- Email: [email protected]
Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin
Molecular and Cellular Endocrinology 313 (2009) 64–69
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin Jiazhong Sun ∗ , Yancheng Xu, Zhe Dai, Yanlei Sun Department of Endocrinology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
a r t i c l e
i n f o
Article history: Received 3 July 2009 Received in revised form 18 August 2009 Accepted 25 August 2009 Keywords: Intermittent high glucose Osteopontin VSMCs Atherosclerosis
a b s t r a c t Objective: Hyperglycemia induces vascular smooth muscle cells (VSMCs) proliferation and may thus contribute to the formation of atherosclerotic lesions. Glucose fluctuations are strong predictor of diabetic vascular complications. We investigate the effects of exposure to constant and intermittent high glucose concentrations on the proliferation and matrix metalloproteinase (MMP)-2 activity of rat aortic VSMCs in culture, as well as the expression of osteopontin (OPN). Methods: Rat aortic VSMCs were grown to confluence and then exposed to 5 mmol/L glucose, 25 mmol/L glucose, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor and 5 integrin receptor. The cell proliferation, MMP-2 activity and the expression of OPN were assessed. Results: In cultured VSMCs, treatment with constant or intermittent high glucose significantly increased [3 H]thymidine incorporation in a time-dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum expression at 48 h. However, [3 H]thymidine incorporation was more pronounced in intermittent high glucose than in constant high glucose. Treatment with constant high glucose for 48 h significantly increase cell number, MMP-2 activation, OPN protein and mRNA expression compared with VSMCs treated with the cells normal glucose, and these effects were further enhanced when VSMCs were treated with intermittent high glucose. In addition, neutralizing antibodies to either OPN or its receptor 3 integrin but not neutralizing antibodies to 5 integrin significantly suppressed increase in [3 H]thymidine incorporation and MMP-2 activity induced by constant or intermittent high glucose. Conclusions: In cultured VSMCs, constant high glucose concentrations enhanced MMP-2 activity, cell proliferation and OPN expression. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis, which is mediated by the stimulation of OPN expression and synthesis. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes is a common human disease, in which cardiovascular complications are the leading causes of morbidity and mortality (Massi-Benedetti and Federici, 1999). Progression of atherosclerotic lesion or alteration of vasculature is the characteristic feature of diabetic complications (Ruderman and Haudenschild, 1984). Diabetes accelerates these processes by stimulating the atherogenic activity of vascular smooth muscle cells (VSMCs)—the integral part in the development of atherosclerosis (Beckman et al., 2002). VSMCs proliferation, followed by migration into the media of vessel wall, is a key step in formation and development of atherosclerotic lesions, which play a crucial role in the degradation and remodel-
∗ Corresponding author. Tel.: +86 27 68713107. E-mail address: [email protected] (J. Sun). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.08.019
ing of vascular basement membrane (Massi-Benedetti and Federici, 1999; Srivastava, 2002). It is clear that some MMP, particularly MMP-2, are dominant factors involved in the migration of VSMCs into the media of vessel wall. Chronic hyperglycemia has been identified as a risk factor for diabetes complications leading to accelerated atherosclerosis. Both fasting and postprandial hyperglycemia contribute to this process. However the acute glucose fluctuations that occur in diabetes, including upward (postprandial) and downward (interprandial) fluctuations can be considered as risk factors for cardiovascular events and should be included in the “dysglycemia” of diabetes in combination with fasting and postprandial hyperglycemia. Intermittent rather than constant hyperglycemia induces an increase in collagen production by cultured mesangial cells (Takeuchi et al., 1995). Hence, the acute changes in plasma glucose concentrations may results in development of microangiopathy. There is increasing evidence that glycemic disorders such as rapid glucose fluctuations
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
over a daily period might play an important role on diabetic complications (Monnier et al., 2006). However, the mechanism and the effects of intermittent hyperglycemia on VSMC proliferation and migration are poorly understood. Osteopontin (OPN) is an arginine–glycine–asparatic acid (RGD)containing protein secreted by a variety of cells, including VSMCs. High levels of OPN mRNA and protein were reported in human atherosclerotic plaque from the aorta, carotid and coronary arteries (Kwon et al., 2000). Some studies have already demonstrated that OPN transgenic mice developed larger atherosclerotic lesions with an atherogenic diet than non-transgenic mice (Isoda et al., 2003). In contrast, OPN-deficient mice develop smaller atherosclerotic lesions than non-transgenic mice (Matsui et al., 2003). A neutralizing antibody against OPN was found to inhibit rat carotid neointimal formation after endothelial denudation (Liaw et al., 1997). These results have suggested that OPN promotes the development of atherosclerosis. It was noted that high glucose concentrations stimulated OPN expression in cultured rat aortic VSMC (Takemoto et al., 1999). The OPN upregulation has also been demonstrated in the aortas of high-fat diet-induced diabetic mice and in the renal cortex of streptozotocin-induced diabetic rats, suggesting a role for OPN in the development of vascular as well as renal complications of diabetes (Towler et al., 1998; Fischer et al., 1998). The aims of our study therefore were to compare the effects of exposure to constant and intermittent high glucose concentrations on matrix metalloproteinase activity and the proliferation of rat aortic VSMCs in culture. We also measured the role of OPN in mediating proliferation and migration of VSMCs. 2. Materials and methods 2.1. Cell culture Primary cultures of rat aortic VSMCs were isolated as described (Lo et al., 2005) by the explant method from adult male Wistar rats weighing about 200 g. Cells were maintained in Dulbeco modified Eagle medium containing 5.5 mmol/L glucose, 10% fetal bovine serum, and 40 g/mL gentamicin (Schering-Plough, Kenilworth, NJ) in a humidified atmosphere at 37 ◦ C in 5% CO2 . Cells at passages 7–9 were used for the present experiments.
65
Proteins were transferred to nylon membranes and blotted for OPN using a 1:10 dilution (5 mg/ml) of the OPN monoclonal antibody MPIIIB10 (R&D Systems). The bound primary antibody was detected with a horseradish peroxidase-conjugated secondary antibody and visualized with an enhanced chemiluminescence method. Quantitations of Western blots were performed by densitometric analysis using an Eagle Eye II video system. 2.5. Assessment of cell proliferation [3 H]thymidine incorporation and cell number were used in the assessment of cell proliferation (Sahai et al., 1997). Briefly, VSMCs were subcultured in six-well plates as described in Section 2.2. Quiescent cultures were then exposed to either constant or intermittent high glucose in serum-free medium for 24 h. [3 H]thymidine (1 mCi/ml, specific activity 20 Ci/mmol) was added to one set of wells in the last 4 h of incubation. The other sets of wells were processed for cell counting. For the assessment of [3 H]thymidine incorporation, medium was removed at the end of incubation, and cells were washed with 10% trichloroacetic acid and digested with 0.5N NaOH. Radioactivity in the cell digest was counted in a Beckman scintillation counter. [3 H]thymidine incorporation is expressed as the total counts per minute per well. 2.6. MMP-2 activity analysis The matrix-degrading activity of MMP-2 was assayed by gelatin zymography as described previously with modifications (Lee et al., 2000). In brief, aliquots of conditioned medium were mixed with sample buffer and applied directly, without prior heating or reduction, to 10% (w/v) acrylamide gels containing 1 mg/mL gelatin. Electrophoresis was then performed at 100 V for approximately 2 h. After removal of sodium dodecyl sulfate (SDS) from the gel by incubation in 2.5% (v/v) Triton X-100 for 30 min, the gels were incubated at 37 ◦ C overnight in development buffer [50 mmol/L Tris-HCl (pH 7.6)], containing 0.2 mol/L NaCl, 5 mmol/L CaCl2 , and 0.2% [w/v] (Brij35). The gel was stained with 0.5% coomassie blue, and then destained in destaining buffer containing 5% methanol and 7% acetic acid, and then photographed. 2.7. Statistical analysis All experimental conditions were replicated fivefold. Protein content and MMP2 activity were expressed as a change from the control value (normal glucose) which was regarded as 100%. Results are expressed as mean ± S.D. Statistical comparisons between groups were made by analysis of variance (ANOVA), with pairwise multiple comparisons made by Fisher’s protected least-significant differences test. Analyses were by the software package, SPSS 13.0. A value of p less than 0.05 was considered significant.
