Chronic hyperglycemia characteristic of type diabetes 2 is responsible for the accelerated atherosclerosis and increased cardiovascular risk [1], it is established that cardiovascular complications are the major causes of diabetic patients morbidity and mortality worldwide [2,3]. Chronic hyperglycemia induces a modulation of the expression of many key proteins involved in glucose toxicity [4]. It induces a dysfunction of the intracellular signal transduction in modulating the activity of kinase C protein, generating oxygen reactive species, activating ERstress, generating advanced glycation end products, activating polyol pathway and hexoamines, increased release of proinflammatory cytokines as well as growth factors. All these alterations lead to endothelial dysfunction which may be regarded as a sign of vascular disease being a key factor in the development of atherosclerosis [1, 4,5].

Given our interest in cardiovascular complications risk factors and knowing that Psammonys obesus, is a choice model for diabetes and atherosclerosis, we found it interesting to analyze the effect of a long-term glucotoxicity in vivo by adding 30% sucrose during 11 months period of experimentation on aortic vascular cells of Psammomys obesus and analyzing the effect of glucotoxicity in vitro on fibroblast aortic subculture.

The long-term administration (11 months) of a highcarbohydrate diet caused both plasma and tissue disorders in Psammomys confirming its high sensitivity to caloric overload [14]. The elevation of body weight is associated with hyperinsulinemia that is lipogenic, the latter appeared in our animals after 3 months. Our results confirm the work of Aouichat et al. (2004) [7] in Psammomys subjected to a high carbohydrates diet, those of Shafrir. (2001) [15] and Ikeda et al. (2001) [16] in Psammomys subjected to a high-calorie diet.

In our study, we found a hyperglycemia from month 4 associated with hypertriglyceridemia, hypercholesterolemia and hyperinsulinemia characteristic of type 2 diabetes [7,15,16]. According to Stentz et al. (2005) [17] hyperglycemia is related to alterations in biochemical pathways responsible for micro-and macrovascular injuries related to endothelial dysfunction. Chronic hyperglycemia is associated with an increase of ROS by activating polyols pathways, hexoamines, activation of PKC family, formation of glycation end products that activate the transcription factor nuclear, NFKB [18]. According to Goel et al. (2007) [19], chronic hyperglycemia generates ROS such as superoxide ions via activation of PKC, which alters vascular permeability by decreasing the bioavailability of NO.  

Our results show hyperinsulinemia in animals subjected to sucrose, associated with hyperglycemia. According to Marquie et al. (1984) [14], Shafrir (2001) [15], Ikeda et al. (2001) [16], progression of diabetes in Psammomys subjected to high-calorie diet increased from hyperinsulinemia to hyperinsulinemia associated with hyperglycemia then to a deficiency of insulin or insulinopenia due to apoptosis and necrosis of β cells. Insulin increases oxidative stress by overproduction of superoxide radical and decreased vascular NO. It activates the transcription factor NF_kB by activating the PI₃K pathway and plays a role in the induction of proinflammatory gene expression and promotion of the inflammatory process in the vascular tissue, the initial event being the recruitment monocytes in lesions where MCP-1 plays a key role [20]. Shafrir’s work (2001) [15] showed an increase in PKCξ in normoglycemic Psammomys, a marker of pre-diabetes compared to Psammomys resistant that do not develop diabetes.  

Our results show the occurrence of dyslipidemia. Indeed, electrophoresis profile analysis of Psammomys plasma lipoproteins subjected to a high-carbohydrate diet showed a significant increase in LDL-VLDL at the end of the experiment, known as highly atherogenic lipopro-

teins since they carry esterified cholesterol to parietal cells [18]. Besides, we observed a decrease of HDL, which allows the efflux of cholesterol to the liver and are classified as cardioprotective molecules [21]. According to Shafrir (2001) [15], dyslipidemia may be responsible for insulin resistance via increased diacylglycerol (DAG), an intermediate product of esterification of fatty acids into triglycerides and the catabolism of the latter into fatty acids and glycerol. Hyperactivation and increased expression of PKC appear to be the consequence of hyperglycemia and the main cause of diabetic complications associated with hypertriglyceridemia [22] and hyperinsulinemia [23].  

According Navab et al. (2002) [24], HDL decreases atherosclerosis by protecting LDL from oxidation. Epidemiological studies show a reverse correlation between the level of HDL-cholesterol and cardiovascular risk [25]. However, chronic hyperglycemia can induce glycation of HDL and impair their protective functions with regard to peroxidation, impair cholesterol transport and increase oxidation threshold, thus enhancing the atherosclerotic process [25].

