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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1283
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1130-1136
Copyright © 2005 by The Endocrine Society

Diabetes Induces p66shc Gene Expression in Human Peripheral Blood Mononuclear Cells: Relationship to Oxidative Stress

Elisa Pagnin, Gianpaolo Fadini, Renzo de Toni, Antonio Tiengo, Lorenzo Calò and Angelo Avogaro

Department of Clinical and Experimental Medicine, University of Padova School of Medicine, 35128 Padova, Italy

Address all correspondence and requests for reprints to: Dr. Angelo Avogaro, Cattedra di Malattie del Metabolismo, Università degli Studi di Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail: angelo.avogaro{at}unipd.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Oxidative stress plays a role in cardiovascular dysfunction. This is of interest in diabetes, a clinical condition characterized by oxidative stress and increased prevalence of cardiovascular disease. The role of p66shc in oxidative stress-related response has been demonstrated by resistance to and reduction of oxidative stress and prolonged lifespan in p66shc–/– mice. In this study we assess p66shc gene expression in peripheral blood mononuclear cells (PBM) from type 2 diabetic patients and healthy subjects. The p66shc mRNA level was assessed using RT-PCR with two sets of primers mapping for different p66shc regions. p66shc is expressed in both monocytes and lymphocytes. The level of p66shc mRNA was significantly higher in type 2 diabetic patients compared with controls (0.38 ± 0.07 densitometric units vs. 0.13 ± 0.08; P < 0.0001). In addition, total plasma 8-isoprostane levels, a marker of oxidative stress, were higher in type 2 diabetics (0.72 ± 0.04 ng/ml) than in normal subjects (0.43 ± 0.04, P < 0.001) and were significantly correlated to the p66shc mRNA level in PBM from type 2 diabetics (r2 = 0.47; P = 0.0284). In conclusion, diabetes induces p66shc gene expression in circulating PBM; this up-regulation in expression is significantly associated with markers of oxidative stress. p66shc gene expression in PBM may represent a useful tool to investigate the oxidative stress involved in the pathogenesis of long-term diabetic complications.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
INCREASED OXIDATIVE STRESS plays an important role in vascular dysfunction and atherogenesis. Both risk factors, such as hypercholesterolemia and hyperglycemia, and local factors, such as the activation of mononuclear cells, may contribute to increase oxidative stress (1). Increased production of reactive oxygen species (ROS) affects vascular reactivity and atherogenesis by modulating multiple signaling pathways and transcriptional events. The generation of ROS has been reported not only in diabetes mellitus (2), but also in normal subjects in response to a glucose challenge and has been attributed not only to elevation of plasma glucose, but also to increased free fatty acid concentrations (3, 4, 5, 6).

In the oxidative stress-mediated responses, a major role is played by growth factor-induced cell signaling. Among these intracellular pathways, Shc proteins, intracellular adaptors involved in the regulation of many cellular functions, seem to play a major role (7, 8). Three Shc genes are present in mammals: ShcA, ShcB, and ShcC. ShcA is expressed in various tissues, but not in brain or neurons, where the latest two isoforms are present (9, 10, 11). The mammalian ShcA adaptor protein has three isoforms of 46, 52, and 66 kDa (p46shc, p52shc, and p66shc). An alternative translation initiation site of the same mRNA causes the production of p46shc and p52shc, whereas p66shc transcription is due to an alternative promoter positioned in the first intron of the Shc locus (12).

It is well established that the stimulation of Shc proteins by different growth factors and cytokines causes phosphorylation on tyrosine residues (13, 14, 15, 16, 17), which, in turn, induces the formation of a stable complex with the adaptor protein Grb2 and the Ras guanine nucleotide exchange factor SOS (Son of Sevenless) (18, 19, 20, 21, 22). These events lead to the activation of the Ras/mitogen-activated protein kinase pathway when p46shc and p52shc are involved. On the contrary, activated p66shc is unable to affect mitogen-activated protein kinase activity, whereas it inhibits c-Fos promoter activation (23).

