation. The inhibitory effect of STO-609 on CaMKKβ was confirmed by detecting Thr177 phosphorylation levels of CaMKI, a downstream substrate of CaMKKβ. The increased AMPK phosphorylation induced by RPT coincided with an augmented AMPK activity, as assessed by SAMS peptide assay (Fig. 2B). Taken together, our results suggest that RPT activates AMPK via its upstream kinase CaMKKβ.
RPT Induces Nrf-2 Nuclear Translocation, HO-1 Expression, and Antioxidant Effect through CaMKKβ/AMPK Pathway in HAECs.
Next, we sought to elucidate the mechanisms underlying RPT-induced antioxidant effect. Confocal microscopic analysis demonstrated that RPT stimulated Nrf-2 nuclear translocation in HAECs (Fig. 3A). Nrf-2 nuclear translocation is a crucial step in the activation of the antioxidant response element (ARE) pathway. Additionally, inhibition of the CaMKKβ/AMPK pathway by STO-609 and compound C reduced the nuclear accumulation of Nrf-2 in RPT-treated HAECs exposed to HG (Fig. 3B), suggesting that CaMKKβ/AMPK activation is responsible for RPT-induced Nrf-2 nuclear translocation. Furthermore, RPT treatment promoted the expression of a key target of the ARE pathway, HO-1 (Fig. 3C), in HG-treated HAEC, and this effect was also blocked by STO-609 and compound C. Accordingly, we demonstrate that RPT significantly blocked HG-induced ROS formation, which was blocked by CaMKKβ/AMPK pathway and HO-1 inhibition (Fig. 3D). Together, RPT exerted its antioxidant effect through activating CaMKKβ/AMPK and subsequently inducing Nrf-2 nuclear translocation and antioxidant enzyme HO-1 expression.
RPL Decreases AGE Serum Concentration and Insulin Resistance in High-Fat Diet/STZ Type 2 Diabetic Rats.
RPL treatment of 8 weeks lowered serum AGE concentration in high-fat diet (HFD)/STZ type 2 diabetic rats (Fig. 4A). In addition, daily treatment with RPL prevented the increase elicited by the HFD diet in plasma insulin levels. A moderate but significant reduction in glucose plasma levels was noted in RPL-treated T2DM rats when compared with the nontreated T2DM rats (Fig. 4, B and C). These results of RPL were blocked by in vivo AMPK inhibition with compound C. Furthermore, our hyperinsulinemic-euglycemic clamp test demonstrated that the GIR for keeping homeostasis of blood glucose in HFD/STZ T2DM rats was decreased versus control (Fig. 4D), implying that HFD induced insulin resistance in rats. RPL treatment significantly restored GIR in T2DM rats. Moreover, the results of short insulin tolerance test using capillary blood glucose revealed that KITT decreased to 3.9 ± 1.1 in T2DM rats (P < 0.05, n = 8). RPL treatment significantly blocked this decrease in T2DM rats (Fig. 4E). Likewise, these results of RPL were ablated by in vivo AMPK inhibition with compound C. Taken together, our results suggest that RPL reduces insulin resistance in T2DM rats via AMPK activation-elicited antioxidant action.
RPL Enhances Endothelium-Dependent Vasorelaxation in T2DM Rat Aortas via AMPK Activation and HO-1 Induction.
Next, to further substantiate the effect of RPT on AMPK and HO-1 observed in cultured HAEC, we detected AMPK phosphorylation, HO-1 expression, and ROS formation in T2DM rat treated with RPL. HO-1, a Nrf2-regulated gene, encodes a crucial antioxidant enzyme and plays a critical role in the prevention of vascular inflammation and atherogenesis (Araujo et al., 2012). As indicated in Figure 5A, RPL treatment was found to promote CaMKKβ/AMPK activity and HO-1 expression seen in aortas isolated from T2DM rats. More importantly, RPL treatment during chronic ingestion significantly prevented the increases in H2O2 formation in vascular tissues from T2DM rats (Fig. 5B). These results of RPL were blocked by in vivo AMPK inhibition with compound C. Our results suggest that RPL shows a significant antioxidant effect in vivo.