3. Results 2.2. Experimental protocol VSMCs at the logarithmic growth phase were trypsinized to a single cell suspension, adjusted to cell concentration 1 × 105 ml−1 , and transferred to a six-well plate. After the cells reached confluence, the medium was replaced with serum-free DMEM and incubated for 24 h for synchronization. Cells were then incubated for a further 48 h in glucose specific basic media. The cells were randomly divided into three groups: (1) constant normal glucose medium (5 mmol/L); (2) constant highglucose medium (25 mmol/L); (3) alternating normal and high-glucose media every 6 h; (4) an osmotic control of 16.5 mmol/L mannose. Neutralizing antibodies to OPN, 3 integrin receptor and 5 integrin receptor (10 pg/ml, R&D Systems) were also added individually to the three media previously described. All groups were otherwise subjected to identical conditions, and the experiment was repeated at least 5 times. 2.3. Northern blot analysis After 24 h of incubation, total RNA was isolated from cells using ISOGEN (Nippon Gene, Tokyo, Japan). Northern hybridization was performed essentially as described (Takemoto et al., 1999) using 32 P-labeled rat OPN cDNA probe (Shanghai Sangon Biological Engineering Co., Ltd.). The blots were stripped and subsequently rehybridized with 32 P-labeled rat GAPDH cDNA probe (Shanghai Sangon Biological Engineering Co., Ltd.) to assess the amount of RNA loaded in each lane. Densitometric analysis of fluorograms and autoradiograms was performed using the imaging scanner (EPSON ES 8000) with the NIH Image 1.44 software. 2.4. Western blot analysis Osteopontin protein levels were assessed by Western blot analysis as previously described (Sodhi et al., 2000). VSMCs were subcultured in 75 cm3 flasks and exposed to high glucose or intermittent high glucose as described in Section 2.2. At the end of 2–24 h of incubation, conditioned medium was removed and centrifuged at 1000 rpm for 5 min to remove any cell debris. Supernatants were then mixed with SDS sample buffer, boiled for 5 min, and subjected to 10% SDS-PAGE.
3.1. Effect of intermittent high glucose on proliferation of VSMCs To examine the effect of high glucose on the proliferation of cultured VSMCs, quiescent cultures were exposed to constant or intermittent high glucose in a serum-free medium for 12, 24, 48, 72 h, and at the end of the respective incubation times, [3 H]thymidine incorporation was assessed. As shown in Fig. 1A, high glucose level stimulated VSMCs proliferation in a timedependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Continuous increasing effect was not found when the cells were treated for the longer time (72 h). Therefore, 48 h was selected as the treated time in subsequent studies. Mannose (16.5 mmol/L), an enhancer of osmotic pressure, did not affect the proliferation of VSMCs, indicating that high glucose-induced VSMCs proliferation was not due to an enhanced osmotic pressure. When VSMCs were exposed to constant high glucose there was an increase in thymidine incorporation compared with cells exposed to normal glucose (a basic level of serum, here as a control) at different time. This effect was further enhanced when cells were exposed to intermittent high glucose concentrations (Fig. 1A). As shown in Fig. 1B, exposure to constant high glucose concentrations for 48 h induced a significant 83% increase in cell number when compared with the results obtained under normal glucose concentrations conditions (3.34 ± 0.06 × 106 per well versus 1.80 ± 0.05 × 106 per well, p < 0.01). This effect was further enhanced to 180% (5.08 ± 0.12 × 106 per well versus
66
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
Fig. 2. Effect of high glucose on MMP-2 activity in VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. MMP-2 activity was determined by zymography analysis. The data expressed as mean ± S.D. of at least five independent experiments. **p < 0.001 vs. NG; # p < 0.001 vs. HG.
This effect was further enhanced to 220% (±6.0%, p < 0.001) when cells were exposed to intermittent high glucose.
3.3. Effect of intermittent high glucose on OPN expression
Fig. 1. High glucose induced proliferation of VSMCs. [3 H]thymidine incorporation (A) and cell number (B) in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. The growth-arrested VSMCs were exposed to glucose at different concentrations for indicated time and total cell lysates were harvested. Cell proliferation was evaluated by the assessment of [3 H]thymidine incorporation and cell number. The data expressed as mean ± S.D. of at least five independent experiments. *p < 0.01 vs. NG; **p < 0.001 vs. NG; # p < 0.01 vs. HG.
1.80 ± 0.05 × 106 per well, p < 0.001) when cells were exposed to intermittent high glucose concentrations compared with normal glucose. There also were statistically significant changes in cell number after exposure to either constant or intermittent high glucose concentrations (p < 0.01). These results indicate that constant or intermittent high glucose directly induces the proliferation of cultured VSMCs, but in the latter condition the proliferation was more marked. 3.2. Effect of intermittent high glucose on MMP-2 activity As shown in Fig. 2, after VSMCs were exposed to constant high glucose, there was a significant increase in MMP-2 activity of 96.3% (±8.5%, p < 0.001), compared with cells exposed to normal glucose.
Because OPN has been shown to play an important role in the development of atherosclerotic lesions, we examined whether constant or intermittent high glucose alters OPN expression in VSMCs. Quiescent cultures of VSMCs were exposed to constant or intermittent high glucose condition for 48 h. OPN protein and mRNA levels were assessed. As shown in Fig. 3A, after exposure to constant high glucose, the concentration of OPN protein was increased by 80% (±3.2%, p < 0.01) compared with cells exposed to normal glucose. This effect was further enhanced following exposure of VSMCs to intermittent high concentrations of glucose with OPN protein secretion increased by 110% (±4.7%, p < 0.01) compared with cells exposed to constant normal glucose. There was a statistically significant increase in the cell protein content of OPN exposed to intermittent high glucose concentrations compared with cells exposed to constant high glucose concentrations (p < 0.01). Both constant and intermittent high glucose significantly increased OPN mRNA expression in VSMCs compared with cells exposed to normal glucose (relative expression: 0.82 ± 0.05 versus 0.40 ± 0.03, p < 0.01; 1.21 ± 0.04 versus 0.40 ± 0.03, p < 0.01, respectively, Fig. 3B). Moreover, this effect was further enhanced in VSMCs exposed to intermittent high concentrations than cells exposed to constant high glucose (1.21 ± 0.04 versus 0.82 ± 0.05, p < 0.01, Fig. 3B). These results suggest that constant and intermittent high glucose treatment not only increases the mRNA level of the OPN gene, but also increases its protein synthesis and secretion from VSMCs, but in the intermittent high glucose condition the increase was more marked.
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
67
Fig. 4. Role of OPN in proliferation of cultured VSMCs induced by high glucose. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h in the absence or presence of anti-OPN antibody (OPNab) or neutralizing antibody to either 3 (3ab) or 5 (5ab) integrin receptor, and [3 H]thymidine incorporation was assessed. The data expressed as mean ± S.D. of at least five independent experiments. * p < 0.01 vs. the respective control.
3 integrin receptor had no effect on [3 H]thymidine incorporation under normal glucose or 16.5 mmol/L mannose conditions (Fig. 4). 3.5. Role of OPN in stimulation of MMP-2 activity in VSMCs induced by intermittent high glucose
Fig. 3. The effect of high glucose on OPN protein (A) and mRNA (B) levels in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/H G: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h. OPN protein and mRNA expression were assessed by Western blot analysis and Northern blot analysis, respectively. OPN protein levels are expressed as a percent of the value of NG (100%). “mRNA relative expression” means the ratio of OPN over -actin. The data expressed as mean ± S.D. of at least five independent experiments. **p < 0.01 vs. NG; # p < 0.01 vs. HG.