It has been reported that p66shc also become phosphorylated at Ser36 of the unique CH2 residue in response to H2O2 treatment (24, 25, 26), and phosphorylation of the unique Ser36 site seems to be crucial for oxidative stress response. Migliaccio and colleagues (27) have demonstrated an increased resistance to oxidative stress in p66shc knockout mice. Moreover, p66shc–/– cells show a reduced oxidative stress-induced apoptosis, which is restored by p66shc overexpression (27). More recently, it has been reported that p66shc is essential for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis (28).

No data are currently available on p66shc expression in human pathology, and its evaluation in clinical conditions with increased oxidative-related responses, such as diabetes, appears to be particularly relevant.

Therefore, the aims of this work were 1) to evaluate p66shc gene expression in peripheral blood mononuclear cells (PBM) from type 2 diabetic patients, compared with normal healthy subjects used as controls; and 2) to correlate the p66shc gene expression in PBM to clinical markers of oxidative stress, such as total plasma 8-isoprostanes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

We recruited for this study 10 patients with a diagnosis of type 2 diabetes who were free from clinical and instrumental evidence of atherosclerotic cardiovascular disease. Diabetic control was achieved with diet alone, diet plus sulfonylurea or biguanide preparations, or both. All pharmacological treatments were stopped at least 3 d before the study to render results independent upon therapy. All participants were asked to fast for at least 12 h before the examination. Ten healthy control volunteers, comparable for age, body mass index, and lifestyle, were recruited from the local community (Table 1Go). All participants underwent a full medical history and physical examination. Systolic and diastolic blood pressures were measured with sphygmomanometers after the patient had been seated for at least 5 min. A patient was defined as hypertensive if he/she had a systolic blood pressure of 140 mm Hg or greater or a diastolic blood pressure of 90 mm Hg or greater or used antihypertensive medication. Alcohol consumption was calculated in equivalent milliliters of wine, taking into account the daily consumption of wine, hard alcohol, liqueurs, and beer; subjects with a daily alcohol intake of more than 20 g/d were excluded. Smoking was also assessed as either actual smoking or previous smoking.


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  		TABLE 1. Clinical characteristics of the study cohorts

 
All subjects followed an isocaloric diet, recorded by a dietitian, with three meals daily (50% carbohydrate, 35% fat, and 15% protein) for at least 30 d before the study. The patients completed a lifestyle questionnaire to obtain information on medical histories, parental history of cardiovascular disease, smoking habits, and physical activity. Peripheral vascular disease was ruled out by both minimum criteria, such as the absence of peripheral pulses of the lower extremity (dorsal pedal, posterior tibial, popliteal, and femoral arteries), which were examined through manual palpation, and ankle-brachial pressure indexes. Atherosclerotic involvement was also excluded by Doppler ultrasound. A resting 12-lead electrocardiogram was performed, and angina was excluded in each patient according to the WHO Rose questionnaire. Patients with proliferative retinopathy or significant renal impairment were also excluded. Smoking and alcohol intakes were prohibited at least 24 h before the study.

The local ethical committee approved the study protocol, and informed written consent was obtained from the study subjects.

Experimental procedures

On the day of the study, at 0700 h after an overnight fast, the study subjects were admitted to the Division of Metabolic Diseases, University of Padova. A 20-gauge butterfly needle was inserted into a dorsal hand vein at 0730 h. Subjects had blood sampling (30 ml) performed for determination of circulating glucose, hemoglobin A1c, lipids, total plasma 8-isoprostanes, and p66shc gene expression in PBM. Diabetic patients were evaluated at their spontaneous fasting plasma glucose concentration.

Mononuclear cell preparation

PBM from 30 ml EDTA anticoagulated blood were isolated by Ficoll-Paque Plus gradient (Amersham Biosciences, Uppsala, Sweden). Differential recovery of lymphocytes and monocytes was obtained by adherence.

Cell culture

As a positive control of p66shc gene expression, human fibroblasts from healthy donors, in which p66shc is known to be expressed (8), obtained as previously described (29), were used. Briefly, human fibroblasts were derived from skin biopsy taken from the anterior surface of the left forearm by excision under topical anesthesia with ethyl chloride and were cultured in Ham’s Nutrient Mixture F-10 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 mmol/liter glutamine. Cells were seeded onto a 25-cm2 flask and incubated at 37 C, and medium was changed every 2–3 d.

RNA extraction

RNA from PBM and fibroblasts was extracted using a commercially available kit (RNABle, Eurobio, France), with l ml product/approximately 5 x 106 cells. The extracted RNA had an OD280/260 ratio between 1.8 and 2.0.