To determine whether RPL treatment regulates the endothelial function in the T2DM rats, we investigated the vascular response to endothelium-dependent and -independent vasodilators. The endothelium-dependent relaxation induced by increasing concentrations of acetylcholine was significantly lower in aortic rings from T2DM rats than those from the control group (Fig. 5C). RPL treatment in T2DM rats prevented the reduction in the vasorelaxing responses to acetylcholine. These results of RPL were blocked by in vivo AMPK inhibition with compound C and ex vivo HO-1 inhibition with Zn(II)PPIX. The endothelium-independent vasodilation to sodium nitroprusside was slightly reduced in T2DM rats, but the RPT-treated T2DM rats showed no significant difference compared with those cotreated with compound C or Zn(II)PPIX (Fig. 5D). Our results suggest that RPL alleviates endothelial dysfunction (i.e., decreased endothelium-dependent vasorelaxation) in T2DM rats via AMPK activation and HO-1 induction.
The present study aimed to explore the effect of RPL treatment on HG-induced oxidative stress in vitro and hyperglycemia-induced endothelial dysfunction in an animal model of T2DM. The HFD/STZ type 2 diabetic rats were used as an animal model to study the effect of RPL because T2DM is the most common form of diabetes and these rats display hyperinsulinemia, hyperglycemia, insulin resistance, and hypertriglycemia (Reed et al., 2000). In this study, we demonstrate that several alterations induced by HFD/STZ treatment in rats can be ameliorated or prevented by RPL administration. First, a marked improvement in insulin sensitivity was observed in RPL-treated T2DM rats. Second, RPL treatment prevented the increases in plasma glucose and insulin levels, as well as the alterations in vascular responses to the endothelium-dependent vasodilator acetylcholine. These improvements were associated with higher levels of HO-1 and marked reductions in ROS formation in vascular endothelium. These results demonstrate that oxidative stress causing harmful effects on vascular endothelium could play a crucial role in the early vascular and metabolic changes in T2DM. Our results propose that an early intervention aiming to control oxidative stress might alleviate, or even prevent, the pathophysiological processes contributing to the development of chronic diseases.
Our studies suggest that RPL treatment has beneficial effects on endothelial dysfunction induced by HG. These results are consistent with previous studies by Becker et al. (1991) reporting that RPL preserves endothelial function in rabbits on a long-term antherogenic diet. We for the first time demonstrated that RPL exerted its beneficial effect via stimulating the CaMKKβ pathway, which phosphorylates AMPK. The activated AMPK induces Nrf-2 nuclear translocation and stimulates ARE pathways in ECs, leading to increase in expression of HO-1, an important antioxidant enzyme. RPL treatment of diabetic rats decreased serum accumulation of AGE and ROS formation in aortas, eventually resulting in alleviation of global insulin resistance and improvement of endothelial function in type 2 diabetic rats (Fig. 6).
HG is a known cause of liberation of ROS and reactive nitrogen species (Cai and Kang, 2001). Oxidative stress induced by ROS and reactive nitrogen species leads to faulty signal transduction and apoptosis of ECs, vascular smooth muscle cells, and cardiomyocytes. The role of these species in HG-mediated apoptotic cell death is relevant to the complications of diabetes, such as neuropathy, nephropathy, and cardiovascular disease. ECs regulate vascular homeostasis through generating paracrine factors that regulate vascular tone, inhibit platelet function, prevent adhesion of leukocytes, and limit proliferation of vascular smooth muscle. The most important weapon of ECs to fight vascular diseases is endothelial NO synthase, an enzyme that generates the vasoprotective molecule NO (NO·) (Forstermann et al., 1994). Vascular NO· dilates all types of blood vessels via stimulating soluble guanylyl cyclase and increasing cyclic guanosine monophosphate in vascular smooth muscle cells (Forstermann et al., 1994). Endothelial dysfunction characterized by enhanced inactivation or reduced synthesis of NO, alone or in combination, is seen in conjunction with risk factors for cardiovascular disease. Increased levels of ROS reduce bioactive NO through chemical inactivation, forming toxic peroxynitrite, which in turn can uncouple endothelial NO synthase to form a dysfunctional superoxide-generating enzyme that contributes further to oxidative stress, leading to impairment of endothelium-dependent vascular relaxation and apoptosis. Thus, the mechanisms underlying the ability of RPL to prevent HG-induced endothelial dysfunction could involve an inhibition in ROS production and an enhanced NO bioactivity, leading to an improvement in endothelium-dependent vascular responses.