3.4. Role of OPN in proliferation of VSMCs induced by intermittent high glucose To examine a role for OPN in proliferation of VSMCs induced by intermittent high glucose, quiescent VSMCs were exposed to constant and intermittent high glucose for 48 h in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor, 5 integrin receptor, and then [3 H]thymidine incorporation was assessed. The blocking of OPN action with neutralizing antibody to either OPN or its 3 integrin receptor completely prevented the increase in [3 H]thymidine incorporation induced by constant and intermittent high glucose (Fig. 4). By contrast, the neutralizing antibody to 5 integrin receptor had no effect on reducing the increase in [3 H]thymidine incorporation induced by constant and intermittent high glucose (Fig. 4). Neutralizing antibodies to OPN or
The quiescent VSMCs were exposed to constant and intermittent high glucose for 48 h in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor, 5 integrin receptor, and then MMP-2 activity was assessed. The blocking of OPN action (neutralizing antibody to either OPN or its 3 integrin receptor) equally inhibited the increase of MMP-2 activity in the constant and intermittent high glucose condition as regards the same conditions where no inhibitor was added (Fig. 5). By contrast, the neutralizing antibody to 5 integrin receptor had no effect on reducing the increase in MMP-2 activity induced by constant or intermittent high glucose (Fig. 5). Neutralizing antibodies to OPN or 3 integrin receptor had no effect on MMP-2 activity under normal glucose or high osmotic pressure conditions (Fig. 5). 4. Discussion This study has shown that cultured rat aortic VSMCs exposed to high glucose concentrations have increased cell proliferation and MMP-2 activity, as well as the expression of OPN. Moreover these effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose concentrations. These findings suggest that variability in glycaemic control could be more deleterious to the VSMCs than constant high concentrations of glucose. Furthermore, neutralizing antibodies to either OPN or its receptor 3 integrin but not neutralizing antibodies to 5 integrin reverse high-glucose-induced expression of OPN, as well as over-production of cell proliferation and MMP-2 activity. These findings suggest an important role of intermittent hyperglycemia in the initiation and/or development of diabetic atherosclerosis and demonstrate an important role for OPN in mediating this process. A large body of work has established that chronic hyperglycemia promotes VSMCs proliferation and migration, and contributes to
68
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
Fig. 5. Role of OPN in high-glucose-induced MMP-2 activity in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h in the absence or presence of anti-OPN antibody (OPNab) or neutralizing antibody to either 3 (3ab) or 5 (5ab) integrin receptor, and MMP-2 activity was assessed by zymography analysis. The data expressed as mean ± S.D. of at least five independent experiments. *p < 0.01 vs. the respective control.
the progress of diabetic atherosclerosis (Kawamura et al., 2004; Krolewski et al., 1991; Sodhi et al., 2001; Stamler et al., 1993). Studies in experimental animal models of diabetes and in humans demonstrated enhanced proliferation of aortic VSMCs (Alipui et al., 1993; Oikawa et al., 1996). Also, porcine aortic VSMCs in culture exhibit increased cell proliferation under hyperglycemic conditions (Natarajan et al., 1992). Therefore, hyperglycemia induced VSM cell proliferation appears to be an important cause for diabetic atherosclerosis. MMP family is an important group of proteolytic enzymes that can digest all the components of the extracellular matrix and is closely involved in the vascular remodeling, migration, and proliferation of VSMCs (Delbosc et al., 2008). MMP-2 is an important member of MMP family and is closely associated with the cardiovascular system. MMP-2 is the main enzyme that degrades collagens, and plays an important role in collagen metabolism. It can completely degrade basement membrane collagens I, III, IV, V, VII, X, and elastic protein (Ambalavanan et al., 2008). Collagen is the most abundant component of extracellular matrix proteins in the vessel wall. VSMCs and endothelial cells are surrounded by a basement membrane. Collagen in this basement membrane forms a migration barrier for VSMCs, and serves as the main component of the thickened intima. There is an increase in the early expression of MMP-2 in model rats, rabbits, and pigs with balloon injury, suggesting its central role in VSMCs migration through the extracellular matrix (Solomatina et al., 2005). Consistent with in vitro studies of Natarajan et al. (1992) in porcine aortic VSMCs, cultured aortic VSMCs exposed to high glucose concentrations have increased cell proliferation and MMP-2 activity. More importantly, these effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose concentrations. These findings suggest that variability in glycaemic control could be more deleterious to the VSMCs than constant high glucose concentrations. These previous findings in combination with the results of our present study indicate a pivotal role for VSMCs exposed to intermittent high glucose concentrations in accelerated VSMCs proliferation in diabetes.
The effects of intermittent high glucose on proliferation and MMP-2 activity in VSMCs are not yet defined. We have shown that the exposure of VSMCs to intermittent high glucose significantly enhanced the effects of the exposure of such cells to constant high glucose concentrations, supporting a pathophysiologic link between intermittent high glucose and increased cardiovascular risk (Ceriello et al., 2008). Intermittent high glucose may be more dangerous for the cells than constant high glucose. Mesangial cells cultured in periodic high glucose concentration increase matrix production more than the cells cultured in stable high glucose (Takeuchi et al., 1995). Similarly, fluctuations of glucose display a more dangerous effect than stable high glucose on both tubulointerstitial cells and human renal cortical fibroblasts, in terms of collagen synthesis and cell growth (Jones et al., 1999). New diabetes therapies focused on reducing postprandial hyperglycemia have become available and may benefit glycemic control and CVD risk factor levels (Bastyr et al., 2000). It is now recognized that both hyperglycemia at 2 h during an oral glucose challenge, as well as glucose fluctuations (Ceriello, 2004) are strong predictor of cardiovascular disease, and microangiophatic complications (Singleton et al., 2003), and it has been suggested that “hyperglycemic spikes” may play a direct and significant role in the pathogenesis of diabetic vascular complications (Ceriello, 1998). OPN is a cell adhesion molecule with a primitive amino acid sequence, which can combine with the VSMCs surface integrin receptor, and facilitate cellular adhesion, proliferation, and migration (Li et al., 2007). A growing body of both in vivo and in vitro evidence indicated an important role of OPN in VSMCs proliferation and atherosclerosis (Ikeda et al., 1993; Giachelli et al., 1993). It was reported that the expression of OPN protein and mRNA increased in the neointima and in calcified atheromatous plaque (Shanahan et al., 1994). A neutralizing antibody against OPN was found to inhibit rat carotid neointimal formation after endothelial denudation (Liaw et al., 1997). Furthermore, OPN overexpression has been shown to stimulate the proliferation of cultured VSMCs, and an exogenous addition of OPN has been found to promote the proliferation of cultured human coronary artery smooth muscle cells (Kwon et al., 2000; Takemoto et al., 2000). Upregulation of OPN expression was found in diabetic human and rat vascular walls (Takemoto et al., 2000). These results have suggested that that OPN plays an important role in accelerated atherogenesis in diabetes mellitus. Our present study showed that proliferation of cultured VSMCs induced by high glucose was also involved in increased expression of OPN. Treatment with both constant and intermittent high glucose significantly increased OPN expression, cell proliferation and MMP-2 activation compared with VSMCs treated with normal glucose. A neutralizing antibody to OPN or its 3 integrin receptor completely blocked the proliferation and MMP-2 activity in VSMCs induced by high glucose, suggesting a functional role of OPN in mediating VSMCs proliferation and migration. Neutralizing antibody to 5 integrin receptor, which binds preferentially to vitronectin (Kawano et al., 2000; Cheng et al., 2000) was unable to prevent the proliferation of VSMCs induced by high glucose. Taken together, these findings suggest that the high glucose and the associated marked increases in OPN expression in diabetes may be the key events responsible for accelerated VSMCs proliferation, migration and the development of diabetic atherosclerosis. In summary, we found that constant high glucose increased the proliferation and MMP-2 activity in cultured aortic VSMCs, which is mediated by the stimulation of OPN synthesis. These effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis. In addition, experimental decrease of OPN (by a neutralizing antibody to OPN or its 3 integrin receptor) inhibited the proliferation and MMP-2 activity in