Deoxyribonuclease treatment

Five micrograms of extracted total RNA were incubated at 37 C for 15 min with RQ1 ribonuclease-free deoxyribonuclease (Promega Corp., Madison, WI) to exclude genomic contaminations. The mixture containing RNA and deoxyribonuclease was extracted with phenol/chlorophorm/isoamylic alcohol (25:24:1, vol/vol/vol) and centrifuged at 12,000 x g for 2 min. The RNA-containing supernatant was mixed with an equal amount of chlorophorm/isoamylic alcohol (24:1, vol/vol). The aqueous phase was precipitated with 0.5% (vol/vol) 3.25 M sodium acetate and 2.5% (vol/vol) 100% ethanol and stored at –80 C for 30 min. After centrifugation at 12,000 x g for 5 min, the pellet was washed with 70% ethanol, dissolved in diethylpyrocarbonate water, and spectrophotometrically quantified.

RT

RT of RNA was performed with the GeneAmp RNA PCR Kit, essentially as described by the manufacturer (PerkinElmer, Norwalk, CT). RNA (1 µg) was reverse transcribed using random hexamer primers and murine leukemia virus reverse transcriptase in a PerkinElmer 2400 thermocycler (15 min at 42 C, 5 min at 99 C, and 5 min at 5 C) as previously reported (30, 31, 32).

PCR

We assessed p66shc expression with two sets of primers, sets A and B, and consequentially used two different PCR protocols to clarify previous contrasting reports about p66shc expression in mononuclear cells (12).

The first set, set A (5'-CAGCCCCTCTTTCACCTCAA-3' and 5'-CATTCCGGAGTGGATTGTACTTG-3'), was designed with primer Express software (Applied Biosystems, Foster City, CA) mapping for a specific p66shc region in exon 2 (170–248 bp). The PCR mixture was 1.5 mM MgCl2, 0.5 µM of each primer, and 1.25 U/50 µl AmpliTaq Gold polymerase (Applied Biosystems), and the amplification protocol was 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec for 50 cycles of amplification.

The second set, set B (5'-CCACTACCCTGTGCTCCTTC-3' and 5'-CTGAGTCCGGGTGTTGAAGT-3'), was designed with Primer3 software (29) spanning from a specific p66shc region in exon 2 to exon 3 (358–752 bp). This set was also used to assess the relative expression of p66shc determined by semiquantitative PCR. The PCR mixture was 1.5 mM MgCl2, 0.8 µM of each primer, and 1.25 U/50 µl AmpliTaq Gold polymerase (Applied Biosystems), and the amplification protocol was 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec for 30 cycles of amplification. The number of cycles used for the p66shc amplifications in relative quantification experiments was obtained from the analysis of a kinetic curve set using an increasing number of cycles from 25–60, in the order of 5, to determine the number of cycles corresponding to the exponential phase. ß-Actin was used as the control gene.

The identities of p66shc PCR products were evaluated by insertion of amplicons in sequencing specific vectors (Invitrogen Life Technologies, Carlsbad, CA). After purification from 1% low melting agarose gel with a Qiaex II Gel Extraction Kit (Qiagen, Frankfurt, Germany), PCR products were inserted in pCR4-TOPO vectors, which were used to transform TOP10 Escherichia coli cells (Invitrogen Life Technologies), and colonies were selected according to the manufacturer’s instructions. Constructs and the identities of PCR products were evaluated using the PRISM Taq Polymerase Dye Terminator fluorescent sequencing kit (PerkinElmer) and were analyzed using an ABI 373 automated sequencer and ABI PRISM analysis software.

Evaluation of p66shc gene expression

p66shc gene expression was quantified using a PCR-based densitometric semiquantitative analysis with NIH image analyzer software, as previously reported (30). The ratio of p66shc to ß-actin PCR products, expressed as pixel density, was used as an index of p66shc gene expression (in densitometric units).

