Selective Rac-1 Inhibition Protects From Diabetes-Induced Vascular Injury
http://www.100md.com
《循环研究杂志》
the Department of Angio-Cardio-Neurology, IRCCS "NEUROMED" (C.V., A.A., G.M., U.B., R.P., A.M., M.T.G., A.N., L.I., G.L.), Pozzilli (IS)
the Department of Genetics, Biology
Biochemistry (M.S., E.H., G.T.), University of Turin
Biochemistry and Medical Biotechnology (L.P.), University of Naples
the Department of Experimental Medicine and Pathology (G.L.), "La Sapienza" University of Rome, Italy.
Abstract
Diabetes mellitus is a main risk factor for vascular diseases. Vascular injury induced by diabetes mellitus is characterized by endothelial dysfunction attributable to an increased oxidative stress. So far, the molecular mechanisms involved in the vasculotoxic effects of diabetes are only partially known. We examined the effect of diabetes mellitus on oxidative stress and Rac-1 activation, a small G-protein involved in the activation of NADPH oxidase. Our results show that oxidative stress in vessels of different murine models of diabetes mellitus and in endothelial cells treated with high glucose is associated with an increased Rac-1/PAK binding and Rac-1 translocation from cytosol to plasma membrane, thus demonstrating an enhanced Rac-1 activity. More important, selective Rac-1 inhibition by an adenoviral vector carrying a dominant negative mutant of Rac-1 protected from oxidative stress and vascular dysfunction induced by diabetes mellitus. Our study demonstrates that Rac-1 plays a crucial role in diabetes-induced vascular injury, and it could be a target of novel therapeutic approaches to reduce vascular risk in diabetes mellitus.
Key Words: Rac-1 free radicals diabetes mellitus vessels
Introduction
Diabetes mellitus represents one of the main risk factors for cardiovascular diseases. All forms of diabetes are characterized by chronic hyperglycemia, responsible for the development and progression of vascular complications leading to higher morbidity and mortality.1eC4
It is well known that hyperglycemia induces an increased production of reactive oxygen species (ROS), particularly on endothelial lining of the vessels.5eC9 This hyperglycemia-induced endothelial oxidative stress plays a crucial role in the development of vascular damage in diabetes mellitus, as demonstrated by the administration of antioxidant agents, which are able to rescue hyperglycemia-induced vascular dysfunction.10eC11 The molecular mechanisms activated by hyperglycemia, leading to endothelial oxidative stress and dysfunction, are not completely clarified.
Among the different sources of ROS reported to be activated by hyperglycemia, NADPH oxidase plays a main role. In fact, higher vascular NADPH oxidase activity has been detected in diabetic patients and, more important, endothelial NADPH oxidase activity is markedly increased by high glucose levels.6eC7,12 Thus, focusing on mechanisms leading to NADPH oxidase activation could unveil further molecular details involved in diabetes-induced vascular injury.
Rac-1, a small G protein, is an important signaling molecule integrating intracellular transduction pathways toward NADPH oxidase activation.13 Rac-1, like other small GTPases, needs lipid modifications, such as isoprenylation, to migrate from cytosol to plasma membrane where its attachment favors the assembly of the several subunits of the NADPH oxidase.14eC15 This assembly is a step necessary to activate the ROS-generating NADPH oxidase enzymatic system. Rac-1 activation, thus, could be a crucial mechanism for the hyperglycemia-evoked vascular oxidative stress.
So far, no data are available on Rac-1 activation in diabetes mellitus. Therefore, in this study, we first analyzed the impact of diabetes mellitus on Rac-1 activation, and, subsequently, we explored whether Rac-1 activation is crucial for the development of diabetes-induced vascular dysfunction.
Materials and Methods
Experimental Murine Models of Diabetes Mellitus
The main part of our study was performed in a model of diabetes mellitus obtained injecting a single high dose of streptozotocin (HDS: 200 mg/Kg, i.p, dissolved in sodium citrate buffer) in C57/BL6 mice16eC17 (Charles River, Wilmington, Mass) fed ad libitum and kept in standard cages under a 12-hour light/dark cycle. In this model we evaluated diverse physiologic parameters such as body weight, glycemia, hematocrit, and systolic/diastolic blood pressure by radiotelemetry. After 14 days from injection the animals were euthanized, and aorta and carotid arteries were excised and cleaned of periadventitial tissue for vascular reactivity and molecular analyses.
Further studies were performed in different models of diabetes mellitus where the onset of the disease is more gradual: mice treated with multiple low doses of streptozotocin18 (LDS: 50 mg/Kg per day for 5 days, i.p) and NOD (nonobese diabetic) mice, a genetic mouse model of diabetes mellitus.19 In these latter models our analysis was managed after 60 days from last streptozotocin injection in LDS mice and at 32 weeks of age in NOD mice. C57/BL6 mice were used as control of NOD mice and when treated with vehicle (sodium citrate buffer alone) as control of HDS and LDS. All the experiments were performed according to our institutional guidelines.
Evaluation of Vascular Reactivity
Vascular reactivity was evaluated in aorta, mesenteric, and carotid arteries as previously described.20eC21 Precontraction was elicited with phenylephrine (0.01 to 1 e蘭ol/L). Phenylephrine concentrations were adjusted to obtain a similar level of precontraction in each ring (80% of initial KCl-induced contraction). Caution was taken to avoid endothelium damage, and the functional integrity of this structure was reflected by the response to acetylcholine (10eC6 mol/L; Sigma). The maximal contraction evoked by phenylephrine was considered as the baseline for subsequent evoked vasorelaxations. Vasorelaxation was expressed as the percent reduction in contraction (the maximal vasorelaxation attained with papaverine being 100% vasorelaxation). Endothelial and smooth muscle function was tested by increasing concentrations of acetylcholine (10eC9 to 10eC5 mol/L) and nitroglycerin (10eC9 to 10eC6 mol/L), respectively. A further evaluation was assessed in presence of Tiron (10eC-3 mol/L), a superoxide scavenger.
Cell Culture
Human aortic endothelial cells (HAEC) were obtained from Clonetics. The cells were cultured with endothelial growth medium (EGM) as previously described.22 Twenty-four hours before the experiments, cells were serum deprived, and then the HAEC were exposed to low (5 mmol/L) or high (30 mmol/L) glucose for 6 hours.
Adenoviral Infection of Carotid Artery and Endothelial Cells
After the assessment of adenoviral titer by measuring plaques forming units on 293 cells (109 pfu/mL), carotid arteries of diabetic mice were infected as previously described.21,23 Carotids were placed in a Mulvany pressure system with DMEM/F12 medium, containing 250 e蘈 of AdN17 or Ad0 at concentration of 1x109 pfu/mL. Vessels were perfused at 100 mm Hg of pressure for 1 hour and, subsequently, at 60 mm Hg for 5 hours. The efficiency of infection was detected by GFP expression in all vascular tissues.