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
VSMCs induced by constant or intermittent high glucose. Thus, our results reveal a novel function of OPN as a regulator of VSMCs proliferation and migration, and provide a potential strategy of treatment for diabetic atherosclerosis. Acknowledgement This study was funded by Hubei Provincial Bureau of Health Science Foundation for Young Scholars (grants QJX2008-29). References Alipui, C., Ramos, K., Tenner, T.E., 1993. Alterations of rabbit aortic smooth muscle cell proliferation in diabetes mellitus. Cardiovasc. Res. 27, 1229–1232. Ambalavanan, N., Nicola, T., Li, P., 2008. Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions. Pediatr. Res. 63, 26–32. Bastyr, E.J., Stuart, C.A., Brodows, R.G., Schwartz, S., Graf, C.J., Zagar, A., Robertson, K.E., 2000. Therapy focused on lowering postprandial glucose, not fasting glucose, may be superior for lowering HbA1c: IOEZStudy Group. Diabetes Care 23, 1236–1241. Beckman, J.A., Creager, M.A., Libby, P., 2002. Diabetes and atherosclerosis. JAMA 287, 2570–2581. Ceriello, A., 1998. The emerging role of post-prandial hyperglycaemic spikes in the pathogenesis of diabetic complications. Diabet. Med. 15, 188–193. Ceriello, A., 2004. Impaired glucose tolerance and cardiovascular disease: the possible role of post-prandial hyperglycemia. Am. Heart. J. 147, 803–807. Ceriello, A., Esposito, K., Piconi, L., Ihnat, M.A., 2008. Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients. Diabetes 57, 1349–1354. Cheng, S.L., Lai, C., Fausto, A., Chellaiah, M., Feng, X., McHugh, K., Tietelbaum, S., Civitelli, R., Hruska, K., Ross, P., Avioli, L.V., 2000. Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells. J. Cell Biochem. 77, 265–276. Delbosc, S., Glorian, M., Le Port, A.S., 2008. The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9. Am. J. Pathol. 172, 1430–1440. Fischer, J.W., Tschope, C., Reinecke, A., Giacehelli, C.M., Unger, T., 1998. Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rat is mediated by bradykinin. Diabetes 47, 1512–1518. Giachelli, C.M., Bee, N., Almeida, M., Denhardt, D.T., Alpers, C.E., Schwartz, S.M., 1993. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J. Clin. Invest. 92, 1686–1696. Ikeda, T., Shirasawa, T., Esaki, Y., Toshiki, S., Hirokawa, K., 1993. Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta. J. Clin. Invest. 92, 2814–2820. Isoda, K., Kamezawa, Y., Ayaori, M., Kusuhara, M., Tada, N., Ohsuzu, F., 2003. Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions. Circulation 107, 679–681. Jones, S.C., Saunders, H.J., Qi, W., 1999. Intermittent high glucose enhances cell growth and collagen synthesis in cultured human tubulointerstitial cells. Diabetologia 42, 1113–1119. Kawamura, H., Yokote, K., Asaumi, S., Kobayashi, K., Fujimoto, M., Maezawa, Y., Saito, Y., Mori, S., 2004. High glucose-induced upregulation of osteopontin is mediated via Rho/Rho kinase pathway in cultured rat aortic smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 24 (2), 276–281. Kawano, H., Cody, R.J., Graf, K., Goetze, S., Kawano, Y., Schnee, J., Law, R.E., Hsueh, W.A., 2000. Angiotensin II enhances integrin and a-actinin expression in adult rat cardiac fibroblasts. Hypertension 35, 273–279. Krolewski, A.S., Warram, J.H., Valsania, P., Martin, B.C., Laffel, L.M., 1991. Evolving natural history of coronary artery disease in diabetes mellitus. Am. J. Med. 90, 56S–61S. Kwon, H.M., Hong, B.K., Kang, T.S., Kwon, K., Kim, H.K., Jang, Y., 2000. Expression of osteopontin in calcified coronary atherosclerotic plaques. J. Korean Med. Sci. 15, 483–493.
69
Lee, E.S., Bauer, G.E., Caldwell, M.P., Santilli, S.M., 2000. Association of artery wall hypoxia and cellular proliferation at a vascular anastomosis. J. Surg. Res. 91, 32–37. Li, J.J., Han, M., Wen, J.K., 2007. Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation. Biochem. Biophys. Res. Commun. 356, 13–19. Liaw, L., Lombardi, D.M., Almeida, M.M., Schwartz, S.M., deBlois, D., Giachelli, C.M., 1997. Neutralizing antibodies directed against osteopontin inhibit rat carotid neointimal thickening after endothelial denudation. Arterioscler. Thromb. Vasc. Biol. l17, 188–193. Lo, I.C., Shih, J.M., Jiang, M.J., 2005. Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration. J. Biomed. Sci. 12 (2), 377–388. Massi-Benedetti, M., Federici, M.O., 1999. Cardiovascular risk factors in type 2 diabetes: the role of hyperglycaemia. Exp. Clin. Endocrinol. Diabetes 107, S120–123. Matsui, Y., Rittling, S.R., Okamoto, H., Inobe, M., Jia, N., Shimizu, T., 2003. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. l23, 1029–1034. Monnier, L., Mas, E., Ginet, C., Michel, F., Villon, L., Cristol, J.P., Colette, C., 2006. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA 295, 1681–1687. Natarajan, R., Gonzales, N., Xu, L., Nadler, J.L., 1992. Vascular smooth muscle cells exhibit increased growth in response to elevated glucose. Biochem. Biophys. Res. Commun. 187, 552–560. Oikawa, S., Hayasaka, K., Hashizume, E., Kotake, H., Midorikawa, H., Sekikawa, A., Kikuchi, A., Toyota, T., 1996. Human arterial smooth muscle proliferation in diabetes. Diabetes 45, S114–S116. Ruderman, N.B., Haudenschild, C., 1984. Diabetes as an atherogenic factor. Prog. Cardiovasc. Dis. 26, 373–412. Sahai, A., Mei, C., Pattison, T., Tannen, R.L., 1997. Chronic hypoxia induces proliferation of cultured rat mesangial cells: role of calcium and protein kinase C. Am. J. Physiol. 273, F954–F960. Shanahan, C.M., Cary, N.R., Metcalfe, J.C., Weissberg, P.L., 1994. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J. Clin. Invest. 93, 2393–2402. Singleton, J.R., Smith, A.G., Russell, J.W., Feldman, E.L., 2003. Microvascular complications of impaired glucose tolerance. Diabetes 52, 2867–2873. Sodhi, C.P., Batlle, D., Sahai, A., 2000. Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38MAPkinase. Kidney Int. 58, 691–700. Sodhi, C.P., Phadke, S.A., Batlle, D., Sahai, A., 2001. Hypoxia stimulates osteopontin expression and proliferation of cultured vascular smooth muscle cells: potentiation by high glucose. Diabetes 50 (6), 1482–1490. Solomatina, M.A., Plekhanova, O.S., Men’shikova, M.Y., 2005. Urokinase increases the content and activity of matrix metalloproteinases 2 and 9 during in vivo constrictive arterial remodelling. Bull. Exp. Biol. Med. 139, 283–286. Srivastava, A.K., 2002. High glucose-induced activation of protein kinase signaling pathways in vascular smooth muscle cells: a potential role in the pathogenesis of vascular dysfunction in diabetes. Int. J. Mol. Med. 9, 85–89. Stamler, J., Vaccaro, O., Neaton, J.D., Wentworth, D., 1993. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 16, 434–444. Takemoto, M., Yokote, K., Nishimura, M., Shigematsu, T., Hasegawa, T., Kon, S., Uede, T., Matsumoto, T., Saito, Y., Mori, S., 2000. Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler. Thromb. Vasc. Biol. 20, 624–628. Takemoto, M., Yokote, K., Yamazaki, M., Ridall, A.L., Butler, W.T., Matsumoto, T., Tamura, K., Saito, Y., Mori, S., 1999. Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 258, 722–726. Takeuchi, A., Throckmorton, D.C., Brogden, A.P., 1995. Periodic high extracellular glucose enhances production of collagens III and IV by mesangial cells. Am. J. Physiol. 268, F13–F19. Towler, D.A., Bidder, M., Latifi, T., Coleman, T., Semenkovich, C.F., 1998. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J. Biol. Chem. 273, 30427–30434.
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Intermittent high glucose enhances proliferation of vascular smooth muscle cells by upregulating osteopontin Jiazhong Sun ∗ , Yancheng Xu, Zhe Dai, Yanlei Sun Department of Endocrinology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China
a r t i c l e
i n f o
Article history: Received 3 July 2009 Received in revised form 18 August 2009 Accepted 25 August 2009 Keywords: Intermittent high glucose Osteopontin VSMCs Atherosclerosis
a b s t r a c t Objective: Hyperglycemia induces vascular smooth muscle cells (VSMCs) proliferation and may thus contribute to the formation of atherosclerotic lesions. Glucose fluctuations are strong predictor of diabetic vascular complications. We investigate the effects of exposure to constant and intermittent high glucose concentrations on the proliferation and matrix metalloproteinase (MMP)-2 activity of rat aortic VSMCs in culture, as well as the expression of osteopontin (OPN). Methods: Rat aortic VSMCs were grown to confluence and then exposed to 5 mmol/L glucose, 25 mmol/L glucose, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor and 5 integrin receptor. The cell proliferation, MMP-2 activity and the expression of OPN were assessed. Results: In cultured VSMCs, treatment with constant or intermittent high glucose significantly increased [3 H]thymidine incorporation in a time-dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum expression at 48 h. However, [3 H]thymidine incorporation was more pronounced in intermittent high glucose than in constant high glucose. Treatment with constant high glucose for 48 h significantly increase cell number, MMP-2 activation, OPN protein and mRNA expression compared with VSMCs treated with the cells normal glucose, and these effects were further enhanced when VSMCs were treated with intermittent high glucose. In addition, neutralizing antibodies to either OPN or its receptor 3 integrin but not neutralizing antibodies to 5 integrin significantly suppressed increase in [3 H]thymidine incorporation and MMP-2 activity induced by constant or intermittent high glucose. Conclusions: In cultured VSMCs, constant high glucose concentrations enhanced MMP-2 activity, cell proliferation and OPN expression. These effects are enhanced following intermittent exposure to high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis, which is mediated by the stimulation of OPN expression and synthesis. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes is a common human disease, in which cardiovascular complications are the leading causes of morbidity and mortality (Massi-Benedetti and Federici, 1999). Progression of atherosclerotic lesion or alteration of vasculature is the characteristic feature of diabetic complications (Ruderman and Haudenschild, 1984). Diabetes accelerates these processes by stimulating the atherogenic activity of vascular smooth muscle cells (VSMCs)—the integral part in the development of atherosclerosis (Beckman et al., 2002). VSMCs proliferation, followed by migration into the media of vessel wall, is a key step in formation and development of atherosclerotic lesions, which play a crucial role in the degradation and remodel-
∗ Corresponding author. Tel.: +86 27 68713107. E-mail address: [email protected] (J. Sun). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.08.019
ing of vascular basement membrane (Massi-Benedetti and Federici, 1999; Srivastava, 2002). It is clear that some MMP, particularly MMP-2, are dominant factors involved in the migration of VSMCs into the media of vessel wall. Chronic hyperglycemia has been identified as a risk factor for diabetes complications leading to accelerated atherosclerosis. Both fasting and postprandial hyperglycemia contribute to this process. However the acute glucose fluctuations that occur in diabetes, including upward (postprandial) and downward (interprandial) fluctuations can be considered as risk factors for cardiovascular events and should be included in the “dysglycemia” of diabetes in combination with fasting and postprandial hyperglycemia. Intermittent rather than constant hyperglycemia induces an increase in collagen production by cultured mesangial cells (Takeuchi et al., 1995). Hence, the acute changes in plasma glucose concentrations may results in development of microangiopathy. There is increasing evidence that glycemic disorders such as rapid glucose fluctuations
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
over a daily period might play an important role on diabetic complications (Monnier et al., 2006). However, the mechanism and the effects of intermittent hyperglycemia on VSMC proliferation and migration are poorly understood. Osteopontin (OPN) is an arginine–glycine–asparatic acid (RGD)containing protein secreted by a variety of cells, including VSMCs. High levels of OPN mRNA and protein were reported in human atherosclerotic plaque from the aorta, carotid and coronary arteries (Kwon et al., 2000). Some studies have already demonstrated that OPN transgenic mice developed larger atherosclerotic lesions with an atherogenic diet than non-transgenic mice (Isoda et al., 2003). In contrast, OPN-deficient mice develop smaller atherosclerotic lesions than non-transgenic mice (Matsui et al., 2003). A neutralizing antibody against OPN was found to inhibit rat carotid neointimal formation after endothelial denudation (Liaw et al., 1997). These results have suggested that OPN promotes the development of atherosclerosis. It was noted that high glucose concentrations stimulated OPN expression in cultured rat aortic VSMC (Takemoto et al., 1999). The OPN upregulation has also been demonstrated in the aortas of high-fat diet-induced diabetic mice and in the renal cortex of streptozotocin-induced diabetic rats, suggesting a role for OPN in the development of vascular as well as renal complications of diabetes (Towler et al., 1998; Fischer et al., 1998). The aims of our study therefore were to compare the effects of exposure to constant and intermittent high glucose concentrations on matrix metalloproteinase activity and the proliferation of rat aortic VSMCs in culture. We also measured the role of OPN in mediating proliferation and migration of VSMCs. 2. Materials and methods 2.1. Cell culture Primary cultures of rat aortic VSMCs were isolated as described (Lo et al., 2005) by the explant method from adult male Wistar rats weighing about 200 g. Cells were maintained in Dulbeco modified Eagle medium containing 5.5 mmol/L glucose, 10% fetal bovine serum, and 40 g/mL gentamicin (Schering-Plough, Kenilworth, NJ) in a humidified atmosphere at 37 ◦ C in 5% CO2 . Cells at passages 7–9 were used for the present experiments.
65
Proteins were transferred to nylon membranes and blotted for OPN using a 1:10 dilution (5 mg/ml) of the OPN monoclonal antibody MPIIIB10 (R&D Systems). The bound primary antibody was detected with a horseradish peroxidase-conjugated secondary antibody and visualized with an enhanced chemiluminescence method. Quantitations of Western blots were performed by densitometric analysis using an Eagle Eye II video system. 2.5. Assessment of cell proliferation [3 H]thymidine incorporation and cell number were used in the assessment of cell proliferation (Sahai et al., 1997). Briefly, VSMCs were subcultured in six-well plates as described in Section 2.2. Quiescent cultures were then exposed to either constant or intermittent high glucose in serum-free medium for 24 h. [3 H]thymidine (1 mCi/ml, specific activity 20 Ci/mmol) was added to one set of wells in the last 4 h of incubation. The other sets of wells were processed for cell counting. For the assessment of [3 H]thymidine incorporation, medium was removed at the end of incubation, and cells were washed with 10% trichloroacetic acid and digested with 0.5N NaOH. Radioactivity in the cell digest was counted in a Beckman scintillation counter. [3 H]thymidine incorporation is expressed as the total counts per minute per well. 2.6. MMP-2 activity analysis The matrix-degrading activity of MMP-2 was assayed by gelatin zymography as described previously with modifications (Lee et al., 2000). In brief, aliquots of conditioned medium were mixed with sample buffer and applied directly, without prior heating or reduction, to 10% (w/v) acrylamide gels containing 1 mg/mL gelatin. Electrophoresis was then performed at 100 V for approximately 2 h. After removal of sodium dodecyl sulfate (SDS) from the gel by incubation in 2.5% (v/v) Triton X-100 for 30 min, the gels were incubated at 37 ◦ C overnight in development buffer [50 mmol/L Tris-HCl (pH 7.6)], containing 0.2 mol/L NaCl, 5 mmol/L CaCl2 , and 0.2% [w/v] (Brij35). The gel was stained with 0.5% coomassie blue, and then destained in destaining buffer containing 5% methanol and 7% acetic acid, and then photographed. 2.7. Statistical analysis All experimental conditions were replicated fivefold. Protein content and MMP2 activity were expressed as a change from the control value (normal glucose) which was regarded as 100%. Results are expressed as mean ± S.D. Statistical comparisons between groups were made by analysis of variance (ANOVA), with pairwise multiple comparisons made by Fisher’s protected least-significant differences test. Analyses were by the software package, SPSS 13.0. A value of p less than 0.05 was considered significant.
