Effects of Downregulation of MicroRNA-181a on H2O2-Induced H9c2 Cell Apoptosis via the Mitochondrial Apoptotic Pathway

Glutathione peroxidase-1 (GPx1) is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. This study aims to identify a microRNA (miRNA) that targets GPx1 to maintain redox homeostasis. Dual luciferase assays combined with mutational analysis and immunoblotting were used to validate the bioinformatically predicted miRNAs. We sought to select miRNAs that were responsive to oxidative stress induced by hydrogen peroxide (H2O2) in the H9c2 rat cardiomyocyte cell line. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-181a in H2O2-treated H9c2 cells was markedly upregulated. The downregulation of miR-181a significantly inhibited H2O2-induced cellular apoptosis, ROS production, the increase in malondialdehyde (MDA) levels, the disruption of mitochondrial structure, and the activation of key signaling proteins in the mitochondrial apoptotic pathway. Our results suggest that miR-181a plays an important role in regulating the mitochondrial apoptotic pathway in cardiomyocytes challenged with oxidative stress. MiR-181a may represent a potential therapeutic target for the treatment of oxidative stress-associated cardiovascular diseases.


Introduction
Growing evidence demonstrates that increased levels of reactive oxygen species (ROS) are associated with a variety of cardiovascular diseases [1]. Although ROS can originate from different organelles, mitochondria are considered to be the main producers of ROS. Approximately 80% of anion superoxide (O 2 • ) is produced by mitochondria [2]. In the heart, approximately 30% of the total volume is occupied by mitochondria, and thus the heart is easily subjected to oxidative damage by ROS [3]. To combat the deleterious effects of ROS, mitochondria have evolved an intrinsic antioxidant defense network that mainly consists of the superoxide dismutase (SOD), NADH, and a complete glutathione redox system, formed by glutathione reductase, reduced glutathione (GSH), and glutathione peroxidase (GPx) [4].
GPx converts H 2 O 2 and lipid hydroperoxides into water, using GSH as an electron donor. GPx1 is the main isoform, produced in all tissues and expressed in both the cytosol and the mitochondrial matrix. GPx1 prevents the formation of the highly reactive hydroxyl radical [5]. Several genetically modified animal models have been used to show the protective role of GPx1 in cardiac damage caused by ischemia-reperfusion injury [6][7][8]. Recent findings also indicate that the lack of GPx1 contributes to the risk of atherosclerosis and cardiovascular disease. Mice deficient in GPx1 (GPx1 −/− /ApoE −/− ) developed significantly more atherosclerosis than the control apolipoprotein E-deficient mice [9,10]. Additionally, transgenic mice overexpressing Gpx1 were protected from aging-related enhanced susceptibility to venous thrombosis compared with wild-type mice [11]. Gpx1 also plays a pivotal role in the protection against angiotensin II-induced vascular dysfunction [12].
The posttranscriptional regulation of GPx1 expression in oxidative stress via nuclear factor B (NF B) and activator protein 1 (AP-1) has been shown by many investigators [13,14]. However, the posttranscriptional mechanism of 2 Oxidative Medicine and Cellular Longevity Gpx1 in response to H 2 O 2 -mediated oxidative stress in cardiomyocytes has not been thoroughly studied. MicroRNAs (miRNAs) are one example of a translational mechanism for the regulation of a large number of developmental and physiological processes in the heart [15]. An miRNA is a small, single-stranded RNA that is approximately 22 nucleotides (nt) long. miRNAs are widely distributed and induce both messenger RNA (mRNA) degradation and the suppression of protein translation based on sequence complementarity between the miRNA and its target [16].
The H9c2 rat cardiomyocyte cell line was chosen in the present study as this cell line retains the characteristics of isolated primary cardiomyocytes [17]. We investigated the role of miR-181a in regulating H9c2 cell apoptosis and modulating the mitochondrial apoptotic pathway in the setting of oxidative stress. We found that miR-181a is upregulated in cardiomyocytes with oxidative stress and that the downregulation of miR-181a significantly inhibited the H 2 O 2induced cellular apoptosis, ROS production, mitochondrial structure disruption, and activation of key signaling proteins in the mitochondrial apoptotic pathway. These protective effects were mediated at least partially through the direct targeting of Gpx1.

Cell Culture and Determination of Cell
Viability. HEK293 cells, myogenic L6 cells, and H9c2 cells (from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 5 g/L glucose and 15% (v/v) fetal bovine serum. The cells were maintained in a humidified 37 ∘ C incubator with 5% CO 2 , supplied with fresh medium every 3 days, and subcultured before reaching confluence. The H9c2 cells were seeded in 96-well microtiter plates and treated with different concentrations of H 2 O 2 for 2 h. The MTT assay was used to determine cell viability, following the manufacturer's protocols.
The 3 UTR of the Gpx1 gene was amplified by PCR from total RNA extracted with a Genomic DNA Extraction kit (TaKaRa Bio Inc., Tokyo, Japan) from myogenic L6 cells. The sequences of the primers for the Gpx1 3 UTR and mutant Gpx1 3 UTR were as follows: R-Gpx1-3 UTR-F, CCGCTCG AGCCTAAGGCATTCCTGGTATCTGG; R-Gpx1-3 UTR-R, GAATGCGGCCG CTTCTTTGACAT-TCAGCACTTTATTC; and R-Gpx1-3 UTR-Mut-R, GAA-TGCGGC CGCTTCTTTGAGAATGAGCACTTTATTCT-TAG. The bold letters in the primers indicate XhoI and SalI restriction sites. The PCR products were excised with XhoI and NotI and cloned into the pmiR-RB-REPORT vector (GuangzhouRuiBio Corp., Guangzhou, China). This plasmid contains hRluc (synthetic renilla luciferase gene), encoding renilla luciferase as the reporter, and hluc (synthetic firefly luciferase gene), encoding firefly luciferase as the internal control. The recombinant plasmid pmiR-RB-Gpx1-3 UTR was confirmed by restriction enzyme digestion and DNA sequencing.

Measurement of Lactate Dehydrogenase (LDH) and
Malondialdehyde (MDA) Levels. H9c2 cells were transfected with miRNAs for 6 h and cultured for 24 h. H 2 O 2 (400 M) was added for the last 2 h. Subsequently, the cells were harvested and lysed. LDH release and MDA content were measured using commercial kits (Jiancheng Bioengineering Institute, China), according to the manufacturer's instructions.

Intracellular Reactive Oxygen Species (ROS) Assay.
H9c2 cells were grown to confluence in a 96-well plate and then transfected with miRNAs for 6 h and cultured for 24 h. H 2 O 2 (400 M) was added for the last 2 h. The cells were then incubated with 2 ,7 -dichlorofluorescin-diacetate (DCFH-DA, Sigma) at 37 ∘ C for 30 min. The DCFH-DA stain detecting ROS production was observed using a fluorescence microscope (Nikon, Japan). Fluorescence was read at 485 nm for excitation and 530 nm for emission with an Infinite M200 Microplate Reader (Tecan, Switzerland).

Annexin V and PI Binding
Assay. The Annexin V and PI Fluorescein Staining kits (Bender MedSystems, Austria) were utilized to measure H9c2 cell apoptosis, according to the manufacturer's instructions. Briefly, a single-cell suspension was prepared and cultured in a six-well plate at a density of 1 × 10 5 /well. Twenty-four hours after transfection, cells from each group were collected and resuspended in two Oxidative Medicine and Cellular Longevity 3 hundred microliters of 1x binding buffer. The cells were then incubated with annexin V (1 : 20) for 3 min followed by incubation with propidium iodide (PI, 1 mg/mL) for 15 min. The apoptosis rate was evaluated by flow cytometry (BD).

Measurement of Mitochondrial Membrane Potential.
H9c2 cells were grown on cover slips and incubated with 5 g/mL JC-1 dye (Enzo Life Sciences, USA) at 37 ∘ C for 30 min. The cells were analyzed immediately with a fluorescence microscope. JC-1 accumulates in the mitochondria, selectively generating an orange J-aggregate emission profile (590 nm) in healthy cells. However, upon cell injury, the membrane potential decreases, and JC-1 monomers are generated, resulting in a shift to green emission (529 nm). The orange and green fluorescence intensities were detected using an Infinite M200 Microplate Reader. The Δ m of the H9c2 cells in each treatment group was calculated as the ratio of orange to green fluorescence and expressed as a multiple of the level in the control group.

Quantitative Real-Time PCR.
Total RNA was extracted from H9c2 cells using the Trizol Reagent (Invitrogen). The RT primers and primer sets specific for each miRNA are shown in Tables 1 and 2. The SYBR green stem-loop RT-PCR method was used to assess the expression levels of the miRNAs. The PCR conditions were as follows: 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 sec and 60 ∘ C for 1 min. Samples were run in duplicate with RNA preparations from three independent experiments. The reactions were conducted on the ABI PRISM 7900 system (Applied Biosystems). The fold change in the expression of each gene was calculated using the 2 −ΔΔ method, with U6 as an internal control.

Western Blot Analysis.
H9c2 cells growing on sixwell plates were transduced with miRNAs and treated with 400 M H 2 O 2 , as indicated. The proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. For immunoblotting, PVDF membranes were blocked and probed with antibodies overnight at 4 ∘ C. Immunocomplexes were visualized with horseradish peroxidasecoupled secondary antibodies.

Immunofluorescence Detection of Intracellular Cytochrome c Localization by Fluorescence Microscopy.
After the appropriate treatment, the cells were washed with PBS and stained with 200 nM MitoTracker Red CMXRos dye (Molecular Probes, Inc., France), followed by incubation for 30 min at room temperature, to visualize the mitochondria. The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked using 3% serum dissolved in PBS for 30 min at room temperature. The cells were then probed with anti-cytochrome c antibody (1 : 40; Santa Cruz Biotechnology) overnight at 4 ∘ C. The cells were washed with PBS twice and incubated with FITCconjugated secondary antibody (1 : 40; Biovision, China) for 2 h in the dark at 37 ∘ C. After washing, images of stained cells were obtained using a fluorescence microscope.
2.11. Statistical Analysis. All data are expressed as the mean ± SEM, unless indicated otherwise. Differences among groups were determined by ANOVA. Differences between groups were determined by Student's t-test, with < 0.05 considered statistically significant.

Effects of H 2 O 2 on Cell Viability and Protein Expression.
The viability of H9c2 cells was determined using the MTT assay by exposing the cells to different concentrations of H 2 O 2 for 2 h. Although low concentrations of H 2 O 2 had no effect on apoptosis and death, high concentrations (100-800 M) increased H9c2 cell death in a dose-dependent manner after a 2 h treatment under our experimental conditions ( Figure 1(a)).
Because GPx1 is a major antioxidant enzyme that catalyzes the breakdown of H 2 O 2 , we hypothesized that exogenous H 2 O 2 must induce endogenous Gpx1 expression changes in H9c2 cells. As shown in Figure 1

Predicted miR-181a as a Negative Regulator of Gpx1.
To focus on the role of miRNAs as regulators of Gpx1, three commonly utilized miRNA target prediction algorithms (TargetScan, miRDB, and MiRanda) were interrogated for possible miRNAs interacting with Gpx1. The results of these three algorithms had four potential miRNA candidates in common: miR-7a, 125a, 181a, and 423 ( Figure 2(a)). Next, to assess the expression of these candidate miRNAs induced by the apoptotic H 2 O 2 concentration, H9c2 cells were exposed to 400 M H 2 O 2 for 2 h, and the expression levels of the miRNAs were determined by real-time PCR. If an miRNA candidate is a negative regulator of Gpx1, then H 2 O 2 should increase its expression in H9c2 cells because the Gpx1 expression was downregulated after stimulation with 400 M H 2 O 2 (Figure 1(b)). At the apoptotic concentration of H 2 O 2 , only miR-181a expression was increased compared with the control group, whereas the expression of the other candidate miRNAs was decreased (Figure 2(b)). To further confirm the expression change, a 2 h exposure of H9c2 cells to varying concentrations of H 2 O 2 resulted in concentrationdependent increases in miR-181a expression, with a peak at approximately 400 M (Figure 2(b)). Taken together, these results suggest that miR-181a plays a key role in oxidative stress-induced cardiomyocyte apoptosis and that miR-181a may be linked with the expression of Gpx1.

Validation of the In Silico Target Analysis of miR-181a.
To experimentally validate the computational data, a pmiR-RB-REPORT luciferase construct with the Gpx1-3 -UTR was generated. The purified gel product of Gpx13 -UTR was inserted into the cloning site downstream of the luciferase gene, as described in Section 2. A mutant version, pmiR-RB-Gpx1-3 -UTR-mut, with a three-base-pair mutation within the seed region (Figure 3(a)), was also generated. A significant decrease ( * < 0.01) in the relative luciferase activity was observed when the pmiR-RB-Gpx1-3 -UTR was cotransfected with a mature miR-181a into HEK293 cells compared with the miR-control. The miR-181a-mediated suppression was abolished by mutation of the 3 -UTR miR-181a binding site, which disrupts the interaction between miR-181a and the Gpx1-3 -UTR (Figure 3(b)).
Western blotting analyses further confirmed the luciferase assay results. The transfection with the mature miR-181a resulted in decreased Gpx1 protein expression ( * < 0.01) compared with the control, whereas the anti-miR-181a protected against the mature miR-181a-mediated inhibition of Gpx1 expression ( * < 0.05) (Figure 3(c)).

Transduction of the Anti-miR-181a
Restored the H 2 O 2 -Altered H9c2 Cell Morphology and Decreased the Levels of LDH and MDA. H9c2 cells were transfected as described in Section 2. As shown in Figure 4(a), the mature miR-181a increased miR-181a expression in H9c2 cells, whereas anti-miR-181a decreased miR-181a expression. The control oligonucleotides had no effect on miR-181a expression.
H 2 O 2 treatment changed the spindle-shaped, wellorganized cell morphology into a shrunken, round, and distorted morphology. Transduction of the anti-miR-181a, however, almost restored the spindle-shaped morphology observed in untreated cells (Figure 4(b)). MDA levels are indicative of cardiomyocyte oxidativedamage. The H 2 O 2 treatment strikingly increased the MDA level, whereas the miR-181a inhibitor significantly decreased the MDA level (Figure 4(c)). LDH release is an indicator of cellular injury. Compared with untreated cells, the LDH levels were markedly increased by H 2 O 2 -induced injuries. Transduction of the anti-miR-181a decreased LDH release, whereas transduction of the mature miR-181a elevated LDH levels compared with the miR-control (Figure 4(d)).

Anti-miR-181a
Reduced ROS Production. Because Gpx1 catalyzes the reduction of H 2 O 2 to water [5] and miR-181a mediates the suppression of Gpx1 expression, we attempted to investigate the effect of miR-181a upon ROS generation. ROS production was detected using an ROS-sensitive dye, 2 ,7 -dichlorofluorescein-diacetate (DCFH-DA). As shown in Figure 5(a), H 2 O 2 treatment led to strong DCFH-DA staining. Transduction of the mature miR-181a increased the staining intensity, whereas the anti-miR-181a produced relatively dim DCFH-DA staining. These data suggest that the anti-miR-181a attenuates the production of ROS. ROS production was quantified by measuring the cellular fluorescence intensities ( Figure 5(b)). anti-miR-181a significantly inhibited H 2 O 2 -induced apoptosis (Figures 6(b)-6(c)). To investigate the mechanism by which the anti-miR-181a attenuates the H 2 O 2 -induced H9c2 apoptosis, we examined the protein levels of Bcl-2 and Bax. Western blot analyses showed that Bcl-2 protein levels were markedly increased in the anti-miR-181a-treated cells compared with the miR-CTL group. As expected, Bax expression was markedly decreased by the anti-miR-181a (Figures 6(d) and 6(f)), suggesting that the anti-miR-181a prevents H9c2  blocked the H 2 O 2 -induced formation of JC-1 monomers (Figure 7(a)), suggesting that the anti-miR-181a can restore the H 2 O 2 -induced loss of the mitochondrial membrane potential. The ratio of orange to green fluorescence was used to quantify the Δ m, with a low ratio representing mitochondrial depolarization. We found that the ratio of the H 2 O 2 group was lower than that of the control group ( * < 0.01), that the anti-miR-181a showed protective effects compared with the anti-CTL-treated groups, and that the upregulation of miR-181a decreased the ratio ( & < 0.05, # < 0.05; Figure 7(b)).  a critical step in the progression of the intrinsic apoptotic pathway [19]. H 2 O 2 treatment for 2 h increased the release of cytochrome c from the mitochondria, as shown by the loss of the colocalization of cytochrome c with the mitochondria (Figure 8). The effect of H 2 O 2 was attenuated by transduction with the anti-miR-181a, whereas the upregulation of miR-181a exacerbated this effect (Figure 8).