Subconfluent endothelial cells were incubated with adenoviral GFP vector containing a Rac1-dominant negative mutant (AdN17) or an empty adenoviral GFP vector (Ad0) in serum-free EGM (Biowhittaker) for 5 hours at 37°C. Afterward, the medium was removed, and the cells were incubated with complete EGM (10% FBS) for at least 8 hours until the experiment was performed. The efficiency of infection was 80% as detected by fluorescent GFP expression.
Evaluation of Rac-1 and p47phox Translocation to Plasma Membrane
Vascular tissue and endothelial cells were lysed and pottered in hypotonic lysis buffer (25 mmol/L Tris, 1 mmol EGTA and EDTA, in presence of proteinase inhibitors, 1 mmol/L PMSF, 1 e蘥/mL leupeptin, 1 e蘥/mL pepstatin A). The extract was sedimented by centrifugation (3000g for 10 minutes), and the membrane fraction was obtained by ultracentrifugation (100 000g for 1 hour). The pellet was defined as the membrane fraction and the supernatant as the cytosolic fraction and subjected to Western blot analysis for Rac-1 and p47phox using polyclonal antibodies (1:100 Santa Cruz). Moreover, Integrin -1 and GAPDH polyclonal antibodies (1:200 Santa Cruz) were used as a markers of membrane and cytosol fraction, respectively.
Evaluation of Oxidative Stress
Lucigenin Assay
Measurements were performed by using chemiluminescence assay and lucigenin (5 e蘭ol/L) as an enhancer in intact aorta and carotid rings, as previously described.22
Dihydroethidium Staining
Dihydroethidium (DHE) was used to evaluate oxidative stress in infected HAEC. Cells were stained with 10 e蘭ol/L DHE for 20 minutes and observed under a fluorescence microscope (Zeiss). Images were acquired by a digital camera system and analyzed by SPOT software.
Dichlorofluorescein Staining
Cells were loaded with 10 e蘭ol/L dihydrodichlorofluorescein (DCHF) diacetate for 15 minutes. Fluorescence images were obtained using a fluorescence microscope (ZEISS excitation 488 nm, emission 512 nm) and an imaging system. Changes in intensity were calculated from the mean intensities of 10 cells from each individual image.22
Evaluation of NADPH-Mediated O2eC Production
NADPH oxidase activity was measured in mouse vessels and in endothelial cells by luminescence assay using 5 e蘭ol/L lucigenin. Vessels were incubated in Krebs buffer and equilibrated for 30 minutes at 37°C. Vessels were homogenized in a buffer containing protease inhibitors (mmol/L: 20 monobasic potassium phosphate, 1 EGTA, 0.01 aprotinin, 0.01 leupeptin, 0.01 pepstatin, 0.5 phenylmethylsulfonyl fluoride, pH 7.0). Protein content was measured in an aliquot of the homogenate by Bradford method. The assay was performed in a 50 mmol/L phosphate buffer (pH 7.0) containing 1 mmol/L EGTA, 150 mmol/L sucrose, 5 e蘭ol/L lucigenin as the electron acceptor, and 100 e蘭ol/L NADPH as the substrate (final volume 2 mL). The reaction was started by the addition of NADPH to homogenate sample. Luminescence was measured every 30 seconds for 30 minutes in a scintillation counter. A buffer blank containing lucigenin and NADPH was subtracted from each reading and results were expressed as cpm/mg protein/minutes.21eC22,24
Evaluation of Nox 2 and Nox 4 Expression
HAEC total RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Total RNA (2 e蘥) from each sample was transcribed into complementary DNA using M-MLV Reverse Transcriptase (Invitrogen) and random hexamer primers (New England Biolabs) according to supplier’s instructions. Two microliters (10% of reverse transcription reaction) of each cDNA preparation were subsequently used as template for 25 e蘈 PCR containing 12.5 e蘈 TaqMan Universal Master Mix (Applied Biosystems), 0.25 e蘈 Nox4 TaqMan Gene Expression Assay (Hs00276431_m1, Applied Biosystems), and 0.25 e蘈 Eukaryotic 18S rRNA Endogenous Control (Applied Biosystems). Real-time PCR was performed using ABI Prism 7300 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C for 2 minutes; 95°C for 10 minutes; 40 cycles 95°C for 15 seconds; 60°C for 1 minute. Relative Nox2 and Nox4 gene expression levels were determined using Relative Quantification (ddCt) Study of 7300 System SDS Software (Applied Biosystems).
Evaluation of Nitric Oxide Production
Nitric oxide production was evaluated by reaction with diaminorhodamine-4 mol/L AM (DAR-4 mol/L AM), as previously described.25eC26 Briefly, endothelial cells were incubated with 10 e蘭ol/L DAR-4 mol/L AM for 30 minutes, and then stimulated with 1e蘭ol/L acetylcholine for 15 minutes. Supernatants were transferred to black microplates and the fluorescence was measured with a fluorescence microplate reader calibrated for excitation 560 nm, emission 575 nm.
Evaluation of Rac-1 Activity
Rac-1 Activity Assay
The activity of Rac-1 can be monitored by the interaction of Rac-1 with PAK, which is realized only when Rac-1 is active. Vessels were excised and incubated in Krebs buffer. Vessels and HAEC were lysed in a buffer containing NP-40. Lysates stimulated with GTPS were considered as a positive control. A PAK-GSH fusion protein bound to agarose beads was added and active Rac-1, binding PAK-GSH, was separated by repetitive centrifugation and washing. Then, the samples were boiled in Laemmli buffer and were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Biorad). The membrane was immunoblotted with antieCRac-1 antibody (Upstate Biotechnology). Rac-1 expression was quantified by Western blotting on flow-through after immunoprecipitation.22
Immunocytochemistry Detection of Rac-1 Translocation to Plasma Membrane
At the end of the incubation time cells were fixed in 4% paraformaldehyde. After blocking with 1% BSA, permeabilization with 0.01% Triton X-100, cells were incubated with antieCRac-1 antibody (1:250 BD Transduction) and as secondary antibodies anti-rabbit Cy3 rhodamine. Images were observed using fluorescence microscope (ZEISS).22
Statistical Analysis
Data are presented as mean±SEM. Statistical analysis was performed by 2-Way ANOVA followed by Bonferroni post hoc test. Differences were considered to be statistically significant at P<0.05.
