Glucagon-Like Peptide 1 Protects against Hyperglycemic-Induced Endothelial-to-Mesenchymal Transition and Improves Myocardial Dysfunction by Suppressing Poly(ADP-Ribose) Polymerase 1 Activity
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Under high glucose conditions, endothelial cells respond by acquiring fibroblast characteristics, that is, endothelial-to-mesenchymal transition (EndMT), contributing to diabetic cardiac fibrosis. Glucagon-like peptide-1 (GLP-1) has cardioprotective properties independent of its glucose-lowering effect. However, the potential mechanism has not been fully clarified. Here we investigated whether GLP-1 inhibits myocardial EndMT in diabetic mice and whether this is mediated by suppressing poly(ADP-ribose) polymerase 1 (PARP-1). Streptozotocin diabetic C57BL/6 mice were treated with or without GLP-1 analog (24 nmol/kg daily) for 24 wks. Transthoracic echocardiography was performed to assess cardiac function. Human aortic endothelial cells (HAECs) were cultured in normal glucose (NG) (5.5 mmol/L) or high glucose (HG) (30 mmol/L) medium with or without GLP-1analog. Immunofluorescent staining and Western blot were performed to evaluate EndMT and PARP-1 activity. Diabetes mellitus attenuated cardiac function and increased cardiac fibrosis. Treatment with the GLP-1 analog improved diabetes mellitus-related cardiac dysfunction and cardiac fibrosis. Immunofluorescence staining revealed that hyperglycemia markedly increased the percentage of von Willebrand factor (vWF)+/alpha smooth muscle actin (α-SMA)+ cells in total α-SMA+ cells in diabetic hearts compared with controls, which was attenuated by GLP-1 analog treatment. In cultured HAECs, immunofluorescent staining and Western blot also showed that both GLP-1 analog and PARP-1 gene silencing could inhibit the HG-induced EndMT. In addition, GLP-1 analog could attenuate PARP-1 activation by decreasing the level of reactive oxygen species (ROS). Therefore, GLP-1 treatment could protect against the hyperglycemia-induced EndMT and myocardial dysfunction. This effect is mediated, at least partially, by suppressing PARP-1 activation.
Longstanding diabetes leads to pathogenesis of fibrotic disorders, such as diabetic cardiac fibrosis and renal fibrosis. The resultant fibrosis disrupts the normal architecture of the affected organs, ultimately leading to their dysfunction and failure (1, 2, 3). Fibroblasts are major contributors to extracellular matrix accumulation in tissue fibrosis. There is increasing evidence to show that a significant fraction of these interstitial fibroblasts is derived from the endothelium, a process called endothelial-to-mesenchymal transition (EndMT) (4, 5, 6). Recent studies suggest that the EndMT could contribute to the progression of diabetic cardiac fibrosis and renal fibrosis. Widyantoro et al. (7) showed that 15–20% of fibroblasts coexpressed both the endothelial marker CD31 and the fibroblast marker fibroblast-specific protein 1 (FSP1) in the hearts of diabetic WT mice. Zeisberg et al. (5) found that 30–50% of fibroblasts in the kidneys of diabetic mice coexpressed the endothelial marker CD31 along with FSP1-specific and alpha smooth muscle actin (α-SMA)-specific markers of fibroblasts and myofibroblasts. The current studies also indicated that blockade of EndMT could prevent the progression of organ fibrosis (7, 8, 9).
Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear enzyme that can be activated by oxidative DNA damage, mainly reactive oxygen species (ROS) (10). On binding to damaged DNA, PARP-1 cleaves nicotine amide adenine dinucleotide (NAD+) to produce nicotin-amide and ADP-ribose (11). When DNA damage is mild, PARP-1 participates in the DNA repair process. However, excessive activation of PARP-1 by stimuli, such as hyperglycemia, leads to intracellular depletion of NAD+ and ATP, thus resulting in cellular energy crisis, irreversible cytotoxicity and cell death (12,13). PARP-1 activation also facilitates diverse inflammatory responses by promoting inflammation-relevant gene expression, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α and endothelin-1 (14, 15, 16). These all play a significant role in fibrotic disorders. Recently, Rieder et al. showed that the combination of transforming growth factor (TGF)-1, IL-1 and TNF-α could induce EndMT in human intestinal microvascular endothelial cells (17). Widyantoro et al. (7) also demonstrated that endothelin-1 gene silencing could inhibit hyperglycemia-induced EndMT in cultured endothelial cells. However, it is still unclear whether PARP-1 inhibition could suppress hyperglycemia-induced EndMT.
Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal L cells in response to food intake. Emerging evidence has shown the antiinflammatory effects of GLP-1 on the cardiovascular system. In fact, it has been reported that GLP-1 analogs could ameliorate cardiac steatosis in diet-induced obesity (DIO) mice or type 2 diabetes mice (18,19). However, in those studies, after systemic administration of GLP-1 analogs, the mice also showed an improvement in blood glucose and insulin sensitivity, both of which play significant roles in cardiac fibrosis (20,21). Thus, the molecular mechanisms by which GLP-1 caused the antifibrotic effects are still controversial. We recently demonstrated that GLP-1 could protect microvascular endothelial cells by inactivating the PARP-1-inducible nitric oxide (NO) synthase-NO pathway (22). On the basis of these findings, we wondered whether GLP-1 could inhibit myocardial EndMT in diabetic mice and whether this was mediated by inactivating PARP-1. Accordingly, we designed this study to determine the protective effects of GLP-1 on hyperglycemia-induced EndMT and to characterize the underlying molecular mechanism.
Materials and Methods
Reagents and Antibodies
Human aortic endothelial cell (HAEC) medium (endothelial cell medium [ECM]) and fetal bovine serum (FBS) were Gibco products (Thermo Fisher Scientific Inc., Waltham, MA, USA); exenatide acetate (GLP-1 analog) was from Huayi BIO-Lab (Shanghai, China); N-acetyl-L-cysteine (NAC) and diacetate (DCFH-DA) were from Sigma-Aldrich (St. Louis, MO, USA); Lipofectamine 2000 was from Life Technologies (Thermo Fisher Scientific); small interfering RNA duplexes against PARP-1 (siPARP-1), small interfering RNA duplexes against Snail (siSnail) and the negative control (si-NC) were from GenePharma (Shanghai, China). The primary antibody β-actin, VE-cadherin and poly(ADP-ribose) (PAR) were from BD Biosciences (San Jose, CA, USA). Matrix metalloproteinase (MMP)-2, MMP-9, and collagen I and III were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were Invitrogen products (Thermo Fisher Scientific).
Cell Culture and Treatment
HAECs (ATCC, Manassas, VA, USA) were cultured in ECM supplemented with 5% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C. The culture medium was changed to a serumfree solution for 12 h, and then cells were treated with 5 mmol/L d–glucose (normal glucose [NG]) or 30 mmol/L d–glucose (high glucose [HG]) for 3 d. The medium was changed every 24 h. siPARP-1, siSnail and si-NC duplexes were transfected into HAECs by using Lipofectamine 2000. Then cells were exposed to HG with or without 50 nmol/L GLP-1, or 10 nmol/L NAC (an inhibitor of ROS) after a 24-h transfection for 72 h. We used cells at no more than passage 4.
Western Blot Analysis
Equal amounts of protein were separated on 10% SDS-PAGE and electrotransferred onto nitro-cellulose membranes (Amersham Biosciences [GE Healthcare Bio-Sciences, Pittsburgh, PA, USA]), which were blocked with 5% nonfat milk for 2 h at room temperature, washed three times for 10 min, and incubated overnight at 4°C with primary antibodies against PARP-1, α-SMA, Snail, β-actin, VE-cadherin, PAR, MMP-2, MMP-9 or collagen I or III and then horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were visualized by chemiluminescence (Millipore, Billerica, MA, USA).
ROS Production Assay
HAECs were washed twice with phosphate-buffered saline (PBS) and then incubated with 2,7-dichlorofluoresceindiacetate (H2DCF-DA) for 30 min at 37°C. ROS level was determined by the oxidative conversion of H2DCF-DA to fluorescent dichlorofluorescein on reaction with ROS in cells. HAECs were incubated with dihydroethidium for 30 min at room temperature. After a washing with PBS, fluorescent signals (ROS, 488 nm) were acquired by using a Zeiss LSM 710 confocal microscope. Three independent experiments were performed.
The relative amount of MMP-2 and MMP-9 protein in HAECs was measured by gelatin zymography. The culture supernatant was harvested and mixed with a gel sample buffer (0.5 mol/L Tris-HCl, glycerol, 10% sodium dodecyl sulfate [SDS], β-mercaptoethanol and 0.5% bromophenol blue). An amount of 10 µg protein was separated by SDS-polyacrylamide gel electrophoresis with 0.1% gelatin (Sigma-Aldrich). After electrophoresis, gels were washed with 50 mmol/L Tris buffer containing 2.5% Triton X-100. Gels were incubated for an additional 24 h in incubation fluid (50 mmol/L Tris buffer, 10 mmol/L CaCl2 and 200 mmol/L NaCl) and then stained with 0.5% Coomassie blue containing 30% methanol and 10% glacial acetic acid and destained in 45% methanol/10% acetic acid/H2O. White bands on a blue background indicated zones of digestion corresponding to the presence of different MMPs. Gels were scanned, and analysis of bands involved use of ImageJ (NIH, Bethesda, MD, USA).
C57BL/6J male mice (n = 80, 8 wks old, 25–30 g) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), housed at a constant temperature (24°C) and given a normal diet with free access to water. Nondiabetic mice were used as controls (n = 26). Diabetes mellitus (DM) was induced by multiple lowdose (50 mg/kg) intraperitoneal injection of streptozotocin (STZ) as described (23). Blood samples for glucose measurements were taken from the tail vein ∼48 h after STZ injection. All mice with nonfasting glucose levels of 13.8 mmol/L were considered diabetic and received no insulin treatment. One month after diabetes onset, diabetic mice were randomly divided into two groups for treatment (n = 26 each): daily saline (DM alone) and GLP-1 analog (24 nmol/kg daily; DM + GLP-1) (18). After 24 wks of diabetes, mice were sacrificed. Plasma samples were collected by heart puncture by using a heparinized syringe after mice were anesthetized. Blood glucose concentrations were measured by using a Breese 2 glucose meter (Bayer HealthCare, Leverkusen, Germany). Plasma insulin levels were measured with a Linco insulin ELISA (enzyme-linked immunosorbent assay) kit. All experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (24) and in compliance with Shandong University.
Cardiac Function Measurement
Transthoracic echocardiography was performed 24 wks after STZ injection. Mice were anesthetized with isoflurane mixed with O2 at a flow rate of 5 psi. Cardiac diameter and function were measured by use of the Vevo770 imaging system equipped with a high-frequency ultrasound probe. M-mode images of the parasternal long- and short-axis views of the left ventricle were obtained. Left ventricular ejection fraction and fractional shortening were calculated by using Vevo 770 software.
Immunocytochemistry and Immunohistochemistry
Frozen heart tissues were transversely cut at 5-µm thickness and fixed in 100% acetone at −20°C for 10 min. HAECs were fixed in 4% paraformaldehyde for 20 min. Then tissue sections or cells were permeabilized in 0.03% Triton X-100 for 15 min. After being blocked in 5% bovine serum albumin for 30 min at room temperature, cells were incubated with primary antibodies for PAR, Snail, α-SMA or VE-cadherin at 4°C overnight and then with a fluorescence dye-conjugated secondary antibody. Nuclei were visualized with 4′6-diamidino-2-phenylindole-2HCl (DAPI) (Santa Cruz Biotechnology).
Paraformaldehyde (4%) fixed hearts were bisected transversely at the midventricular level, embedded in paraffin and cut into 5-µm-thick sections and then stained with Masson staining. Dark green-stained collagen fibers were quantified as a measure of fibrosis and examined by light microscopy in a blinded manner. To normalize the perivascular collagen area around vessel with different sizes, perivascular fibrosis was calculated as the ratio of the fibrotic area surrounding the vessels to the total vessel area. The intramyocardial and perivascular collagen area were quantified digitally by using Image-Pro Plus 6.2 (Media Cybernetics Inc., Rockville, MD, USA).
Quantitation of Fibroblasts of Endothelial Cell Origin
Cells that coexpressed both the endothelial marker von Willebrand factor (vWF) and the myofibroblast marker α-SMA were counted in the hearts. Five areas were randomly selected and analyzed at 400× magnification in each of five sections from each heart. The percentage of vWF+/α-SMA+ cells in total α-SMA+ cells and the percentage of α-SMA+ cells in total cells were determined.
Cell Migration Assay
Cell motility was examined by scratch assay as described (25). Cells were seeded in 12-well plates at 1.0 × 105 cells per well and incubated plates at 37°C until cells reached 100% confluence. An artificial gap was generated by scratching with use of a 200-µm pipette tip. Photographic images were taken immediately and after 12 h with a digital camera system. The software program HMIAS-2000 was used to calculate the cell migration distance (µm). Each experiment was repeated at least three times.
Data are expressed as mean ± standard deviation (SD). An independent Student t test was used for comparison between groups and one-way analysis of variance for comparison among multiple groups with post hoc Bonferroni correction. SPSS v12.0 for Windows (SPSS, Chicago, IL) was used for analysis. p < 0.05 was considered statistically significant.
Basic Characteristics of Mice
Basic parameters of mice with DM and GLP-1 analog treatment.
Controls (n = 9)
DM (n = 9)
DM+GLP-1 (n =9)
Fasting blood glucose (mmol/L)
56 ± 1.8
16.1 ± 4.5a
15.5 ± 3.5b
Nonfasting blood glucose (mmol/L)
8.3 ± 2.3
25.9 ± 7.7a
24.3 ± 4.6b
Systolic blood pressure (mmHg)
112 ± 5.1
130 ± 9.2a
125 ± 7.4b
Diastolic blood pressure (mmHg)
87 ± 5.2
102 ± 4.6a
99 ± 72b
Body weight (g)
28.7 ± 3.4
21.8 ± 5.1a
22.6 ± 4.5b
Fasting insulin (ng/mL)
0.82 ± 0.19
0.15 ± 0.07a
0.18 ± 0.05b
Nonfasting insulin (ng/mL)
2.11 ± 0.55
0.21 ± 0.15a
0.25 ± 0.12b
GLP-1 Attenuates Myocardial Fibrosis and Cardiac Dysfunction in Mice with DM
GLP-1 Inhibits HG-Induced EndMT In Vitro and In Vivo
GLP-1 Decreased HG-Induced PARP-1 Expression and Activity by Suppressing ROS Production in HAECs
GLP-1 Inhibited HG-Induced EndMT by Suppressing PARP-1 Activation
GLP-1 Inhibits HG-Induced EndMT by Suppressing PARP-1-Snail Interaction
GLP-1 Reduced HG-Induced Collagen I and III and MMP-2 and MMP-9 Expression by Inhibiting EndMT
GLP-1 Inhibits HG-Induced Cell Migration by Suppressing EndMT
Glucagon-like peptide-1 (GLP-1) has cardioprotective properties independent of its glucose-lowering effect. However, the underlying mechanisms have not been fully elucidated. Recent studies showed that EndMT plays a significant role in diabetes mellitus-induced cardiac dysfunction. Here we report that GLP-1 treatment could protect against hyper-glycemia-induced EndMT and myocardial dysfunction. This effect is mediated, at least partially, by suppressing PARP-1 activation.
GLP-1, an intestinally derived hormone, reduces blood glucose levels by increasing the secretion of insulin (28). We and others have previously observed that GLP-1 can exert significant antiinflammatory effects through GLP-1-specific receptors that are found in the heart, vascular smooth cells and endothelial cells (29, 30, 31). In fact, it has been reported that GLP-1 analogs can ameliorate cardiac steatosis in DIO mice or type 2 diabetes mice (18,19). However, in those studies, after systemic administration of GLP-1 analog, the mice also showed an improvement in blood glucose and insulin sensitivity, both of which play significant roles in cardiac fibrosis (20,21). Thus, the molecular mechanisms by which GLP-1 caused the antifibrotic effects are still controversial. In this study, we hypothesize that GLP-1 analog treatment has a protective effect against cardiac fibrosis via an insulin- and glucose-independent mechanism in a STZ-induced type 1 diabetes model. Similar to previous studies (7,9), the DM group showed significantly increased intramyocardial and perivascular fibrosis and impaired cardiac diastolic function compared with controls. Chronic GLP-1 analog administration markedly reduced myocardial fibrosis and cardiac dysfunction in STZ-induced diabetic mice.
To elucidate the mechanism through which GLP-1 reduces myocardial fibrosis and improves cardiac function, we focused on myofibroblasts in the fibrotic area. Increasing evidence shows that a significant fraction of these myofibroblasts are derived from the endothelium via a process called endothelial-to-mesenchymal transition (EndMT) (32,33). Fibroblasts derived from the endothelial lineages can also function as resident tissue fibroblasts that produce massive ECM proteins and ECM degrading enzymes in fibrotic disorders. Chronic hyperglycemia is an important initiator of EndMT. In this study, we observed that in the cardiac tissue of diabetic mice, the proportion of α-SMA+ cells that coexpress vWF was much greater than that in nondiabetic mice. In addition, GLP-1 treatment significantly reduced the percentage of vWF+/α-SMA+ cells. The reason that we chose vWF instead of VE-cadherin in vivo is because vWF is an endothelial cell marker expressed in plasma, which could reflect the proportion of transitioned cells more clearly in vivo compared with the markers expressed on the membrane under a confocal microscope. Several previous studies also used this marker to detect EndMT in vivo (8,17), which could clearly support our results. To further confirm our in vivo observations, we conducted in vitro experiments with HAECs incubated with HG. Similar to our in vivo study, incubation of HAECs with HG resulted in decreased levels of the endothelial marker VE-cadherin (also endothelial cell-to-cell regulator) and increased expression of the myofibroblast (the functional fibroblast) marker α-SMA. These effects were markedly reversed with GLP-1 analog treatment.