It is imperative to develop a new generation of antioxidant-based therapies for treatment of cardiovascular complications of diabetes (Czernichow et al., 2006). The traditional antioxidants, including vitamin C and E, have been suggested to be disadvantageous due to inhibition of exercise-induced decreases in insulin resistance (Ristow et al., 2009). RPL represents a new generation of antioxidants that exert additional beneficial effects in addition to ROS-scavenging activity. In the present study, we demonstrate that RPL can activate the CaMKKβ/AMPK and ARE pathway in ECs, increasing expression of the antioxidant enzyme, HO-1. Barbagallo et al. (2013) suggest that HO-1 inducers can be used as a potential therapeutic strategy to protect the cardiovascular system against various stressors in several pathologic conditions. More importantly, AMPK pathways play a key role in development of insulin resistance and endothelial damage. Activation of AMPK by exercise or pharmacological inducers can improve insulin sensitivity and endothelial function by increasing glucose transport and oxidation, fatty acid oxidation, and subsequent decrease in lipid accumulation in nonadipose tissues (Wu et al., 2007). These additional merits make RPL an attractive candidate medication for future clinical trials in diabetic subjects. A potential limitation of the present study is the heavy reliance on pharmacological agents to dissect signaling pathways [e.g., compound C, STO-609, and Zn(II)PPIX], leaving open the possibility for off-target effects. However, at the concentrations used, the compounds [STO-609 (5 μM), compound C (10 μM), and Zn(II)PPIX (5 μM)] are likely to have acted relatively specifically (Zhou et al., 2007; Nowis et al., 2008; Reihill et al., 2011; Guo et al., 2014).
Oxidative stress, apoptosis, and endothelial dysfunction were reported in a variety of populations at risk for metabolic syndrome and cardiovascular disease and could play an important role in the pathophysiology of the vasculopathy associated with the disease (Tracy, 2003; Caballero, 2005; Galili et al., 2007; Rizvi, 2007). The present study, using a treatment with RPL in a T2DM animal model, suggests that an early intervention aiming to control one of these initiating factors, oxidative stress, may prevent the development of long-term and irreversible vascular and metabolic complications. In conclusion, RPL is worthy of further research as a drug that may be therapeutically valuable in the inhibition of diabetes-related cardiovascular complications.
Participated in research design: Tian, Ge, Wu, Yang, Liu.
Conducted experiments: Tian, Ge, Wu.
Performed data analysis: Yang.
Wrote or contributed to the writing of the manuscript: Tian, Liu.
- Received January 14, 2014.
- Accepted April 16, 2014.
Y.L. is the guarantor of this work, had full access to all the data, and takes full responsibility for the integrity of data and the accuracy of data analysis. S.T. and K.W. contributed equally to this work.
- angiotensin-converting enzyme inhibitor
- advanced glycation end product
- AMP-activated protein kinase
- antioxidant response element
- Ca2+/calmodulin-dependent protein kinase kinase
- 2′,7′-dihydrodichlorofluorescein diacetate
- endothelial cell
- glucose injection rate
- human aortic EC
- high glucose
- heme oxygenase-1
- nitric oxide
- nuclear factor erythroid 2–related factor-2
- reactive oxygen species
- synthetic peptide substrate HMRSAMSGLHLVKRR
- type 2 diabetes
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics