Exendin-4 restores glucolipotoxicity-induced gene expression in human coronary artery endothelial cells

Biochemical and Biophysical Research Communications 419 (2012) 790–795

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Exendin-4 restores glucolipotoxicity-induced gene expression in human coronary artery endothelial cells Özlem Erdogdu, Linnéa Eriksson, Thomas Nyström, Åke Sjöholm ⇑, Qimin Zhang ⇑ Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, SE-11883 Stockholm, Sweden

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Article history: Received 16 February 2012 Available online 27 February 2012 Keywords: Exendin-4 GLP-1 Endothelial cell Diabetes Vascular disease Endothelial function

a b s t r a c t Exendin-4, a stable GLP-1 receptor agonist, has been shown to stimulate insulin secretion. It has also been shown to exert beneficial effects on endothelial function that are independent of its glycemic effects. The molecular mechanisms underlying the protective actions of exendin-4 against diabetic glucolipotoxicity in endothelial cells largely remain elusive. We have investigated the long-term in vitro effect of palmitate or high glucose (simulating the diabetic milieu) and the role of exendin-4 on gene expression in human coronary artery endothelial cells. Gene expression profiling in combination with Western blotting revealed that exendin-4 regulates expression of a number of genes involved in angiogenesis, inflammation and thrombogenesis under glucolipotoxic conditions. Our results indicate that exendin-4 may improve endothelial cell function in diabetes through regulating expression of the genes, whose expression was disrupted by glucolipotoxicity. As endothelial dysfunction appears to be an early indicator of vascular damage, and predicts both progression of atherosclerosis and incidence of cardiovascular events, exendin-4 and possibly other incretin-based strategies may confer additional cardiovascular benefit beyond improved glycemic control. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Endothelial dysfunction is associated with insulin resistance and type 2 diabetes, and considered an early event in the pathogenesis of atherosclerosis [1]. Hyperglycemia and lipid disturbances can induce endothelial dysfunction [2], endothelial injury and death [3] and impairment of endothelial regeneration [2], contributing to the premature and widespread cardiovascular disease in diabetes. Exendin-4, a stable GLP-1 receptor agonist, has been shown to promote insulin secretion [4]. It has also been shown to exert beneficial actions on endothelial function. A recent study demonstrated that exendin-4 suppresses macrophage adhesion to the endothelium, an initial step in formation of atherosclerotic plaques [5]. Moreover, several studies in various animal models have shown that exendin-4 may reduce myocardial infarct size [6,7]. We recently reported that human coronary artery endothelial cells (HCAECs) express the GLP-1 receptor and that exendin-4 is able to activate the receptor, resulting in an enhanced cell proliferation [8]. Acute administration of GLP-1 improves endothelial dysfunction in type 2 diabetes patients with coronary heart disease [9], suggesting an important role of GLP-1 and exendin-4 in endothelial function. Agents that promote HCAEC proliferation and viability

might ‘‘patch up’’ an early endothelial lesion occurring in the highly pro-atherogenic diabetic milieu by rapidly covering it with endothelial cells and thus preventing further atherothrombotic events. However, the precise nature of the protective actions of exendin-4 against glucolipotoxicity in the endothelial cells remains unclear. The present study was undertaken to investigate the long-term in vitro effect of palmitate or high glucose, and the role of exendin4 on gene expression in human coronary artery endothelial cells. 2. Materials and methods Exendin-4, alpha-tubulin and sodium palmitate were from Sigma–Aldrich (St Louis, MO). Antibodies against TPA and angiopoietin 1 were purchased from Abcam. Antibodies against Tie-2, fibronectin, eNOS (NOS3), horseradish peroxidase-labeled goat anti-rabbit IgG, horseradish peroxidase-labeled goat anti-mouse IgG and the enhanced chemiluminescence ECL kit were from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitor cocktail was from Roche Diagnostics (Mannheim, Germany). D-Glucose was purchased from Merck (Germany). Bicinchoninic acid (BCA) kit was from Pierce Chemical Co. (Rockford, IL). 2.1. Cell culture

⇑ Corresponding authors. E-mail addresses: [email protected] (Å. Sjöholm), [email protected] (Q. Zhang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.106

Human coronary artery endothelial cells (HCAECs), purchased from Lonza (Walkersville, MD), were grown as described previously

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[8]. To examine the effects of in vitro diabetic glucolipotoxicity on gene expression, and the influence of exendin-4 thereupon, HCAECs were grown to 80% confluence, followed by incubation overnight in EGM medium containing 2% FBS and 2 mM L-glutamine. Exendin-4 (10 nM) was added 1 h prior to high glucose or palmitate (0.125 mM palmitate/0.25%BSA or vehicle) and the incubation was continued for 48 h. At the end of the incubation, the cells were washed and harvested. 2.2. RNA extraction and sample preparation High quality RNA was extracted from the cell lysate samples using Aurum total RNA mini kit (Bio-Rad, Stockholm, Sweden) according to the manufacturer’s instruction. The quality and purity of the extracted RNA was confirmed by agarose gel electrophoresis and analysis using the NanoDrop ND-1000 UV–Vis Spectrophotometer (Nano Technologies Wilmington, DE) [10]. 2.3. Gene expression profiling Gene expression was examined using the Human Endothelial Cell Biology RT2 Profiler™ PCR Array (SABiosciences, Qiagen) to detect the expression of 84 genes involved in permeability and vascular tone, angiogenesis, endothelial cell activation and endothelial cell injury by real-time PCR with a set of optimized real-time PCR primers. The PCR array performs gene expression analysis with real-time PCR sensitivity and the multi-gene profiling capability of a microarray. The cDNA template prepared from highly purified RNA (10 lg) was mixed with the ready-to-use PCR master mix, aliquot equal volumes to each well of the same plate, and then run the real-time PCR cycling program. The RT2 Profiler™ PCR Arrays includes built-in positive control elements for the proper normalization of the data (using data from species-specific housekeeping genes included in the pre-set wells), detection of genomic DNA contamination (using genomic DNA control primer set present in the wells to detect non-transcribed genomic contaminations), quality of the RNA samples (using reverse transcription controls), and general PCR performance (using positive PCR controls). 2.4. Western blotting Cells were washed with PBS and lysed in ice-cold lysis buffer containing 1 mM sodium fluoride, 1 mM sodium orthovanadate, protease inhibitor cocktail and 2% Triton X-100 in PBS, pH 7.5, on ice for 30 min. The cell lysate was then processed for Western blotting as described in [8]. 3. Results We recently showed that exendin-4 activates HCAECs, resulting in an enhanced cell proliferation [8] and a protection against lipotoxicity-induced apoptosis [11]. To gain insight into the molecular mechanisms of the beneficial actions of exendin-4, we performed gene expression profiling studies. The expression of approximately 40 genes with known or inferred functions were affected by high glucose or palmitate exposure, effects that were reversed by exendin-4, as demonstrated by microarray analyses. A large number of differentially expressed genes belong to several functional categories, such as vascular development and signal transduction (Table 1). Among these genes, glucose down-regulated Tie-2 (14-fold) and up-regulated its ligand angiopoietin-1 (Ang1, 3.8-fold), while both genes were up-regulated 11-fold and 4-fold, respectively, by exendin-4 at high glucose. In the presence of high glucose, exendin-4 significantly

Table 1 Exendin-4 regulates glucolipotoxicity-induced gene expression in HCAECs. Cells were incubated for 48 h in medium containing 5 mM or 30 mM glucose, or 5 mM glucose with palmitate (0.125 mM) or vehicle in the presence or absence of exendin-4. Gene expression profiling was analyzed by microarray analysis. Results are derived from five independent experiments and normalized by Interquartile Normalization. Fold changes in gene expression up-regulated or down-regulated under the different conditions are shown. Accession no

Gene name

Change in fold

Gene expression NM_000603 NM_001146 NM_000459 NM_001147 NM_000930 NM_000602 NM_000633 NM_000584 NM_000442 NM_001795 NM_002026 NM_006528

upregulated by exendin-4 at high glucose (30 mM) eNOS 3 Angiopoetin-1 4 Angiopoetin-receptor (TEK/Tie-2) 11 Angiopoetin-2 2.3 Plasminogen activator (PLAT/TPA) 39 Plasminogen activator inhibitor-1 (PAI-1) 25 BCL-2 4.8 IL8 6 PECAM (CD31) 10 Cadherin 5 (CDH5) 10 Fibronectin (FN1) 88 Tissue factor pathway inhibitor-2 (TFPI2) 2.2

Gene expression NM_000789 NM_004324 NM_000576 NM_001716 NM_000212 NM_002421 NM_004530 NM_000873 NM_002162

downregulated by exendin-4 at high glucose ACE (Angiotensin I-converting enzyme) BAX (BCL-2-associated X protein) IL-1 beta BLR1 (Burkitt lymphoma receptor 1) Integrin beta 3 (ITGB3) MMP1 (matrix metalloproteinase-1) MMP2 ICAM2 ICAM3

Gene expression NM_000459 NM_000930 NM_000602 NM_000584 NM_002026 NM_006528 NM_000442 NM_001795

downregulated by high glucose versus 5 mM glucose TEK (Tie-2) 14 Plasminogen activator (PLAT/TPA) 25 Plasminogen activator inhibitor-1 (PAI-1) 50 IL8 3.7 Fibronectin (FN1) 100 TFPI2 (Tissue factor pathway inhibitor-2) 6 PECAM (CD31) 17 Cadherin 5 (CDH5) 11

Gene expression NM_000379 NM_001146 NM_001147 NM_000552 NM_004324 NM_000576 NM_000201 NM_000873 NM_002162 NM_001716 NM_000212 NM_002421 NM_004530 NM_000789 NM_001275

up-regulated by high glucose versus 5 mM glucose Xantine dedydrogenase/ oxidase 8 Angiopoetin-1 (ANGPT1) 3.8 Angiopoetin-2 (ANGPT2) 1.7 von Willebrand factor (vWF) 9 BCL-2-associated X protein (BAX) 5.4 IL-1 beta 7.6 ICAM1 1.6 ICAM2 5.8 ICAM3 5.8 Burkitt lymphoma receptor 1 12 Integrin beta 3 (ITGB3) 19 Matrix metalloproteinase-1 (MMP1) 3.2 MMP2 4.3 Angiotensin I-converting enzyme (ACE) 4 Chromogranin A (CHGA) 13

Gene expression NM_000603 NM_000930 NM_000459

down-regulated by palmitate eNOS Plasminogen activator (PLAT/TPA) TEK (Tie-2)

3 5.6 4 17 17 4 4 5 4

3 2.2 2

Gene expression downregulated by exendin-4 with palmitate NM_000584 IL8

1.5

Gene expression upregulated by palmitate NM_000584 IL8 NM_000600 IL6

4.8 1.7

Gene expression upregulated by exendin-4 with palmitate NM_000603 eNOS

1.7

enhanced expression of the endothelial nitric oxide synthase (eNOS). Glucose was also shown to inhibit the expression of plasminogen activator (TPA or PLAT, 25-fold), which was enhanced 39-fold with exendin-4. Expression of platelet endothelial cell adhesion molecule (PECAM, 17-fold) and cadherin 5 (11-fold) were

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suppressed by high glucose. In the presence of exendin-4, the expression of both genes was enhanced 10 times, compared to high glucose alone. In addition, high glucose silenced the expression of the matrix protein fibronectin (100-fold), which was up-regulated 88-fold by exendin-4 under these conditions. Furthermore, at high glucose, exendin-4 up-regulated the apoptosis regulator protein Bcell lymphoma 2 (BCL-2, 4.8-fold), but inhibited the BCL-2-associated X protein BAX (5.6-fold), which was up-regulated by high glucose alone. Inflammatory factors, such as interleukin-1b (IL-1b, 7.6-fold) and intercellular adhesion molecule (ICAM-1–3) were up-regulated when the cells were incubated in high glucose. Exendin-4 inhibited the glucose-induced upregulation of IL-1b and ICAM-2 and -3 4 to 5 times under these conditions. The glucoseinduced expression of the inflammatory factors, together with the increased expression of chromogranin A (CHGA, 13-fold that induces ET-1 secretion), xantine dehydrogenase/oxidase (8-fold that mediates reactive oxygen species [ROS] production) and von Willebrand factor (vWF, 9-fold) that regulates platelet adhesion and fibrin formation observed in the present study may contribute to the glucotoxicity in endothelial cells [12], [13]. In contrast to high glucose, palmitate showed modest effects on gene expression in the HCAECs, except eNOS, which was down-regulated 3-fold by palmitate. Co-incubation of palmitate with exendin-4 resulted in a 1.7-fold increase in eNOS expression, compared to palmitate alone. Palmitate also down-regulated TPA (2.2-fold) and Tie-2 (2-fold), but up-regulated interleukins (IL-8, 4.8-fold; IL-6, 1.7-fold). Select protein products of the genes which were shown to be significantly affected by exendin-4 at high glucose or palmitate in the microarray analyses were evaluated by Western blotting. Consistent with the results from gene profiling studies, exendin4 significantly enhanced the expression of eNOS protein in the HCAECs in the presence of either high glucose or palmitate. After incubation of the cells with exendin-4 for 48 h, eNOS was increased approximately 40% at high glucose and 30% in the presence of palmitate, while expression of eNOS tended to be suppressed by palmitate, but not significantly affected by high glucose (Fig. 1). Incubation with high glucose also enhanced Ang1, but inhibited Tie-2 expression, although statistical significance was not reached. In the presence of exendin-4, Ang1 was significantly increased by approximately 48% at high glucose, compared to control (Fig. 2A).

A

Similarly, exendin-4 enhanced the expression of Tie-2 up to 131% at high glucose, compared to high glucose alone (Fig. 2B). Compared to the results from microarray analysis, Western blot did not reveal any significant change in TPA and fibronectin levels by high glucose (Fig. 3). Incubation of the cells with exendin-4 at high glucose induced more than 70% increase in expression of TPA (Fig. 3A). Under these conditions, the expression of fibronectin (Fig. 3B) was doubled in the presence of exendin-4, compared to controls. 4. Discussion Exendin-4 is a stable GLP-1 receptor agonist that improves glycemia in diabetes [14]. In addition to the actions on glucose homeostasis, exendin-4 exhibits beneficial effects on endothelial and cardiac functions [15,18]. Here we show that exendin-4 modulates the expression of multiple genes in human coronary endothelial cells, involving regulation of endothelial and cardiovascular functions under glucolipotoxic conditions, mechanisms that may contribute to the beneficial effects of the drug in diabetic patients with vascular complications.eNOS is one of the key regulators of endothelial function. Our data show that the expression of eNOS was up-regulated by exendin-4 in the presence of either palmitate or high glucose, as demonstrated by both microarray and Western blotting analyses. However, microarray analysis showed a suppressed eNOS expression by palmitate, which was not observed in Western blot. It has been reported that the half-life of eNOS protein can be altered in certain circumstances [16]. In addition, posttranscriptional regulation of eNOS mRNA stability plays an important role in the protein expression of eNOS [17]. Many factors have been found to be able to destabilize eNOS mRNA [17,18]. Whether palmitate treatment prolongs the half-life of the eNOS protein or influences its mRNA degradation in HCAECs requires further investigation. Release of NO is crucial to the function of the endothelium both under physiological and pathological conditions [19], [20]. Endothelial dysfunction, characterized by decreased NO production, is one of the early pathophysiologic events in development of atherosclerosis [21]. Endothelial-derived NO corrects tissue redox imbalance [22], mediates vasodilation [23], suppresses platelet

B - eNOS - -tubulin -

* Band density (% of control)

Band density (% of control)

100

50

0 Glucose Ex-4

*

150

150

5 -

30 -

30 +

mM

100

50

0 Palmitate Ex-4

-

+ -

+ +

Fig. 1. Exendin-4 upregulates eNOS protein expression of HCAECs at high glucose or in the presence of palmitate. Cells were incubated as described in Table 1. At the end of the incubation, cells were lysed and proteins were analyzed by Western blotting. The upper panels show representative blots. The bar plots are the summarized data derived from four (A) or seven (B) independent experiments after being normalized to a-tubulin bands from the same blots, and expressed as percentage changes ± SEM compared to controls (5 mM glucose).  denotes P < 0.05 for a chance difference vs controls by ANOVA.

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A

B

- Ang1

- Tie-2 - -tubulin

- -tubulin

150

*

*

*

300

Ang1

Band density (% of control)

Band density (% of control)

200

100 50 0 Glucose Ex-4

5 -

30 -

30 +

Tie-2 200

100

0 Glucose Ex-4

mM

5 -

30 -

30 +

mM

Fig. 2. Exendin-4 stimulates protein expression of Ang1 and its receptor Tie-2. Cells were incubated as described in Table 1. Protein expression of Ang1 and Tie-2 was analyzed by Western blotting. The upper panels show representative blots. The bar plots are the summarized data derived from seven (A) or six (B) independent experiments after being normalized to a-tubulin bands from the same blots, and expressed as percentage changes ± SEM compared to controls (5 mM glucose).  denotes P < 0.05 for a chance difference vs controls by ANOVA.

A

B

- TPA

- Fibronectin

- α-tubulin

* TPA

150 100 50 0 Glucose Ex-4

*

250

Band density (% of control)

Band density (% of control)

200

- α-tubulin

200

Fibronectin

150 100 50

5

30

30

-

-

+

0 Glucose Ex-4

5 -

30 -

30 +

Fig. 3. Exendin-4 stimulates protein expression of TPA and fibronectin. Cells were incubated as described in Table 1. Protein expression of TPA and fibronectin was analyzed by Western blotting. The upper panels show representative blots. The bar plots are the summarized data derived from five (A) or seven (B) independent experiments after being normalized to a-tubulin bands from the same blots, and expressed as percentage changes ± SEM compared to controls (5 mM glucose).  denotes P < 0.05 for a chance difference vs controls by ANOVA.

aggregation [24] and inhibits adhesion of neutrophils [25], thus preventing atherogenesis [26]. NO protects endothelial cells against proinflammatory and proatherosclerotic factors, such as ROS and angiotensin II [27]. Hence, the stimulatory effect of exendin-4 on eNOS expression under the glucolipotoxic conditions shown herein suggests advantageous effects of the drug in diabetic patients with cardiovascular diseases. Endothelial cell proliferation is an important step in angiogenesis as observed in response to ischemia [28]. The present study shows that exendin-4 enhances the expression of genes promoting angiogenesis at high glucose. These include Ang1 and its receptor Tie-2 (also known as TEK) [29], a trans-membrane tyrosine kinase that is uniquely expressed in endothelial cells, essential to angiogenesis [30] and endothelial cell survival [31]. Tie-2 expression is attenuated in diabetic db/db mice subjected to myocardial ischemia [32]. The suppressed expression of Tie-2 in diabetic mouse model is supposed to be due to hyperglycemia [32].

Entirely consistent with this finding, incubation of HCAECs with high glucose resulted in down-regulation of Tie-2 expression. Importantly, exendin-4 enhanced the expression of Ang1 and reversed the suppressed expression of Tie-2 at high glucose by over-expression of the receptor, indicating a beneficial role of exendin-4 on angiogenesis in diabetes. This effect suggests a role of exendin-4 in compensation of the blood supply through establishment of collateral circulation during ischemia, especially with hyperglycemia. The possible action of exendin-4 on angiogenesis is also supported by its effect on expression of cell adhesion molecules involved in angiogenesis (such as cadherin, platelet endothelial cell adhesion molecule [PECAM]) and matrix proteins (such as fibronectin) whose expression was inhibited by high glucose in the HCAECs. Some of these changes were also confirmed at the protein level by Western analysis. Integrin-mediated cell-cell interactions and fibronectin play major roles in cell adhesion, migration,

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differentiation, growth and survival in different cell types [33], including endothelial cells [34]. Fibronectin contributes to angiogenesis, as angiogenic process is strongly dependent on the interaction of endothelial cells with fibronectin to stabilize endothelial cell-cell connection [35]. Another ligand of integrins up-regulated by exendin-4 is PECAM-1, which is required for the trans-endothelial migration of leukocytes [36]. Signaling through PECAM-1 has been implicated in thrombus formation in the circulation [37]. Exendin-4 also enhanced the expression level of calcium-dependent adhesion molecule cadherin 5, the main structural protein of the intercellular junctions between endothelial cells [38]. Exendin-4 normalized the suppressed expression of these genes under such conditions, indicating extensive actions of the drug on vascular function in diabetes. Angiotensin I-converting enzyme (ACE) expression was up-regulated by high glucose in the endothelial cells, while exendin-4 suppressed expression of this gene at high glucose. Such effect of exendin-4 may be clinically significant as increased ACE activity is associated with thrombosis and coronary heart disease, following an increased production of plasminogen activator inhibitor-I (PA-1) and platelet aggregability [39]. To this end, human ACE has long been used as a target for treatment of hypertension and its related cardiovascular diseases [40]. Our results also revealed that expression of plasminogen activator in HCAECs was suppressed by high glucose and exendin-4 reversed this effect. The fibrinolytic compensation for hypercoagulability is impaired in obese patients with type 2 diabetes [41]. Type 2 diabetic patients have increased hypercoagulability [42] and reduced fibrinolysis [43]. The hypercoagulability in hyperglycemia is believed to be due to an increased formation of advanced glycation end products (AGE) and diminished endothelial NO [44], [45]. Downregulation of plasminogen activator expression by high glucose may promote the reduced degradation of fibrin-containing thrombi in hyperglycemia. Apoptosis of vascular endothelial cells plays an important role in the development of atherosclerosis [46]. Endothelial disruption and damage subject the arterial wall at greater risk for vascular disease [47] [48]. In addition, inflammatory processes are involved in evolution of atherosclerosis and plaque development [49]. Our data show that exendin-4 regulates expression of the genes involved in endothelial cell apoptosis and inflammation. BCL-2–associated X protein, Bax, is a pro-apoptotic protein responsible for induction of caspase activation and apoptosis through interaction with mitochondrial membrane and the consequent release of pro-apoptotic proteins and ER Ca2+ [50]. The expression of Bax was enhanced at high glucose in the HCAECs and was completely normalized by exendin-4. Endothelial cells express cytokines and CAMs [51] that participate in molecule interactions and are pivotal to the inflammatory response [51], during thrombosis. The normalizing effect of exendin-4 on the expression of these genes may suggest a salutary role of the drug in anti-thrombosis. Taken together, our findings suggest that exendin-4 may improve endothelial function in part through regulating expression of genes involved in angiogenesis, inflammation and thrombogenesis by reversing glucolipotoxic gene dysregulation. As endothelial dysfunction appears to be an early indicator of vascular damage, and predicts both progression of atherosclerosis [52] and incidence of cardiovascular events [53], exendin-4 and possibly other incretin-based strategies may provide additional cardiovascular treatment benefits beyond improved glycemic control. Acknowledgments The gracious financial support from Stiftelsen Olle Engkvist Byggmästare, the Juvenile Diabetes Research Foundation Interna-

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