According to Oldegren et al. (2003) [26], elevation of CPK is an indicator of cardiac complications in particular, marked by cellular necrosis. Our histological sections show SMCs and apoptotic or necrotic nuclei fibroblasts. In a state of necrosis, SMCs release enzymes and proteins in particular creatine phosphokinase into the blood stream. During a cardiac episode, increased CPK is systematic [27]. The latter remains a very sensitive marker since in myocardial infarction, its rate rises at the 4th hour, reaching 7 to 12 times its basic rate. Increased CPK reflecting cell lysis could be related to the increase of NO due to hyperglycemia and hyperlipidemia. NO induces apoptosis depending on cell type and concentration. At low doses, it is pro-apoptotique, whereas at higher concentrations, it induces necrosis [28].

In the intima, we observed a thickening of this layer, a hypertrophy of endothelial cells thus an alteration of their structure and functioning, a thickening of the subendothelial space presenting a fibrous material, indicating the formation of young plaques rich notably with collagen. These findings corroborate those of Orbe et al. (2003) [29] suggesting that chronic hyperglycemia induces endothelial dysfunction through reduced bioavailability of NO. Endothelial damage and ruptures of elastica interna are observed, which would increase intimal permeability allowing an increased passage of macromolecules into the intima thus contributing to its thickening [20].

In the media, we notice alterations characterized by thinning, loss of ondulation, disorganization and disruption of elastic laminae, reflecting vascular remodeling. These results are consistent with those of Lusis (2002) [30]. The elastolysis of the elastic plates and the the elastica interna may be due to the activation of several proteases. According to Lusis (2002) [30], degradation of elastin peptides may contribute to increased proteinemia, and to this end, our results showed that proteinemia in animals subjected to sucrose is significantly increased at the end of experimentation. Moreover, in regions close to the intima, we noted CML migration zones between interlamellar spaces within a completely disorganized matrix, extended interlamellar spaces with accumulations of collagen and probably lipid bodies. Hyperglycemia, by inducing endothelial dysfunction promotes the synthesis of adhesion molecule such as ICAM-1, VCAM-1, Eselectin and fibronectin, which facilitate the adhesion of monocytes to endothelium [30].

In the adventitia, we noticed a matrix rich in collagen and an important fibroblasts densification. These appear to undergo a phenotypic change associated with their migration to the media. Lesions of the vascular endothelium are responsible for the proliferation and dedifferentiation of fibroblasts ans CLM’s via activation of NADPH oxydase, for the release of ROS and that of the growth factors in particular PDGF, βFGF, IGF [31].  

Our results on the proliferation of adventitial fibroblasts schoe those of Aouichat et al. (2004) [7] on aortic SMCs of Psammomys in the presence of 15 mM DGlucose for 48h and 4 passages. According to Neumann et al. (2002) [32], Heo et al. (2008) [33], the proliferative effect of D-Glucose is not related to changes in osmolarity, but rather to the activation of MAPKinase via the pathway ERK1/2, which regulates the activity of Erg-1 and which plays a role in proliferation and dedifferentiation of vascular cells [34].

In addition, our morphometric results show a significant increase in the number of nucleoli. This increase is indicative of increased synthesis of cellular protein. Benazzoug, (1998) [35] noted little change in total protein synthesis of incubated human fibroblasts 15Mm Glc. Hoffman et al. (1998) [36] noted that hyperglycemia stimulates the synthesis of many growth factors including TGF-β in mice mesangial cells. According to Ryoo et al. (2004) [37], activation of PKC_θ after hyperglycemia activates the p38 pathway and the production of IL-8 via activation of AP-1 and the involvement of NFkB which increase DNA synthesis. Our morphologic resuls show an hypertrophy of fibroblasts submitted to D-Glucose. According to Grimm et al. (2002) [38], hyperglycemia induces a phenotypic change of SMC into hypertrophied phenotype. Liu et al. (2006) [39] have also observed a phenotypic change in cardiac myocyte into hypertrophied phenotype in the presence of D-Glucose, it is linked to increased ROS and would correspond to an adaptation phenomenon.

The hypercondensation of chromatin and nuclear fragmentations that we observed are signs of apoptosis. According to Babich et al. (1996) [40], ROS are responsible for hypercondensation of chromatin. ROS in particular superoxide ion () allows interaction with the NO⁻ and the genesis of peroxynitrite (ONOO-) a cause for damage to DNA. These are detected by the P53 tumor suppressor, to be overexpressed [41]. P53 is a molecular switch that detects DNA breaks and commits cells in the process of repair or apoptosis [41].

Cytoplasmic vacuolization that we observed likely to be linked to increased reactive oxygen species following glucotoxicity is the oncosis and it precedes apoptosis. Babich et al. (1996) [40] noted an important vacuolization characteristic of oncosis in fibroblasts exposed to ROS-producing agents. According to Babich and al. (2007) [42], induction of oncosis and apoptosis in fibroblasts treated with an agent that releases ROS is very important. Chen et al. (2000) [2] noted the increase in apoptosis in the myocardium of diabetic rats. Boumaza,(2011) [43] observed the oncosis and apoptosis in  aortic fibroblasts of cultured  Psammomys treated with hydrogen peroxide to 1.2 mM for 7d.  

The protein contained in the CEC shows an increase compared to corresponding controls. Our findings corroborate those of Chen et al. (2000) [2], who noted an increase in matrix proteins, Benazzoug et al. (1998) [35] noted an increase in total collagen in human cutaneous fibroblasts incubated in 15 Mm Glc for 48 h. Aouichat et al. (2004) [7] also noted an increase in total collagen in SMCs of Psammomys incubated in 15 mM Glc for 48 h and 4 passages. The Typing of collagen showed an increase and intensification of bands corresponding to the γ fragments representing the trimer chain α1 (coll I and III), the β fragments representing the dimeric chain α1 (coll I and III), α1 chains (coll I and III) and α2 chain (coll I). Chen et al. (2000) [2] noted an increase in α1 chain after diabetes onset, Grimm et al. (2001) [38] have shown that hyperglycemia increases the extracellular matrix remodeling of cardiomyocytes. Andreea et al. (2008) [44] showed an increase of procollagen α2 (I) and procollagen α1 (III) at D-Glc concentrations of  11mM and 22mM but decreased procollagen α1 (III) at 33 mM of D-Glc. Hyperglycemia activates PKCβ, involved in the increased expression of TGF betal which initiates the accumulation of ECM [45].

In our study we assessed the effect of a glucotoxicity on the production of MDA, a marker of lipid peroxidation in the intracellular compartments of Psammomys adventitial fibroblasts in culture. According to Hayek et al. (2007) [46], D-Glc induces, in macrophages, oxidative stress by activating NADPH oxidase, thus increasing the production of superoxide ion which increases lipid peroxidation, including LDL. According to Whiteside and Dulgasz, (2002) [45], hyperglycemia increases the genesis of ROS via activation of the polyol pathway and glycation, leading to lipid peroxidation [46]. Lipid peroxidation mediated by ROS, mainly targets the polyunsaturated fatty acids located at the cell membrane fluidity modifying and altering membrane permeability, which also affects the functions of membrane proteins, inactivation of receptors of which and activation of some proteases lead to cell damage [47].

Glucotoxicity induced on the adventitial fibroblasts in the presence of 0.6% D-Glc for 7 days induces an increase in production of total nitrite in the intracellular compartments. According to Stentz et al. (2005) [17], hyperglycemia induced on T cells, increased synthesis of proinflammatory cytokines, which are responsible for the increase of NO by activation of iNOS. Kolb et al. (2001) [41], iNOS, present in all cell types require transcriptional induction, particularly by cytokines; iNOS, once synthesized, smoothly binds to calmodulin and produces larger quantities of NO and within a longer period of time (hours to days) than constitutive NOS which synthesize NO through short bursts in a response to rapid changes in intracellular Ca²⁺. The redox state of the cellular environment plays a key role in the anti-or proapoptotic NO effect.

The induction in vivo of long term glucotoxicity by addition of sucrose to 30% in Psammomys obesus establishes a type 2 diabetes, characterized by increased VLDLLDL, atherogenic factors, CPK, a marker of cardiovascular complications, as well as functional and structural alterations characterized by the remodeling of the intima and the media. Furthermore, the induction in vitro of glucotoxicity by addition of D-Glucose at supraphysiological dosages creates an oxidative stress characterized by an overproduction of MDA, marker of membrane lipid peroxidation and total nitrites. Morphological changes highlight the deleterious effects of glucotoxicity on adventitial fibroblasts in culture. The increased proliferation and the number of nucleoli and protein synthesis and collagens are linked to cell activation following glucotoxicity. The increase in collagen may be related to extracellular matrix remodeling in response to glucotoxicity. All these changes are factors inducing cardiovascular complications and accelerated atherosclerosis.