Results
Diabetes Mellitus-Induced Vascular Oxidative Stress Is Associated to an Increased Rac-1 Activity
In HDS model of diabetes mellitus blood glucose levels started to be higher at 5th day after the injection (Figure 1C), and at sacrifice time the glycemic levels continued to be markedly elevated in HDS as compared with vehicle-treated mice (533±27 versus 94±7 mg/dL, n=94; P<0.01). The onset of diabetes mellitus was accompanied by a body weight loss (20±0.9 versus 24±0.5 g, n=5; P<0.05), whereas no change of systolic (117±4 versus 120±4 mm Hg, n=8; n.s.) and diastolic blood pressures (88±2 versus 89±3 mm Hg, n=8; n.s) and hematocrit (39±7 versus 42±12% n=8; n.s.) were observed between diabetic and control mice after 14 days of drug injection.
The analysis of diabetes impact on vascular function revealed an impaired endothelial vasorelaxation, as demonstrated by reduced response to acetylcholine in mice treated with streptozotocin, in both aorta (% max vasorelaxation: 51±2 versus 82±3%, n=15; P<0.01) and mesenteric arteries (% max vasorelaxation: 54±3 versus 80±4%, n=10; P<0.02). In contrast, smooth muscle relaxation evoked by nitroglycerine was unaffected by diabetes mellitus (% max aortic vasorelaxation: 84±3 versus 87±2%, n=14, n.s; % max mesenteric vasorelaxation: 81±2 versus 85±3%, n=11, n.s). Furthermore, aorta from streptozotocin-treated mice showed higher ROS production (Figure 1A), associated with a higher NADPH oxidase activity (Figure 1B), as compared with vehicle-treated mice. The oxidative stress was abolished by apocynin treatment, indicating that the increased ROS production in diabetes mellitus is dependent on increased NADPH oxidase activity. More important, diabetic mice showed an increased Rac-1/PAK binding, expression of an increased Rac-1 activity. Rac-1 activation started at the 5th day after streptozotocin treatment, when glycemic levels began to increase, and remained activated along the observation period (Figure 1C and 1D). Furthermore, in diabetic mice vascular Rac-1 was mainly localized on plasma membrane, whereas in vehicle-treated mice Rac-1 was in cytosolic fraction (Figure 1E). This different cellular fractioning induced by diabetes mellitus was similar to that observed for another subunit of NADPH oxidase, such as p47phox (Figure 1E). Altogether, our results demonstrate that diabetes evokes Rac-1 activation by inducing its translocation to plasma membrane.
These results obtained in HSD mice were further supported by those obtained in LSD, a further experimental model of diabetes mellitus, where streptozotocin was injected at low dose. Even in this case, diabetic mice showed an impaired acetylcholine-induced vasorelaxation (% max aortic vasorelaxation: 56±2 versus 86±3%, n=12, P<0.02), an increased superoxide anion (Figure 2A), and Rac-1/PAK binding (Figure 2B). Finally, similar results showing vascular endothelial dysfunction (% max aortic vasorelaxation: 63±1 versus 87±2%, n=10, P<0.02), increased oxidative stress (Figure 2A), and Rac-1 activation (Figure 2B) were also observed in diabetic NOD mice.
Selective Adenoviral Rac-1 Inhibition Protects From Diabetes-Induced Endothelial Dysfunction
To definitively clarify the role of Rac-1 in diabetes-induced vascular toxicity we selectively inhibited this GTPase using an adenoviral vector carrying a dominant negative Rac-1 mutant (AdN17). The efficiency of infection was >80%, as evaluated by the presence of coexpressed green fluorescent protein in vascular tissues (Figure 3A) and no morphological vascular changes were detected after infection. More important, carotid arteries infected with AdN17 were protected against NADPH oxidase activation (Figure 3B) and oxidative stress (lucigenin chemiluminescence: 8±5 versus 53±10 cpm/mg per min; P<0.01, n=10) induced by diabetes mellitus. In contrast, vessels infected with an empty adenovirus (Ad0) still showed enhanced NADPH oxidase activation (Figure 3B) and ROS production (lucigenin chemiluminescence: 57±6 versus 53±10 cpm/mg per min, P=n.s, n=8). Furthermore, AdN17, but not Ad0 infection, prevented the increase in Rac-1/PAK binding observed in diabetic vessels (Figure 3C), thus suggesting that the blunted oxidative stress observed in diabetic mice treated with AdN17 has to be ascribed to interference with Rac-1 signaling converging on NADPH oxidase activity. Infection with both AdN17 and Ad0 did not influence Rac-1/PAK binding in vehicle-treated mice.
Analysis of vascular function revealed that in vessels from HSD mice (Figure 3D) only infection with AdN17 was able to rescue acetylcholine-induced vasorelaxation, not influencing nitroglycerine relaxation (data not shown).
These data demonstrate that selective Rac-1 inhibition significantly improves endothelial function in diabetes mellitus. Interestingly, the administration of Tiron, a superoxide scavenger, induced in diabetic vessels, but not in vehicle-treated mice (data not shown), a significant improvement in acetylcholine vasorelaxation (% max vasorelaxation: 80±5 versus 59±3%; P<0.01, n=4), not altering nitroglycerine response. These results were comparable to that obtained with AdN17 treatment. Smooth muscle relaxation was unaffected by Tiron (data not shown). Finally, in vehicle-treated mice, acetylcholine-induced vasorelaxation was not affected by Ad0 (% max vasorelaxation: 82±5 versus 84±6%; P=n.s, n=8) and AdN17 (% max vasorelaxation: 83±7 versus 86±9%; P=n.s, n=8). The effect of Tiron was detectable also in diabetic vessels of LSD and NOD mice (data not shown).
High Glucose Induces Oxidative Stress and Rac-1 Activation in Endothelial Cells
High glucose plasma levels are the main feature of diabetes mellitus, so that a further challenge was to explore the effects of high glucose per se on endothelial cells, and to verify whether Rac-1 activity is crucial for ROS production.
High glucose levels were able to enhance ROS production (Figure 4A) as compared with cells exposed to low glucose levels. High glucoseeCinduced ROS generation was blunted by apocynin, thus suggesting that the increased oxidative stress induced by high glucose on endothelial cells is dependent on increased NADPH oxidase activity. This conclusion is further supported by the evidence that exposure to high glucose induced p47phox plasma membrane translocation and the expression of Nox4, the main endothelial isoform of gp91phox NADPH oxidase subunit27eC28 (Table). More important, in endothelial cells high glucose was able to activate Rac-1 as demonstrated by its translocation to plasma membrane (Figure 4B and 4C) and the formation of Rac-1/PAK complex (Figure 4D).
Interestingly, infection with AdN17, but not with Ad0, was able to prevent high glucoseeCinduced ROS production (Figure 5A), NADPH oxidase activation (Figure 5B), p47phox translocation to plasma membrane (Figure 5C), and impairment of nitric oxide production (Figure 5D).
Altogether our data reveal that high glucose induces ROS production through the Rac-1/NADPH oxidase pathway even in isolated endothelial cells.
Discussion
In the present study we demonstrate that Rac-1 activation is involved in the vascular damage induced by high glucose levels and, more important, that selective inhibition of Rac-1, obtained by an adenoviral vector carrying a dominant negative mutant of Rac-1, protects from vasculotoxic effects evoked by diabetes mellitus.
Vascular dysfunction represents the leading cause of increased mortality in diabetic population.1eC2 The main cause of vascular abnormalities in diabetes mellitus is the excess of oxidative stress on endothelium, which alters its function.8,29 The integrity of endothelium is necessary to maintain a balance between vasodilation and vasoconstriction, an adequate perfusion to various tissues and, more in general, protects the vessels from atherogenic insults. The excess of ROS scavenges vasoprotective mechanisms like nitric oxide and, inducing an alteration of local redox conditions, provokes vascular injury.
This study was focused on the importance of Rac-1 in diabetes-induced vascular dysfunction. Rac-1 has been reported to be adaptive or maladaptive in a diverse array of cardiovascular conditions by initiating redox-dependent signaling cascades.13eC14 This process requires translocation of Rac-1 from cytosol to plasma membrane, and the isoprenylation of Rac-1 is necessary for this trafficking.15 So far it is still unknown the role of Rac-1 in vascular dysfunction induced by diabetes mellitus. For this aim we used murine models of diabetes mellitus.
Firstly, we confirmed that diabetes mellitus induces vascular endothelial dysfunction through an increased ROS production. Such vascular abnormalities are dependent on the onset of diabetes mellitus because other pathological conditions commonly associated to endothelial dysfunction, such as arterial hypertension, are absent in the murine model of diabetes mellitus considered in our study. More important, in vessels of diabetic mice we observed an increased Rac-1 activation associated to enhanced NADPH oxidase enzymatic activity, suggesting that the sequence of molecular events leading to NADPH oxidase generation of ROS formation in diabetes mellitus involves Rac-1 activation. Importantly, the evidence that vascular oxidative stress and Rac-1 activation are commonly observed in different model of diabetes mellitus excludes that the pharmacological or genetic way handled to attain diabetic conditions could have played per se a mechanistic role in our results. Therefore, oxidative stresseCmediated vascular endothelial dysfunction and Rac-1 activation are direct consequence of diabetic hyperglycemic conditions.
Our next purpose was to inhibit selectively Rac-1 by a genetic approach and clarify its role in diabetes-induced vascular dysfunction. For this aim we infected vessels with an adenoviral vector carrying a dominant negative mutant of Rac-1. In these experimental conditions, in which Rac-1 is selectively impaired, our results demonstrate that high glucoseeCtreated diabetic vessels are protected from oxidative stress and vascular endothelial dysfunction. Thus, selective Rac-1 blockade rescues the vasculotoxic effects evoked by diabetes mellitus, and reveals that Rac-1 signaling plays a crucial role in hyperglycemia-evoked vascular damage.
A previous study reported a low expression of Rac-1 in enterocytes of diabetic rats.30 Although this result could seem conflicting with our data, a careful analysis reveals that the two studies are not comparable. First of all intracellular signaling varies significantly among cell types, and it is easy to imagine that the pathophysiological significance of Rac-1 expression in enterocytes could be very different to that observed in vascular endothelium, thus justifying different responses to diabetes mellitus. It is also noteworthy to emphasize that in our analysis we did better than expression analysis, because we measured Rac-1 protein activity, which analyzes with greater accuracy the real impact of diabetes mellitus on Rac-1 signaling pathway.
Recent observations indicate that high glucose levels hold the main responsibility for diabetes-induced endothelial dysfunction, because improved metabolic control in diabetic patients is associated with a dramatic amelioration of endothelial function.1 Thus, to characterize whether the effect of diabetes mellitus on Rac-1 activation could be reproduced by high glucose levels, we extended our analyses to endothelial cells. In this latter experimental system, we were able to recapitulate the hyperglycemia-induced oxidative stress with the consequent impairment of nitric oxide bioavailability. In endothelial cells exposed to high glucose we observed increased expression of Nox4 and translocation of p47phox plasma to membrane, which gives evidence of an involvement of NADPH oxidase in the increased oxidative stress even in the cellular system. More important, in endothelial cells high glucose induced Rac-1 translocation from cytoplasm to plasma membrane, a clear hallmark of Rac-1 activation. Thus, our observations reveal that diabetes mellitus and, more in particular, high glucose levels generate an enhanced endothelial oxidative stress by inducing Rac-1 activation. More important, even in endothelial cells the selective inhibition of Rac-1 prevents high glucose induced oxidative stress and rescued nitric oxide bioavailability.
So far a main role for high glucose activation of NADPH oxidase has been attributed to p47phox, because its selective inhibition by antisense is able to prevent high glucoseeCinduced ROS production.31eC32 Our data confirms that p47phox expression is increased in plasma membrane of diabetic vessels and endothelial cells exposed to high glucose. However, during Rac-1 inhibition, p47phox did not translocate to plasma membrane, and remained in the cytosolic fraction. Thus, the results of the present study reveal that Rac-1 intracellular signaling is a prerequisite for induction of ROS production, leading to the assembly of NADPH oxidase subunits within the plasma membrane. It should be noted that our results were obtained in human endothelial cells, allowing us to conclude that our observations are not confined to mice pathophysiology but can be extended to human pathology.
Previous studies have reported that hyperglycemia-induced vascular dysfunction has to be ascribed to the activation of PKC-dependent signaling, because its inhibition blunts oxidative stress and endothelial dysfunction in diabetes mellitus.33eC34 Recently, the II isoform of PKC has been identified as the molecular mechanism through which high glucose induces oxidative stress in endothelial cells. Interestingly, PKCII isoform has been linked to Rac-1, because the transfection of PKCII activates Rac-1 signaling.35 Therefore, these latter evidence coupled to our results suggest that high glucose could induce endothelial oxidative stress via a PKCII/Rac-1 signaling pathway.
In conclusion, the results of our study improve our knowledge about the molecular mechanisms of oxidative stresseCrelated damage in diabetes mellitus, demonstrating for the first time the involvement of Rac-1 in the vascular injury induced by diabetes mellitus. In the light of our results we can speculate that Rac-1 could be a target of more focused therapeutical approaches reducing vascular complications in diabetes mellitus. In particular, a selective inhibitor of Rac-1, blocking a key step in oxidative stress generation, could help to blunt hyperglycemia-induced vascular damage and reduce cardiovascular risk in diabetic patients.
Acknowledgments
This work was supported in part by grants from Italian Ministry of University and Research and Ministry of Health. We thank Tiziana Neri and Angela Pitisci for their technical support
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the Department of Genetics, Biology
Biochemistry (M.S., E.H., G.T.), University of Turin
Biochemistry and Medical Biotechnology (L.P.), University of Naples
the Department of Experimental Medicine and Pathology (G.L.), "La Sapienza" University of Rome, Italy.
Abstract
Diabetes mellitus is a main risk factor for vascular diseases. Vascular injury induced by diabetes mellitus is characterized by endothelial dysfunction attributable to an increased oxidative stress. So far, the molecular mechanisms involved in the vasculotoxic effects of diabetes are only partially known. We examined the effect of diabetes mellitus on oxidative stress and Rac-1 activation, a small G-protein involved in the activation of NADPH oxidase. Our results show that oxidative stress in vessels of different murine models of diabetes mellitus and in endothelial cells treated with high glucose is associated with an increased Rac-1/PAK binding and Rac-1 translocation from cytosol to plasma membrane, thus demonstrating an enhanced Rac-1 activity. More important, selective Rac-1 inhibition by an adenoviral vector carrying a dominant negative mutant of Rac-1 protected from oxidative stress and vascular dysfunction induced by diabetes mellitus. Our study demonstrates that Rac-1 plays a crucial role in diabetes-induced vascular injury, and it could be a target of novel therapeutic approaches to reduce vascular risk in diabetes mellitus.
Key Words: Rac-1 free radicals diabetes mellitus vessels
Introduction
Diabetes mellitus represents one of the main risk factors for cardiovascular diseases. All forms of diabetes are characterized by chronic hyperglycemia, responsible for the development and progression of vascular complications leading to higher morbidity and mortality.1eC4
It is well known that hyperglycemia induces an increased production of reactive oxygen species (ROS), particularly on endothelial lining of the vessels.5eC9 This hyperglycemia-induced endothelial oxidative stress plays a crucial role in the development of vascular damage in diabetes mellitus, as demonstrated by the administration of antioxidant agents, which are able to rescue hyperglycemia-induced vascular dysfunction.10eC11 The molecular mechanisms activated by hyperglycemia, leading to endothelial oxidative stress and dysfunction, are not completely clarified.
Among the different sources of ROS reported to be activated by hyperglycemia, NADPH oxidase plays a main role. In fact, higher vascular NADPH oxidase activity has been detected in diabetic patients and, more important, endothelial NADPH oxidase activity is markedly increased by high glucose levels.6eC7,12 Thus, focusing on mechanisms leading to NADPH oxidase activation could unveil further molecular details involved in diabetes-induced vascular injury.
Rac-1, a small G protein, is an important signaling molecule integrating intracellular transduction pathways toward NADPH oxidase activation.13 Rac-1, like other small GTPases, needs lipid modifications, such as isoprenylation, to migrate from cytosol to plasma membrane where its attachment favors the assembly of the several subunits of the NADPH oxidase.14eC15 This assembly is a step necessary to activate the ROS-generating NADPH oxidase enzymatic system. Rac-1 activation, thus, could be a crucial mechanism for the hyperglycemia-evoked vascular oxidative stress.
So far, no data are available on Rac-1 activation in diabetes mellitus. Therefore, in this study, we first analyzed the impact of diabetes mellitus on Rac-1 activation, and, subsequently, we explored whether Rac-1 activation is crucial for the development of diabetes-induced vascular dysfunction.
Materials and Methods
Experimental Murine Models of Diabetes Mellitus
The main part of our study was performed in a model of diabetes mellitus obtained injecting a single high dose of streptozotocin (HDS: 200 mg/Kg, i.p, dissolved in sodium citrate buffer) in C57/BL6 mice16eC17 (Charles River, Wilmington, Mass) fed ad libitum and kept in standard cages under a 12-hour light/dark cycle. In this model we evaluated diverse physiologic parameters such as body weight, glycemia, hematocrit, and systolic/diastolic blood pressure by radiotelemetry. After 14 days from injection the animals were euthanized, and aorta and carotid arteries were excised and cleaned of periadventitial tissue for vascular reactivity and molecular analyses.
Further studies were performed in different models of diabetes mellitus where the onset of the disease is more gradual: mice treated with multiple low doses of streptozotocin18 (LDS: 50 mg/Kg per day for 5 days, i.p) and NOD (nonobese diabetic) mice, a genetic mouse model of diabetes mellitus.19 In these latter models our analysis was managed after 60 days from last streptozotocin injection in LDS mice and at 32 weeks of age in NOD mice. C57/BL6 mice were used as control of NOD mice and when treated with vehicle (sodium citrate buffer alone) as control of HDS and LDS. All the experiments were performed according to our institutional guidelines.
Evaluation of Vascular Reactivity
Vascular reactivity was evaluated in aorta, mesenteric, and carotid arteries as previously described.20eC21 Precontraction was elicited with phenylephrine (0.01 to 1 e蘭ol/L). Phenylephrine concentrations were adjusted to obtain a similar level of precontraction in each ring (80% of initial KCl-induced contraction). Caution was taken to avoid endothelium damage, and the functional integrity of this structure was reflected by the response to acetylcholine (10eC6 mol/L; Sigma). The maximal contraction evoked by phenylephrine was considered as the baseline for subsequent evoked vasorelaxations. Vasorelaxation was expressed as the percent reduction in contraction (the maximal vasorelaxation attained with papaverine being 100% vasorelaxation). Endothelial and smooth muscle function was tested by increasing concentrations of acetylcholine (10eC9 to 10eC5 mol/L) and nitroglycerin (10eC9 to 10eC6 mol/L), respectively. A further evaluation was assessed in presence of Tiron (10eC-3 mol/L), a superoxide scavenger.
Cell Culture
Human aortic endothelial cells (HAEC) were obtained from Clonetics. The cells were cultured with endothelial growth medium (EGM) as previously described.22 Twenty-four hours before the experiments, cells were serum deprived, and then the HAEC were exposed to low (5 mmol/L) or high (30 mmol/L) glucose for 6 hours.
Adenoviral Infection of Carotid Artery and Endothelial Cells
After the assessment of adenoviral titer by measuring plaques forming units on 293 cells (109 pfu/mL), carotid arteries of diabetic mice were infected as previously described.21,23 Carotids were placed in a Mulvany pressure system with DMEM/F12 medium, containing 250 e蘈 of AdN17 or Ad0 at concentration of 1x109 pfu/mL. Vessels were perfused at 100 mm Hg of pressure for 1 hour and, subsequently, at 60 mm Hg for 5 hours. The efficiency of infection was detected by GFP expression in all vascular tissues.
Subconfluent endothelial cells were incubated with adenoviral GFP vector containing a Rac1-dominant negative mutant (AdN17) or an empty adenoviral GFP vector (Ad0) in serum-free EGM (Biowhittaker) for 5 hours at 37°C. Afterward, the medium was removed, and the cells were incubated with complete EGM (10% FBS) for at least 8 hours until the experiment was performed. The efficiency of infection was 80% as detected by fluorescent GFP expression.
Evaluation of Rac-1 and p47phox Translocation to Plasma Membrane
Vascular tissue and endothelial cells were lysed and pottered in hypotonic lysis buffer (25 mmol/L Tris, 1 mmol EGTA and EDTA, in presence of proteinase inhibitors, 1 mmol/L PMSF, 1 e蘥/mL leupeptin, 1 e蘥/mL pepstatin A). The extract was sedimented by centrifugation (3000g for 10 minutes), and the membrane fraction was obtained by ultracentrifugation (100 000g for 1 hour). The pellet was defined as the membrane fraction and the supernatant as the cytosolic fraction and subjected to Western blot analysis for Rac-1 and p47phox using polyclonal antibodies (1:100 Santa Cruz). Moreover, Integrin -1 and GAPDH polyclonal antibodies (1:200 Santa Cruz) were used as a markers of membrane and cytosol fraction, respectively.
Evaluation of Oxidative Stress
Lucigenin Assay
Measurements were performed by using chemiluminescence assay and lucigenin (5 e蘭ol/L) as an enhancer in intact aorta and carotid rings, as previously described.22
Dihydroethidium Staining
Dihydroethidium (DHE) was used to evaluate oxidative stress in infected HAEC. Cells were stained with 10 e蘭ol/L DHE for 20 minutes and observed under a fluorescence microscope (Zeiss). Images were acquired by a digital camera system and analyzed by SPOT software.
Dichlorofluorescein Staining
Cells were loaded with 10 e蘭ol/L dihydrodichlorofluorescein (DCHF) diacetate for 15 minutes. Fluorescence images were obtained using a fluorescence microscope (ZEISS excitation 488 nm, emission 512 nm) and an imaging system. Changes in intensity were calculated from the mean intensities of 10 cells from each individual image.22
Evaluation of NADPH-Mediated O2eC Production
NADPH oxidase activity was measured in mouse vessels and in endothelial cells by luminescence assay using 5 e蘭ol/L lucigenin. Vessels were incubated in Krebs buffer and equilibrated for 30 minutes at 37°C. Vessels were homogenized in a buffer containing protease inhibitors (mmol/L: 20 monobasic potassium phosphate, 1 EGTA, 0.01 aprotinin, 0.01 leupeptin, 0.01 pepstatin, 0.5 phenylmethylsulfonyl fluoride, pH 7.0). Protein content was measured in an aliquot of the homogenate by Bradford method. The assay was performed in a 50 mmol/L phosphate buffer (pH 7.0) containing 1 mmol/L EGTA, 150 mmol/L sucrose, 5 e蘭ol/L lucigenin as the electron acceptor, and 100 e蘭ol/L NADPH as the substrate (final volume 2 mL). The reaction was started by the addition of NADPH to homogenate sample. Luminescence was measured every 30 seconds for 30 minutes in a scintillation counter. A buffer blank containing lucigenin and NADPH was subtracted from each reading and results were expressed as cpm/mg protein/minutes.21eC22,24
Evaluation of Nox 2 and Nox 4 Expression
HAEC total RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. Total RNA (2 e蘥) from each sample was transcribed into complementary DNA using M-MLV Reverse Transcriptase (Invitrogen) and random hexamer primers (New England Biolabs) according to supplier’s instructions. Two microliters (10% of reverse transcription reaction) of each cDNA preparation were subsequently used as template for 25 e蘈 PCR containing 12.5 e蘈 TaqMan Universal Master Mix (Applied Biosystems), 0.25 e蘈 Nox4 TaqMan Gene Expression Assay (Hs00276431_m1, Applied Biosystems), and 0.25 e蘈 Eukaryotic 18S rRNA Endogenous Control (Applied Biosystems). Real-time PCR was performed using ABI Prism 7300 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C for 2 minutes; 95°C for 10 minutes; 40 cycles 95°C for 15 seconds; 60°C for 1 minute. Relative Nox2 and Nox4 gene expression levels were determined using Relative Quantification (ddCt) Study of 7300 System SDS Software (Applied Biosystems).
Evaluation of Nitric Oxide Production
Nitric oxide production was evaluated by reaction with diaminorhodamine-4 mol/L AM (DAR-4 mol/L AM), as previously described.25eC26 Briefly, endothelial cells were incubated with 10 e蘭ol/L DAR-4 mol/L AM for 30 minutes, and then stimulated with 1e蘭ol/L acetylcholine for 15 minutes. Supernatants were transferred to black microplates and the fluorescence was measured with a fluorescence microplate reader calibrated for excitation 560 nm, emission 575 nm.
Evaluation of Rac-1 Activity
Rac-1 Activity Assay
The activity of Rac-1 can be monitored by the interaction of Rac-1 with PAK, which is realized only when Rac-1 is active. Vessels were excised and incubated in Krebs buffer. Vessels and HAEC were lysed in a buffer containing NP-40. Lysates stimulated with GTPS were considered as a positive control. A PAK-GSH fusion protein bound to agarose beads was added and active Rac-1, binding PAK-GSH, was separated by repetitive centrifugation and washing. Then, the samples were boiled in Laemmli buffer and were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Biorad). The membrane was immunoblotted with antieCRac-1 antibody (Upstate Biotechnology). Rac-1 expression was quantified by Western blotting on flow-through after immunoprecipitation.22
Immunocytochemistry Detection of Rac-1 Translocation to Plasma Membrane
At the end of the incubation time cells were fixed in 4% paraformaldehyde. After blocking with 1% BSA, permeabilization with 0.01% Triton X-100, cells were incubated with antieCRac-1 antibody (1:250 BD Transduction) and as secondary antibodies anti-rabbit Cy3 rhodamine. Images were observed using fluorescence microscope (ZEISS).22
Statistical Analysis
Data are presented as mean±SEM. Statistical analysis was performed by 2-Way ANOVA followed by Bonferroni post hoc test. Differences were considered to be statistically significant at P<0.05.