Determination of total plasma 8-isoprostane

Total 8-isoprostane evaluation was performed using a commercially available kit (Oxford Biomedical Research, Oxford, MI). Because 8-isoprostanes are present in plasma as both free acid and esters of lipoproteins, to assess total plasma 8-isoprostanes it was necessary to release the free acid from the lipoprotein-bound molecules by alkaline hydrolysis [15% (wt/vol) KOH for 60 min at 40 C], and proteins were then precipitated with absolute ethanol (0.01% butylated-hydroxy-toluene). After centrifugation, supernatants were purified using an 8-isoprostane affinity volume (Cayman Chemicals, Ann Arbor, MI). Total plasma 8-isoprostane levels were measured by enzyme immunoassay as described by the manufacturer (Oxford Biomedical Research). The sensitivity of the total plasma 8-isoprostane assay was 0.10 ng/ml, and inter- and intraassay coefficients of variation were 10% or less.

Statistical analysis

Data were evaluated on a Macintosh G4 computer (Apple Computer, Cupertino, CA) using the StatView II statistical package (BrainPower, Inc., Calabasas, CA). Data are expressed as the mean ± SD and were analyzed using the Mann-Whitney test for nonparametric analysis of unpaired data. The inclusion of 10 patients in each group, for an {alpha} value of 0.05 (two-tailed; a difference in densitometric unit of 0.12), has the power to detect a significant difference of 90%. Values at a 5% level or less (P < 0.05) were considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Monocyte and lymphocyte p66shc gene expression

Assessment of p66shc gene expression in PBM was obtained using two sets of primers. Using primer set A (see Subjects and Methods), p66shc PCR products were obtained in both monocytes and lymphocytes. Their identities were confirmed by insertion of amplicons in vectors, followed by sequencing. Deoxyribonuclease treatment excluded the amplification of a genomic contamination. Figure 1Go shows representative experiments of three performed.



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  		FIG. 1. A representative experiment of three performed of p66shc gene expression in monocytes and lymphocytes evaluated with primer set A (see Subjects and Methods). p66shc RT-PCR products were detected in both monocytes (lane 1) and lymphocytes (lane 2) of healthy subjects. Lane 3, p66shc negative with no cDNA control; lane 4, blank. Mwm, Molecular weight marker.

 
p66shc mRNA in PBM was also demonstrated using primer set B (see Subjects and Methods). This set of primers was able to amplify PCR products corresponding to the select p66shc mRNA region in both monocytes and lymphocytes (Fig. 2Go). The figure also shows p66shc PCR products obtained from cultured fibroblasts, which were used as a positive control.



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  		FIG. 2. p66shc gene expression in monocytes and lymphocytes evaluated with primer set B (see Subjects and Methods). p66shc RT-PCR products were detected in both monocytes (lane 1) and lymphocytes (lane 2) of healthy subjects. Fibroblasts were used as a positive control (lane 3). A representative experiment of three performed is shown.

 
p66shc gene expression in PBM from diabetic patients

Figure 3Go shows the pattern of p66shc gene expression in diabetic patients compared with controls. In PBM of diabetic patients, p66shc mRNA level is significantly higher (0.38 ± 0.07 vs. 0.13 ± 0.08 densitometric units; P < 0.0001).



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  		FIG. 3. Densitometric analysis of p66shc gene expression in PBM of type 2 diabetic patients and healthy controls. Gene expression of p66shc was assessed by RT-PCR, using primer set B, after analysis of a kinetic curve set using an increasing number of cycles from 25–60, in order of 5, to determine the number of cycles corresponding to the exponential phase, as reported in Subjects and Methods. Values were adjusted for expression of the housekeeping gene ß-actin and expressed as densitometric units (d.u.). *, P < 0.0001. Top, Representative polyacrylamide silver-stained gel of p66shc PCR products from two healthy controls (lanes 1 and 2) and two type 2 diabetic patients (lanes 3 and 4).

 
Total plasma 8-isoprostanes

Figure 4Go shows the single values of plasma 8-isoprostanes; their concentration was significantly higher in type 2 diabetic patients (0.72 ± 0.045 ng/ml; 95% confidence interval, 0.62–0.83) than in normal controls (0.43 ± 0.039; 95% confidence interval, 0.34–0.52; P < 0.001). Moreover, a significant correlation (Fig. 5Go) was observed between 8-isoprostanes and p66shc mRNA level in PBM (r2 = 0.47; P = 0.0284).



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  		FIG. 4. Single total plasma 8-isoprostane levels in both controls and type 2 diabetic patients.

 


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  		FIG. 5. Linear regression between total plasma 8-isoprostane levels and p66shc gene expression in diabetics (r2 = 0.47; P = 0.0284).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This is the first study to show that p66shc mRNA can be detected in PBM from both normal subjects and type 2 diabetic patients. We also show that the amount of p66shc mRNA was significantly higher in diabetic patients than in healthy subjects using RT-PCR.

Diabetes mellitus can be considered one of the most common oxidative stress-related diseases. Diabetic patients have higher plasma glucose levels than normal subjects, and it seems that the hyperglycemic damage is mediated by the excessive production of superoxide anion from the mitochondrial electron transport chain (33, 34). The causal relationship between diabetes and oxidative stress has been substantiated by Jain and colleagues (35), who found an increased peroxidation of membrane lipids and accumulation of malonyldialdeyde in erythrocytes of diabetic patients, possibly due either to the increased blood levels of ketone bodies during periods of poor metabolic control (36) or to the effect of increased levels of circulating cytokines, such as TNF-{alpha} and IL-6 (37).

We recently reported that p22phox gene expression, the key subunit of NAD(P)H oxidase for O2·– generation, is increased in mononuclear cells from type 2 diabetic patients (38). Our finding of increased gene expression of p66shc in mononuclear cells of type 2 diabetic patients provides additional insight into the mechanism resulting in the altered oxidative stress and oxidative stress-related responses, such as cardiovascular remodeling and atherogenesis in diabetes.

A pivotal role of p66shc in the oxidative stress-related response has been recently demonstrated. p66shc–/– mice demonstrate a major resistance to oxidative stress and a prolonged life span (27). p66shc–/– mice, compared with wild-type mice, also show a reduction in systemic oxidative stress as well as plasma low density lipoprotein oxidation and early atherogenic lesions, confirming p66shc as a potent inducer of oxidation-sensitive mechanisms. In addition, p66shc induction increases the intracellular ROS availability, which, in turn, affects the rate of oxidative damage. p66shc action on ROS is known to be a key step in appropriate p53-dependent apoptosis (28). Indeed, a dog’s ventricular pacing-induced cardiomyopathy, characterized by increased ROS production and mitochondrial dysfunction, induces a progressive p66shc overexpression that correlates with parameters of ventricular dysfunction, cytochrome c release and procaspases activation (39). In contrast, reduced p66shc and tolerance to oxidative stress were associated with the presence of increased Bcl-2, which protects against apoptosis, in a model of preconditioning stress induced by a transient ischemia (40). More recently, the deletion of p66shc has been demonstrated to be associated with reduced vascular cell apoptosis (41) and tissue damage (42), and to protect against age-dependent, ROS-mediated endothelial dysfunction (43).

It has been demonstrated that diabetes is associated with the up-regulation of endothelial cell genes such as intracellular adhesion molecule-1, TNF-{alpha}, IL-8, collagen, monocyte chemoattractant protein-1, ß2 integrin, and lectin-like oxidized low-density lipoprotein receptor-1 (44, 45, 46, 47). Nonetheless, monocytes play a major role in the earliest events of atherogenesis; evidence indicates that atherosclerotic lesion development is dramatically compromised if monocytes are prevented from entering the blood vessel (48). In this perspective, we have recently demonstrated in monocytes from diabetic patients an increased protein kinase C activation (49), which enhances their initial adhesion to the vascular wall and fibrinogen binding (50) and their differentiation into macrophages (51). This type of cells, therefore, is a useful tool to investigate the processes involved in diabetic complications, oxidative stress, and remodeling to gather proof of generalized phenomena that could be extended to vascular smooth muscle or endothelial cells involved in the cardiovascular remodeling, but not as easily accessible as circulating blood cells. In support of this hypothesis, we also assessed a clinical surrogate of oxidative stress, such as isoprostanes, in plasma from our subjects. We found that type 2 diabetic patients not only have higher levels of total plasma 8-isoprostanes, markers of oxidative stress, but these markers are significantly associated with higher p66shc gene expression in PBM, as depicted in Fig. 5Go. In contrast, we were not able to observe any correlation (data not shown) between p66shc gene expression and other markers of metabolic control, such as hemoglobin A1c and fructosamine. Unfortunately, we did not have the opportunity to assess a direct oxidative damage to proteins, amino acids, and DNA, as previously shown by Dandona and his group in patients with diabetes (52). However, our findings support the hypotheses that 1) during hyperglycemia in type 2 diabetes, there is an ongoing, free radical-mediated oxidative damage (53, 54, 55); and 2) monocytes from diabetic patients are exposed to higher levels of ROS (27), and this could lead to the increased expression of p66shc.

The results of this study show that p66shc is not only expressed at the mRNA level in human mononuclear cells, but also that its expression is higher in mononuclear cells from diabetic patients compared with those from healthy controls. The causative link for this increased p66shc gene expression in mononuclear cells from diabetic patients is unclear. p66shc expression, unlike p46shc and p52shc, which are ubiquitously expressed, is restricted to certain tissues and cell lines. The presence of two distinct promoters can explain the different expression patterns of these isoforms. Ventura and colleagues (12) recently proposed that stable chromatin changes, such as those imposed by DNA hypermethylation, might represent a permanent mechanism of silencing p66shc expression in tissues where apoptosis might be particularly harmful, suggesting that the p66shc gene is silenced through epigenetic modifications of the alternative promoter. Therefore, it could be possible that factors induced by diabetes, such as ROS and/or AGEs (advanced glycation endproducts), acting upstream, could alter the mechanism of silencing of p66shc expression, thus amplifying p66shc-mediated, oxidative stress-related responses. In contrast, it has also been proposed that p66shc is involved in the apoptosis of lymphoid cells, an event accompanied by marked up-regulation of p66shc expression. This could suggest that modifications of p66shc promoter methylation might also occur as an additional mechanism of p66shc regulation (12). Such a mechanism could, therefore, provide a rational explanation for the gene expression we found in mononuclear cells from normal subjects.

Oxidative stress can also influence cytokine levels, which are elevated especially in patients with type 2 diabetes (56); therefore, these compounds may potentially contribute to alter the p66shc gene expression in monocytes. Unfortunately, we did not include their determination in the present study; as far as we know, only one study shows that TNF{alpha} has the potential to increase p66shc phosphorylation (57).

Type 2 diabetic patients enrolled in our study also had associated hypertension and dyslipidemia. These conditions typically cluster in the metabolic syndrome, and it is widely established that associated metabolic alterations act synergically to increase the risk for cardiovascular diseases. We cannot rule out potential interfering effects of these covariables on our results, such as hypertension and hyperlipidemia. However, the only slightly higher blood pressure presented by our patients compared with controls and the lack of difference in the prevalence of hypertension between patients and controls make the role of hypertension in the increased induction of p66shc expression in our patients of low relevance compared to the role of diabetes. The same interpretation could be made for hyperlipidemia. Our patients have, in fact, only slightly higher cholesterol and triglyceridemia; the latter is also known to be mostly a secondary phenomenon in diabetes, making it unlikely that there is a significant influence of such lipid levels on the increased p66shc expression shown by our patients.

Finally, the impact of drugs such as thiazolidinediones, angiotensin-converting enzyme inhibitors, and statins, on p66shc expression has not been addressed by our study, because all therapy was stopped to avoid influences of therapy on the outcome of the study. Clearly, additional studies in this area would be of particular value to obtain direct information on the effect of drugs known to influence ROS generation and oxidative stress status (4, 58, 59) on p66shc expression.

In conclusion, we have shown using ex vivo PBM from diabetic patients and healthy controls that the p66shc mRNA level is significantly increased in type 2 diabetic patients. Our study also shows that, in type 2 diabetic patients, p66shc is positively correlated with total plasma 8-isoprostanes, a validated marker of oxidative stress. To our knowledge this is the first report of increased p66shc gene expression in diabetic patients. Given the critical role played by p66shc in the induction of oxidative stress-related responses, the demonstration of p66shc gene expression in PBM makes these cells a useful tool to investigate the processes involved in diabetic complications such as cardiovascular remodeling and atherogenesis.


   Footnotes
 
First Published Online November 23, 2004

Abbreviations: PBM, Peripheral blood mononuclear cell; ROS, reactive oxygen species.

Received July 9, 2004.

Accepted November 11, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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