3. Results 2.2. Experimental protocol VSMCs at the logarithmic growth phase were trypsinized to a single cell suspension, adjusted to cell concentration 1 × 105 ml−1 , and transferred to a six-well plate. After the cells reached confluence, the medium was replaced with serum-free DMEM and incubated for 24 h for synchronization. Cells were then incubated for a further 48 h in glucose specific basic media. The cells were randomly divided into three groups: (1) constant normal glucose medium (5 mmol/L); (2) constant highglucose medium (25 mmol/L); (3) alternating normal and high-glucose media every 6 h; (4) an osmotic control of 16.5 mmol/L mannose. Neutralizing antibodies to OPN, 3 integrin receptor and 5 integrin receptor (10 pg/ml, R&D Systems) were also added individually to the three media previously described. All groups were otherwise subjected to identical conditions, and the experiment was repeated at least 5 times. 2.3. Northern blot analysis After 24 h of incubation, total RNA was isolated from cells using ISOGEN (Nippon Gene, Tokyo, Japan). Northern hybridization was performed essentially as described (Takemoto et al., 1999) using 32 P-labeled rat OPN cDNA probe (Shanghai Sangon Biological Engineering Co., Ltd.). The blots were stripped and subsequently rehybridized with 32 P-labeled rat GAPDH cDNA probe (Shanghai Sangon Biological Engineering Co., Ltd.) to assess the amount of RNA loaded in each lane. Densitometric analysis of fluorograms and autoradiograms was performed using the imaging scanner (EPSON ES 8000) with the NIH Image 1.44 software. 2.4. Western blot analysis Osteopontin protein levels were assessed by Western blot analysis as previously described (Sodhi et al., 2000). VSMCs were subcultured in 75 cm3 flasks and exposed to high glucose or intermittent high glucose as described in Section 2.2. At the end of 2–24 h of incubation, conditioned medium was removed and centrifuged at 1000 rpm for 5 min to remove any cell debris. Supernatants were then mixed with SDS sample buffer, boiled for 5 min, and subjected to 10% SDS-PAGE.
3.1. Effect of intermittent high glucose on proliferation of VSMCs To examine the effect of high glucose on the proliferation of cultured VSMCs, quiescent cultures were exposed to constant or intermittent high glucose in a serum-free medium for 12, 24, 48, 72 h, and at the end of the respective incubation times, [3 H]thymidine incorporation was assessed. As shown in Fig. 1A, high glucose level stimulated VSMCs proliferation in a timedependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Continuous increasing effect was not found when the cells were treated for the longer time (72 h). Therefore, 48 h was selected as the treated time in subsequent studies. Mannose (16.5 mmol/L), an enhancer of osmotic pressure, did not affect the proliferation of VSMCs, indicating that high glucose-induced VSMCs proliferation was not due to an enhanced osmotic pressure. When VSMCs were exposed to constant high glucose there was an increase in thymidine incorporation compared with cells exposed to normal glucose (a basic level of serum, here as a control) at different time. This effect was further enhanced when cells were exposed to intermittent high glucose concentrations (Fig. 1A). As shown in Fig. 1B, exposure to constant high glucose concentrations for 48 h induced a significant 83% increase in cell number when compared with the results obtained under normal glucose concentrations conditions (3.34 ± 0.06 × 106 per well versus 1.80 ± 0.05 × 106 per well, p < 0.01). This effect was further enhanced to 180% (5.08 ± 0.12 × 106 per well versus
66
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
Fig. 2. Effect of high glucose on MMP-2 activity in VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. MMP-2 activity was determined by zymography analysis. The data expressed as mean ± S.D. of at least five independent experiments. **p < 0.001 vs. NG; # p < 0.001 vs. HG.
This effect was further enhanced to 220% (±6.0%, p < 0.001) when cells were exposed to intermittent high glucose.
3.3. Effect of intermittent high glucose on OPN expression
Fig. 1. High glucose induced proliferation of VSMCs. [3 H]thymidine incorporation (A) and cell number (B) in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. The growth-arrested VSMCs were exposed to glucose at different concentrations for indicated time and total cell lysates were harvested. Cell proliferation was evaluated by the assessment of [3 H]thymidine incorporation and cell number. The data expressed as mean ± S.D. of at least five independent experiments. *p < 0.01 vs. NG; **p < 0.001 vs. NG; # p < 0.01 vs. HG.
1.80 ± 0.05 × 106 per well, p < 0.001) when cells were exposed to intermittent high glucose concentrations compared with normal glucose. There also were statistically significant changes in cell number after exposure to either constant or intermittent high glucose concentrations (p < 0.01). These results indicate that constant or intermittent high glucose directly induces the proliferation of cultured VSMCs, but in the latter condition the proliferation was more marked. 3.2. Effect of intermittent high glucose on MMP-2 activity As shown in Fig. 2, after VSMCs were exposed to constant high glucose, there was a significant increase in MMP-2 activity of 96.3% (±8.5%, p < 0.001), compared with cells exposed to normal glucose.
Because OPN has been shown to play an important role in the development of atherosclerotic lesions, we examined whether constant or intermittent high glucose alters OPN expression in VSMCs. Quiescent cultures of VSMCs were exposed to constant or intermittent high glucose condition for 48 h. OPN protein and mRNA levels were assessed. As shown in Fig. 3A, after exposure to constant high glucose, the concentration of OPN protein was increased by 80% (±3.2%, p < 0.01) compared with cells exposed to normal glucose. This effect was further enhanced following exposure of VSMCs to intermittent high concentrations of glucose with OPN protein secretion increased by 110% (±4.7%, p < 0.01) compared with cells exposed to constant normal glucose. There was a statistically significant increase in the cell protein content of OPN exposed to intermittent high glucose concentrations compared with cells exposed to constant high glucose concentrations (p < 0.01). Both constant and intermittent high glucose significantly increased OPN mRNA expression in VSMCs compared with cells exposed to normal glucose (relative expression: 0.82 ± 0.05 versus 0.40 ± 0.03, p < 0.01; 1.21 ± 0.04 versus 0.40 ± 0.03, p < 0.01, respectively, Fig. 3B). Moreover, this effect was further enhanced in VSMCs exposed to intermittent high concentrations than cells exposed to constant high glucose (1.21 ± 0.04 versus 0.82 ± 0.05, p < 0.01, Fig. 3B). These results suggest that constant and intermittent high glucose treatment not only increases the mRNA level of the OPN gene, but also increases its protein synthesis and secretion from VSMCs, but in the intermittent high glucose condition the increase was more marked.
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
67
Fig. 4. Role of OPN in proliferation of cultured VSMCs induced by high glucose. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h in the absence or presence of anti-OPN antibody (OPNab) or neutralizing antibody to either 3 (3ab) or 5 (5ab) integrin receptor, and [3 H]thymidine incorporation was assessed. The data expressed as mean ± S.D. of at least five independent experiments. * p < 0.01 vs. the respective control.
3 integrin receptor had no effect on [3 H]thymidine incorporation under normal glucose or 16.5 mmol/L mannose conditions (Fig. 4). 3.5. Role of OPN in stimulation of MMP-2 activity in VSMCs induced by intermittent high glucose
Fig. 3. The effect of high glucose on OPN protein (A) and mRNA (B) levels in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/H G: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h. OPN protein and mRNA expression were assessed by Western blot analysis and Northern blot analysis, respectively. OPN protein levels are expressed as a percent of the value of NG (100%). “mRNA relative expression” means the ratio of OPN over -actin. The data expressed as mean ± S.D. of at least five independent experiments. **p < 0.01 vs. NG; # p < 0.01 vs. HG.