Results
Diabetes Mellitus-Induced Vascular Oxidative Stress Is Associated to an Increased Rac-1 Activity
In HDS model of diabetes mellitus blood glucose levels started to be higher at 5th day after the injection (Figure 1C), and at sacrifice time the glycemic levels continued to be markedly elevated in HDS as compared with vehicle-treated mice (533±27 versus 94±7 mg/dL, n=94; P<0.01). The onset of diabetes mellitus was accompanied by a body weight loss (20±0.9 versus 24±0.5 g, n=5; P<0.05), whereas no change of systolic (117±4 versus 120±4 mm Hg, n=8; n.s.) and diastolic blood pressures (88±2 versus 89±3 mm Hg, n=8; n.s) and hematocrit (39±7 versus 42±12% n=8; n.s.) were observed between diabetic and control mice after 14 days of drug injection.
The analysis of diabetes impact on vascular function revealed an impaired endothelial vasorelaxation, as demonstrated by reduced response to acetylcholine in mice treated with streptozotocin, in both aorta (% max vasorelaxation: 51±2 versus 82±3%, n=15; P<0.01) and mesenteric arteries (% max vasorelaxation: 54±3 versus 80±4%, n=10; P<0.02). In contrast, smooth muscle relaxation evoked by nitroglycerine was unaffected by diabetes mellitus (% max aortic vasorelaxation: 84±3 versus 87±2%, n=14, n.s; % max mesenteric vasorelaxation: 81±2 versus 85±3%, n=11, n.s). Furthermore, aorta from streptozotocin-treated mice showed higher ROS production (Figure 1A), associated with a higher NADPH oxidase activity (Figure 1B), as compared with vehicle-treated mice. The oxidative stress was abolished by apocynin treatment, indicating that the increased ROS production in diabetes mellitus is dependent on increased NADPH oxidase activity. More important, diabetic mice showed an increased Rac-1/PAK binding, expression of an increased Rac-1 activity. Rac-1 activation started at the 5th day after streptozotocin treatment, when glycemic levels began to increase, and remained activated along the observation period (Figure 1C and 1D). Furthermore, in diabetic mice vascular Rac-1 was mainly localized on plasma membrane, whereas in vehicle-treated mice Rac-1 was in cytosolic fraction (Figure 1E). This different cellular fractioning induced by diabetes mellitus was similar to that observed for another subunit of NADPH oxidase, such as p47phox (Figure 1E). Altogether, our results demonstrate that diabetes evokes Rac-1 activation by inducing its translocation to plasma membrane.
These results obtained in HSD mice were further supported by those obtained in LSD, a further experimental model of diabetes mellitus, where streptozotocin was injected at low dose. Even in this case, diabetic mice showed an impaired acetylcholine-induced vasorelaxation (% max aortic vasorelaxation: 56±2 versus 86±3%, n=12, P<0.02), an increased superoxide anion (Figure 2A), and Rac-1/PAK binding (Figure 2B). Finally, similar results showing vascular endothelial dysfunction (% max aortic vasorelaxation: 63±1 versus 87±2%, n=10, P<0.02), increased oxidative stress (Figure 2A), and Rac-1 activation (Figure 2B) were also observed in diabetic NOD mice.
Selective Adenoviral Rac-1 Inhibition Protects From Diabetes-Induced Endothelial Dysfunction
To definitively clarify the role of Rac-1 in diabetes-induced vascular toxicity we selectively inhibited this GTPase using an adenoviral vector carrying a dominant negative Rac-1 mutant (AdN17). The efficiency of infection was >80%, as evaluated by the presence of coexpressed green fluorescent protein in vascular tissues (Figure 3A) and no morphological vascular changes were detected after infection. More important, carotid arteries infected with AdN17 were protected against NADPH oxidase activation (Figure 3B) and oxidative stress (lucigenin chemiluminescence: 8±5 versus 53±10 cpm/mg per min; P<0.01, n=10) induced by diabetes mellitus. In contrast, vessels infected with an empty adenovirus (Ad0) still showed enhanced NADPH oxidase activation (Figure 3B) and ROS production (lucigenin chemiluminescence: 57±6 versus 53±10 cpm/mg per min, P=n.s, n=8). Furthermore, AdN17, but not Ad0 infection, prevented the increase in Rac-1/PAK binding observed in diabetic vessels (Figure 3C), thus suggesting that the blunted oxidative stress observed in diabetic mice treated with AdN17 has to be ascribed to interference with Rac-1 signaling converging on NADPH oxidase activity. Infection with both AdN17 and Ad0 did not influence Rac-1/PAK binding in vehicle-treated mice.
Analysis of vascular function revealed that in vessels from HSD mice (Figure 3D) only infection with AdN17 was able to rescue acetylcholine-induced vasorelaxation, not influencing nitroglycerine relaxation (data not shown).
These data demonstrate that selective Rac-1 inhibition significantly improves endothelial function in diabetes mellitus. Interestingly, the administration of Tiron, a superoxide scavenger, induced in diabetic vessels, but not in vehicle-treated mice (data not shown), a significant improvement in acetylcholine vasorelaxation (% max vasorelaxation: 80±5 versus 59±3%; P<0.01, n=4), not altering nitroglycerine response. These results were comparable to that obtained with AdN17 treatment. Smooth muscle relaxation was unaffected by Tiron (data not shown). Finally, in vehicle-treated mice, acetylcholine-induced vasorelaxation was not affected by Ad0 (% max vasorelaxation: 82±5 versus 84±6%; P=n.s, n=8) and AdN17 (% max vasorelaxation: 83±7 versus 86±9%; P=n.s, n=8). The effect of Tiron was detectable also in diabetic vessels of LSD and NOD mice (data not shown).
High Glucose Induces Oxidative Stress and Rac-1 Activation in Endothelial Cells
High glucose plasma levels are the main feature of diabetes mellitus, so that a further challenge was to explore the effects of high glucose per se on endothelial cells, and to verify whether Rac-1 activity is crucial for ROS production.
High glucose levels were able to enhance ROS production (Figure 4A) as compared with cells exposed to low glucose levels. High glucoseeCinduced ROS generation was blunted by apocynin, thus suggesting that the increased oxidative stress induced by high glucose on endothelial cells is dependent on increased NADPH oxidase activity. This conclusion is further supported by the evidence that exposure to high glucose induced p47phox plasma membrane translocation and the expression of Nox4, the main endothelial isoform of gp91phox NADPH oxidase subunit27eC28 (Table). More important, in endothelial cells high glucose was able to activate Rac-1 as demonstrated by its translocation to plasma membrane (Figure 4B and 4C) and the formation of Rac-1/PAK complex (Figure 4D).
Interestingly, infection with AdN17, but not with Ad0, was able to prevent high glucoseeCinduced ROS production (Figure 5A), NADPH oxidase activation (Figure 5B), p47phox translocation to plasma membrane (Figure 5C), and impairment of nitric oxide production (Figure 5D).
Altogether our data reveal that high glucose induces ROS production through the Rac-1/NADPH oxidase pathway even in isolated endothelial cells.