PARP-1, the DNA repair-associated nuclear enzyme, is involved in the development of various diabetic complications, including diabetic cardiomyopathy, diabetic nephropathy, diabetic cystopathy and diabetic retinopathy (23,34,35). Hyperglycemia can lead to overproduction of mitochondrial ROS, which then activates PARP-1 (13). PARP-1 activation can induce inflammation-relevant factors such as IL-1, endothelin-1, TGF-1 and Smad3 (14,15), which are closely related to EndMT. In the present study, we found that HG not only increased PARP-1 activation and Snail (the key regulator that mediates EndMT) expression, it also facilitated the colocalization of PAR and Snail in the nuclei. Furthermore, PARP-1 gene silencing reduced Snail expression and inhibited hyperglycemia-induced EndMT in HAECs. According to a recent study, PARP-1, but not PARP-2, increased Snail protein stability by poly(ADP-ribosyl)ation of Snail in human melanoma cells (36). Thus, our results suggest that PARP-1 may act as an upstream regulator in hyperglycemia-induced EndMT.
On the basis of our previous study, GLP-1 can effectively protect MS1 cells against oxidized low-density lipoprotein-induced apoptosis by inactivating PARP-1 (22). There is also growing evidence to support that GLP-1 can reduce hyperglycemia-induced ROS accumulation via protein kinase A-mediated inhibition of NAD(P)H oxidases (37, 38, 39) and that ROS is a significant stimulus for PARP-1 activation (13). These findings led us to further investigate the potential mechanisms of GLP-1 in HG-induced PARP-1 activation and EndMT. In this study, we found that GLP-1 attenuated PARP-1 activity and Snail expression in HAECs under HG conditions. In addition, immunofluorescent staining showed that GLP-1 inhibited hyperglycemia-induced PAR and Snail colocalization in the nuclei of HAECs. This inhibitory effect of GLP-1 on PARP-1 may be secondary to reduced ROS production.
Although endothelial cells acquired a myofibroblast-phenotype under HG conditions, we were not sure whether these transitioned cells could function as fibroblasts. Therefore, we performed Western blot and gelatin zymography to test the function of these transitioned cells in vitro. As predicted, endothelial cells incubated under HG conditions could produce massive amounts of ECM proteins (collagen I and III) and ECM degradative enzymes (MMP2 and MMP9). After Snail, the key regulator of EndMT, was knocked out, all these effects were abolished. These results confirm our hypothesis that transitioned cells not only acquire the myofibroblast-phenotype, but also the myofibroblast-function. Similar results were obtained when cells were treated with GLP-1 or PARP-1 siRNA.
EndMT was preceded by loss of endothelial cell-to-cell contact, as shown by decreased VE-cadherin expression and increased ECM degrading enzymes such as MMP-2 and MMP-9 (7,27). The disaggregated endothelial cells then start to alter their structure, migrate to surrounding sites, decrease expression of endothelial marker and acquire mesenchymal characteristics. Therefore, we performed scratching assay to evaluate the cell migration ability. In concordance with previous data (7), we observed that hyperglycemia promotes endothelial cell migration, with the transitioned cells having the highest migration capability. Our results also indicated that GLP-1 treatment or PARP-1 gene silencing significantly prevented cell migration. This effect is probably associated with increased VE-cadherin and decreased MMP-2 and MMP-9 activity.
GLP-1 analogs can protect against hyperglycemia-induced EndMT and myocardial dysfunction. This effect may be mediated, at least partially, by suppressing PARP-1 activation. These findings offer an insight into the pathogenesis of diabetic organ fibrosis and may potentially provide a new therapeutic approach to prevent or retard diabetic cardiac fibrosis.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the National 973 Basic Research program of China (no. 2015CB553604), the National Natural Science Foundation of China (81170275),81370412, and 91439201) and the State Program of National Natural Science Foundation of China for Innovative Research Group (81321061).
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