3.4. Role of OPN in proliferation of VSMCs induced by intermittent high glucose To examine a role for OPN in proliferation of VSMCs induced by intermittent high glucose, quiescent VSMCs were exposed to constant and intermittent high glucose for 48 h in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor, 5 integrin receptor, and then [3 H]thymidine incorporation was assessed. The blocking of OPN action with neutralizing antibody to either OPN or its 3 integrin receptor completely prevented the increase in [3 H]thymidine incorporation induced by constant and intermittent high glucose (Fig. 4). By contrast, the neutralizing antibody to 5 integrin receptor had no effect on reducing the increase in [3 H]thymidine incorporation induced by constant and intermittent high glucose (Fig. 4). Neutralizing antibodies to OPN or
The quiescent VSMCs were exposed to constant and intermittent high glucose for 48 h in the absence or presence of neutralizing antibodies to OPN, 3 integrin receptor, 5 integrin receptor, and then MMP-2 activity was assessed. The blocking of OPN action (neutralizing antibody to either OPN or its 3 integrin receptor) equally inhibited the increase of MMP-2 activity in the constant and intermittent high glucose condition as regards the same conditions where no inhibitor was added (Fig. 5). By contrast, the neutralizing antibody to 5 integrin receptor had no effect on reducing the increase in MMP-2 activity induced by constant or intermittent high glucose (Fig. 5). Neutralizing antibodies to OPN or 3 integrin receptor had no effect on MMP-2 activity under normal glucose or high osmotic pressure conditions (Fig. 5). 4. Discussion This study has shown that cultured rat aortic VSMCs exposed to high glucose concentrations have increased cell proliferation and MMP-2 activity, as well as the expression of OPN. Moreover these effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose concentrations. These findings suggest that variability in glycaemic control could be more deleterious to the VSMCs than constant high concentrations of glucose. Furthermore, neutralizing antibodies to either OPN or its receptor 3 integrin but not neutralizing antibodies to 5 integrin reverse high-glucose-induced expression of OPN, as well as over-production of cell proliferation and MMP-2 activity. These findings suggest an important role of intermittent hyperglycemia in the initiation and/or development of diabetic atherosclerosis and demonstrate an important role for OPN in mediating this process. A large body of work has established that chronic hyperglycemia promotes VSMCs proliferation and migration, and contributes to
68
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
Fig. 5. Role of OPN in high-glucose-induced MMP-2 activity in cultured VSMCs. NG: constant normal glucose (5 mmol/L); OC: osmotic control (16.5 mmol/L mannose was used as a negative control); HG: constant high glucose (25 mmol/L); N/HG: 5 mmol/L alternating with 25 mmol/L glucose. Quiescent cells were exposed to glucose at different concentrations for 48 h in the absence or presence of anti-OPN antibody (OPNab) or neutralizing antibody to either 3 (3ab) or 5 (5ab) integrin receptor, and MMP-2 activity was assessed by zymography analysis. The data expressed as mean ± S.D. of at least five independent experiments. *p < 0.01 vs. the respective control.
the progress of diabetic atherosclerosis (Kawamura et al., 2004; Krolewski et al., 1991; Sodhi et al., 2001; Stamler et al., 1993). Studies in experimental animal models of diabetes and in humans demonstrated enhanced proliferation of aortic VSMCs (Alipui et al., 1993; Oikawa et al., 1996). Also, porcine aortic VSMCs in culture exhibit increased cell proliferation under hyperglycemic conditions (Natarajan et al., 1992). Therefore, hyperglycemia induced VSM cell proliferation appears to be an important cause for diabetic atherosclerosis. MMP family is an important group of proteolytic enzymes that can digest all the components of the extracellular matrix and is closely involved in the vascular remodeling, migration, and proliferation of VSMCs (Delbosc et al., 2008). MMP-2 is an important member of MMP family and is closely associated with the cardiovascular system. MMP-2 is the main enzyme that degrades collagens, and plays an important role in collagen metabolism. It can completely degrade basement membrane collagens I, III, IV, V, VII, X, and elastic protein (Ambalavanan et al., 2008). Collagen is the most abundant component of extracellular matrix proteins in the vessel wall. VSMCs and endothelial cells are surrounded by a basement membrane. Collagen in this basement membrane forms a migration barrier for VSMCs, and serves as the main component of the thickened intima. There is an increase in the early expression of MMP-2 in model rats, rabbits, and pigs with balloon injury, suggesting its central role in VSMCs migration through the extracellular matrix (Solomatina et al., 2005). Consistent with in vitro studies of Natarajan et al. (1992) in porcine aortic VSMCs, cultured aortic VSMCs exposed to high glucose concentrations have increased cell proliferation and MMP-2 activity. More importantly, these effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose concentrations. These findings suggest that variability in glycaemic control could be more deleterious to the VSMCs than constant high glucose concentrations. These previous findings in combination with the results of our present study indicate a pivotal role for VSMCs exposed to intermittent high glucose concentrations in accelerated VSMCs proliferation in diabetes.
The effects of intermittent high glucose on proliferation and MMP-2 activity in VSMCs are not yet defined. We have shown that the exposure of VSMCs to intermittent high glucose significantly enhanced the effects of the exposure of such cells to constant high glucose concentrations, supporting a pathophysiologic link between intermittent high glucose and increased cardiovascular risk (Ceriello et al., 2008). Intermittent high glucose may be more dangerous for the cells than constant high glucose. Mesangial cells cultured in periodic high glucose concentration increase matrix production more than the cells cultured in stable high glucose (Takeuchi et al., 1995). Similarly, fluctuations of glucose display a more dangerous effect than stable high glucose on both tubulointerstitial cells and human renal cortical fibroblasts, in terms of collagen synthesis and cell growth (Jones et al., 1999). New diabetes therapies focused on reducing postprandial hyperglycemia have become available and may benefit glycemic control and CVD risk factor levels (Bastyr et al., 2000). It is now recognized that both hyperglycemia at 2 h during an oral glucose challenge, as well as glucose fluctuations (Ceriello, 2004) are strong predictor of cardiovascular disease, and microangiophatic complications (Singleton et al., 2003), and it has been suggested that “hyperglycemic spikes” may play a direct and significant role in the pathogenesis of diabetic vascular complications (Ceriello, 1998). OPN is a cell adhesion molecule with a primitive amino acid sequence, which can combine with the VSMCs surface integrin receptor, and facilitate cellular adhesion, proliferation, and migration (Li et al., 2007). A growing body of both in vivo and in vitro evidence indicated an important role of OPN in VSMCs proliferation and atherosclerosis (Ikeda et al., 1993; Giachelli et al., 1993). It was reported that the expression of OPN protein and mRNA increased in the neointima and in calcified atheromatous plaque (Shanahan et al., 1994). A neutralizing antibody against OPN was found to inhibit rat carotid neointimal formation after endothelial denudation (Liaw et al., 1997). Furthermore, OPN overexpression has been shown to stimulate the proliferation of cultured VSMCs, and an exogenous addition of OPN has been found to promote the proliferation of cultured human coronary artery smooth muscle cells (Kwon et al., 2000; Takemoto et al., 2000). Upregulation of OPN expression was found in diabetic human and rat vascular walls (Takemoto et al., 2000). These results have suggested that that OPN plays an important role in accelerated atherogenesis in diabetes mellitus. Our present study showed that proliferation of cultured VSMCs induced by high glucose was also involved in increased expression of OPN. Treatment with both constant and intermittent high glucose significantly increased OPN expression, cell proliferation and MMP-2 activation compared with VSMCs treated with normal glucose. A neutralizing antibody to OPN or its 3 integrin receptor completely blocked the proliferation and MMP-2 activity in VSMCs induced by high glucose, suggesting a functional role of OPN in mediating VSMCs proliferation and migration. Neutralizing antibody to 5 integrin receptor, which binds preferentially to vitronectin (Kawano et al., 2000; Cheng et al., 2000) was unable to prevent the proliferation of VSMCs induced by high glucose. Taken together, these findings suggest that the high glucose and the associated marked increases in OPN expression in diabetes may be the key events responsible for accelerated VSMCs proliferation, migration and the development of diabetic atherosclerosis. In summary, we found that constant high glucose increased the proliferation and MMP-2 activity in cultured aortic VSMCs, which is mediated by the stimulation of OPN synthesis. These effects were further enhanced in cells that were exposed to intermittent rather than constant high glucose, indicating that short lived excursions in glycaemic control have important pathological effects on the development of diabetic atherosclerosis. In addition, experimental decrease of OPN (by a neutralizing antibody to OPN or its 3 integrin receptor) inhibited the proliferation and MMP-2 activity in