Discussion
In the present study we demonstrate that Rac-1 activation is involved in the vascular damage induced by high glucose levels and, more important, that selective inhibition of Rac-1, obtained by an adenoviral vector carrying a dominant negative mutant of Rac-1, protects from vasculotoxic effects evoked by diabetes mellitus.
Vascular dysfunction represents the leading cause of increased mortality in diabetic population.1eC2 The main cause of vascular abnormalities in diabetes mellitus is the excess of oxidative stress on endothelium, which alters its function.8,29 The integrity of endothelium is necessary to maintain a balance between vasodilation and vasoconstriction, an adequate perfusion to various tissues and, more in general, protects the vessels from atherogenic insults. The excess of ROS scavenges vasoprotective mechanisms like nitric oxide and, inducing an alteration of local redox conditions, provokes vascular injury.
This study was focused on the importance of Rac-1 in diabetes-induced vascular dysfunction. Rac-1 has been reported to be adaptive or maladaptive in a diverse array of cardiovascular conditions by initiating redox-dependent signaling cascades.13eC14 This process requires translocation of Rac-1 from cytosol to plasma membrane, and the isoprenylation of Rac-1 is necessary for this trafficking.15 So far it is still unknown the role of Rac-1 in vascular dysfunction induced by diabetes mellitus. For this aim we used murine models of diabetes mellitus.
Firstly, we confirmed that diabetes mellitus induces vascular endothelial dysfunction through an increased ROS production. Such vascular abnormalities are dependent on the onset of diabetes mellitus because other pathological conditions commonly associated to endothelial dysfunction, such as arterial hypertension, are absent in the murine model of diabetes mellitus considered in our study. More important, in vessels of diabetic mice we observed an increased Rac-1 activation associated to enhanced NADPH oxidase enzymatic activity, suggesting that the sequence of molecular events leading to NADPH oxidase generation of ROS formation in diabetes mellitus involves Rac-1 activation. Importantly, the evidence that vascular oxidative stress and Rac-1 activation are commonly observed in different model of diabetes mellitus excludes that the pharmacological or genetic way handled to attain diabetic conditions could have played per se a mechanistic role in our results. Therefore, oxidative stresseCmediated vascular endothelial dysfunction and Rac-1 activation are direct consequence of diabetic hyperglycemic conditions.
Our next purpose was to inhibit selectively Rac-1 by a genetic approach and clarify its role in diabetes-induced vascular dysfunction. For this aim we infected vessels with an adenoviral vector carrying a dominant negative mutant of Rac-1. In these experimental conditions, in which Rac-1 is selectively impaired, our results demonstrate that high glucoseeCtreated diabetic vessels are protected from oxidative stress and vascular endothelial dysfunction. Thus, selective Rac-1 blockade rescues the vasculotoxic effects evoked by diabetes mellitus, and reveals that Rac-1 signaling plays a crucial role in hyperglycemia-evoked vascular damage.
A previous study reported a low expression of Rac-1 in enterocytes of diabetic rats.30 Although this result could seem conflicting with our data, a careful analysis reveals that the two studies are not comparable. First of all intracellular signaling varies significantly among cell types, and it is easy to imagine that the pathophysiological significance of Rac-1 expression in enterocytes could be very different to that observed in vascular endothelium, thus justifying different responses to diabetes mellitus. It is also noteworthy to emphasize that in our analysis we did better than expression analysis, because we measured Rac-1 protein activity, which analyzes with greater accuracy the real impact of diabetes mellitus on Rac-1 signaling pathway.
Recent observations indicate that high glucose levels hold the main responsibility for diabetes-induced endothelial dysfunction, because improved metabolic control in diabetic patients is associated with a dramatic amelioration of endothelial function.1 Thus, to characterize whether the effect of diabetes mellitus on Rac-1 activation could be reproduced by high glucose levels, we extended our analyses to endothelial cells. In this latter experimental system, we were able to recapitulate the hyperglycemia-induced oxidative stress with the consequent impairment of nitric oxide bioavailability. In endothelial cells exposed to high glucose we observed increased expression of Nox4 and translocation of p47phox plasma to membrane, which gives evidence of an involvement of NADPH oxidase in the increased oxidative stress even in the cellular system. More important, in endothelial cells high glucose induced Rac-1 translocation from cytoplasm to plasma membrane, a clear hallmark of Rac-1 activation. Thus, our observations reveal that diabetes mellitus and, more in particular, high glucose levels generate an enhanced endothelial oxidative stress by inducing Rac-1 activation. More important, even in endothelial cells the selective inhibition of Rac-1 prevents high glucose induced oxidative stress and rescued nitric oxide bioavailability.
So far a main role for high glucose activation of NADPH oxidase has been attributed to p47phox, because its selective inhibition by antisense is able to prevent high glucoseeCinduced ROS production.31eC32 Our data confirms that p47phox expression is increased in plasma membrane of diabetic vessels and endothelial cells exposed to high glucose. However, during Rac-1 inhibition, p47phox did not translocate to plasma membrane, and remained in the cytosolic fraction. Thus, the results of the present study reveal that Rac-1 intracellular signaling is a prerequisite for induction of ROS production, leading to the assembly of NADPH oxidase subunits within the plasma membrane. It should be noted that our results were obtained in human endothelial cells, allowing us to conclude that our observations are not confined to mice pathophysiology but can be extended to human pathology.
Previous studies have reported that hyperglycemia-induced vascular dysfunction has to be ascribed to the activation of PKC-dependent signaling, because its inhibition blunts oxidative stress and endothelial dysfunction in diabetes mellitus.33eC34 Recently, the II isoform of PKC has been identified as the molecular mechanism through which high glucose induces oxidative stress in endothelial cells. Interestingly, PKCII isoform has been linked to Rac-1, because the transfection of PKCII activates Rac-1 signaling.35 Therefore, these latter evidence coupled to our results suggest that high glucose could induce endothelial oxidative stress via a PKCII/Rac-1 signaling pathway.
In conclusion, the results of our study improve our knowledge about the molecular mechanisms of oxidative stresseCrelated damage in diabetes mellitus, demonstrating for the first time the involvement of Rac-1 in the vascular injury induced by diabetes mellitus. In the light of our results we can speculate that Rac-1 could be a target of more focused therapeutical approaches reducing vascular complications in diabetes mellitus. In particular, a selective inhibitor of Rac-1, blocking a key step in oxidative stress generation, could help to blunt hyperglycemia-induced vascular damage and reduce cardiovascular risk in diabetic patients.
Acknowledgments
This work was supported in part by grants from Italian Ministry of University and Research and Ministry of Health. We thank Tiziana Neri and Angela Pitisci for their technical support
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