J. Sun et al. / Molecular and Cellular Endocrinology 313 (2009) 64–69
VSMCs induced by constant or intermittent high glucose. Thus, our results reveal a novel function of OPN as a regulator of VSMCs proliferation and migration, and provide a potential strategy of treatment for diabetic atherosclerosis. Acknowledgement This study was funded by Hubei Provincial Bureau of Health Science Foundation for Young Scholars (grants QJX2008-29). References Alipui, C., Ramos, K., Tenner, T.E., 1993. Alterations of rabbit aortic smooth muscle cell proliferation in diabetes mellitus. Cardiovasc. Res. 27, 1229–1232. Ambalavanan, N., Nicola, T., Li, P., 2008. Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions. Pediatr. Res. 63, 26–32. Bastyr, E.J., Stuart, C.A., Brodows, R.G., Schwartz, S., Graf, C.J., Zagar, A., Robertson, K.E., 2000. Therapy focused on lowering postprandial glucose, not fasting glucose, may be superior for lowering HbA1c: IOEZStudy Group. Diabetes Care 23, 1236–1241. Beckman, J.A., Creager, M.A., Libby, P., 2002. Diabetes and atherosclerosis. JAMA 287, 2570–2581. Ceriello, A., 1998. The emerging role of post-prandial hyperglycaemic spikes in the pathogenesis of diabetic complications. Diabet. Med. 15, 188–193. Ceriello, A., 2004. Impaired glucose tolerance and cardiovascular disease: the possible role of post-prandial hyperglycemia. Am. Heart. J. 147, 803–807. Ceriello, A., Esposito, K., Piconi, L., Ihnat, M.A., 2008. Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients. Diabetes 57, 1349–1354. Cheng, S.L., Lai, C., Fausto, A., Chellaiah, M., Feng, X., McHugh, K., Tietelbaum, S., Civitelli, R., Hruska, K., Ross, P., Avioli, L.V., 2000. Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells. J. Cell Biochem. 77, 265–276. Delbosc, S., Glorian, M., Le Port, A.S., 2008. The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9. Am. J. Pathol. 172, 1430–1440. Fischer, J.W., Tschope, C., Reinecke, A., Giacehelli, C.M., Unger, T., 1998. Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rat is mediated by bradykinin. Diabetes 47, 1512–1518. Giachelli, C.M., Bee, N., Almeida, M., Denhardt, D.T., Alpers, C.E., Schwartz, S.M., 1993. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J. Clin. Invest. 92, 1686–1696. Ikeda, T., Shirasawa, T., Esaki, Y., Toshiki, S., Hirokawa, K., 1993. Osteopontin mRNA is expressed by smooth muscle-derived foam cells in human atherosclerotic lesions of the aorta. J. Clin. Invest. 92, 2814–2820. Isoda, K., Kamezawa, Y., Ayaori, M., Kusuhara, M., Tada, N., Ohsuzu, F., 2003. Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions. Circulation 107, 679–681. Jones, S.C., Saunders, H.J., Qi, W., 1999. Intermittent high glucose enhances cell growth and collagen synthesis in cultured human tubulointerstitial cells. Diabetologia 42, 1113–1119. Kawamura, H., Yokote, K., Asaumi, S., Kobayashi, K., Fujimoto, M., Maezawa, Y., Saito, Y., Mori, S., 2004. High glucose-induced upregulation of osteopontin is mediated via Rho/Rho kinase pathway in cultured rat aortic smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 24 (2), 276–281. Kawano, H., Cody, R.J., Graf, K., Goetze, S., Kawano, Y., Schnee, J., Law, R.E., Hsueh, W.A., 2000. Angiotensin II enhances integrin and a-actinin expression in adult rat cardiac fibroblasts. Hypertension 35, 273–279. Krolewski, A.S., Warram, J.H., Valsania, P., Martin, B.C., Laffel, L.M., 1991. Evolving natural history of coronary artery disease in diabetes mellitus. Am. J. Med. 90, 56S–61S. Kwon, H.M., Hong, B.K., Kang, T.S., Kwon, K., Kim, H.K., Jang, Y., 2000. Expression of osteopontin in calcified coronary atherosclerotic plaques. J. Korean Med. Sci. 15, 483–493.
69
Lee, E.S., Bauer, G.E., Caldwell, M.P., Santilli, S.M., 2000. Association of artery wall hypoxia and cellular proliferation at a vascular anastomosis. J. Surg. Res. 91, 32–37. Li, J.J., Han, M., Wen, J.K., 2007. Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation. Biochem. Biophys. Res. Commun. 356, 13–19. Liaw, L., Lombardi, D.M., Almeida, M.M., Schwartz, S.M., deBlois, D., Giachelli, C.M., 1997. Neutralizing antibodies directed against osteopontin inhibit rat carotid neointimal thickening after endothelial denudation. Arterioscler. Thromb. Vasc. Biol. l17, 188–193. Lo, I.C., Shih, J.M., Jiang, M.J., 2005. Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration. J. Biomed. Sci. 12 (2), 377–388. Massi-Benedetti, M., Federici, M.O., 1999. Cardiovascular risk factors in type 2 diabetes: the role of hyperglycaemia. Exp. Clin. Endocrinol. Diabetes 107, S120–123. Matsui, Y., Rittling, S.R., Okamoto, H., Inobe, M., Jia, N., Shimizu, T., 2003. Osteopontin deficiency attenuates atherosclerosis in female apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. l23, 1029–1034. Monnier, L., Mas, E., Ginet, C., Michel, F., Villon, L., Cristol, J.P., Colette, C., 2006. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA 295, 1681–1687. Natarajan, R., Gonzales, N., Xu, L., Nadler, J.L., 1992. Vascular smooth muscle cells exhibit increased growth in response to elevated glucose. Biochem. Biophys. Res. Commun. 187, 552–560. Oikawa, S., Hayasaka, K., Hashizume, E., Kotake, H., Midorikawa, H., Sekikawa, A., Kikuchi, A., Toyota, T., 1996. Human arterial smooth muscle proliferation in diabetes. Diabetes 45, S114–S116. Ruderman, N.B., Haudenschild, C., 1984. Diabetes as an atherogenic factor. Prog. Cardiovasc. Dis. 26, 373–412. Sahai, A., Mei, C., Pattison, T., Tannen, R.L., 1997. Chronic hypoxia induces proliferation of cultured rat mesangial cells: role of calcium and protein kinase C. Am. J. Physiol. 273, F954–F960. Shanahan, C.M., Cary, N.R., Metcalfe, J.C., Weissberg, P.L., 1994. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J. Clin. Invest. 93, 2393–2402. Singleton, J.R., Smith, A.G., Russell, J.W., Feldman, E.L., 2003. Microvascular complications of impaired glucose tolerance. Diabetes 52, 2867–2873. Sodhi, C.P., Batlle, D., Sahai, A., 2000. Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38MAPkinase. Kidney Int. 58, 691–700. Sodhi, C.P., Phadke, S.A., Batlle, D., Sahai, A., 2001. Hypoxia stimulates osteopontin expression and proliferation of cultured vascular smooth muscle cells: potentiation by high glucose. Diabetes 50 (6), 1482–1490. Solomatina, M.A., Plekhanova, O.S., Men’shikova, M.Y., 2005. Urokinase increases the content and activity of matrix metalloproteinases 2 and 9 during in vivo constrictive arterial remodelling. Bull. Exp. Biol. Med. 139, 283–286. Srivastava, A.K., 2002. High glucose-induced activation of protein kinase signaling pathways in vascular smooth muscle cells: a potential role in the pathogenesis of vascular dysfunction in diabetes. Int. J. Mol. Med. 9, 85–89. Stamler, J., Vaccaro, O., Neaton, J.D., Wentworth, D., 1993. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 16, 434–444. Takemoto, M., Yokote, K., Nishimura, M., Shigematsu, T., Hasegawa, T., Kon, S., Uede, T., Matsumoto, T., Saito, Y., Mori, S., 2000. Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler. Thromb. Vasc. Biol. 20, 624–628. Takemoto, M., Yokote, K., Yamazaki, M., Ridall, A.L., Butler, W.T., Matsumoto, T., Tamura, K., Saito, Y., Mori, S., 1999. Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 258, 722–726. Takeuchi, A., Throckmorton, D.C., Brogden, A.P., 1995. Periodic high extracellular glucose enhances production of collagens III and IV by mesangial cells. Am. J. Physiol. 268, F13–F19. Towler, D.A., Bidder, M., Latifi, T., Coleman, T., Semenkovich, C.F., 1998. Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice. J. Biol. Chem. 273, 30427–30434.