Downregulation of miR-181a Blocks the Mitochondrial
Caspase-3 mediates apoptosis by regulating many important events that lead to the completion of apoptosis [20]. To further investigate the effects of mature miR-181a and  anti-miR-181aon cleaved caspase-3 activation, we measured cleaved caspase-3 protein expression (Figures 6(d)-6(e)) by western blots in five different groups. The H 2 O 2 treatment increased the generation of cleaved caspase-3. This effect was attenuated by transduction with the anti-miR-181a, whereas the upregulation of miR-181a increased cleaved caspase-3 expression (Figure 6(e)). These data suggest that the anti-miR-181a reduces H 2 O 2 -induced apoptosis by blocking caspase-3-dependent cardiomyocyte apoptosis.

Discussion
Increased ROS levels are a hallmark of oxidative stressinduced cardiomyocyte apoptosis. Compared with other ROS, H 2 O 2 is a relatively long-lived molecule commonly used in models of oxidative stress in H9c2 cardiomyocytes [21,22]  necrosis in adult rat ventricular myocytes [23].  miRNAs are endogenous regulators of gene expression. Previous reports have shown that the binding sites of target mRNAs with as few as seven complimentary base pairs (the seed sequence) to the miRNA 5 end are sufficient for miRNA regulation in animals [25]. More recently, several studies indicated that miRNA expression levels were sensitive to H 2 O 2 in cardiac myocytes. miRNAs can be used as powerful tools to modulate a functional phenotype that involves the participation of multiple proteins, as in the case of ROSmediated events [26][27][28][29]. This finding raises the question of whether the oxidative stress-responsive miRNAs play a role in altering the expression of antioxidant genes that quench ROS to maintain redox homeostasis. With the help of current bioinformatics tools, we predicted several miRNAs that may target Gpx1. Although potential miRNAs can be predicted by computational analysis, these miRNAs must be experimentally verified in cells because the targets and functions of miRNAs are cell-specific and each single proteincoding gene can be regulated by multiple miRNAs [30,31]. To test which miRNAs target Gpx1 mRNA in H9c2 cells, we first confirmed that the 400 M H 2 O 2 treatment decreased Gpx1 expression. Considering the negative relationship between miRs and Gpx1, the selected miRs were expected to be upregulated in H 2 O 2 -treated H9c2 cells. By qPCR analyses, we validated that the miR-181a expression was upregulated approximately 4-fold in H 2 O 2 -treated cells compared with the controls. In addition, Gpx1 expression in H9c2 cells was regulated by miR-181a in unstimulated cells, as determined both by gain-of-function and loss-of-function approaches. To further confirm this effect, we utilized a luciferase vector with the cloned target 3 -UTR region of Gpx1 mRNA. We demonstrated that the negative effect of miR-181a on the Gpx1 levels in H9c2 cells was the result of the direct targeting of Gpx1 mRNA by miR-181a. Hutchison et al. used microarray analysis to assess the effects of miR-181 on the transcriptome in primary astrocytes. Pathway and signaling pathway analyses demonstrated that miR-181 targets genes encoding antioxidant enzymes, including glutathione peroxidases 1 and 4 (Gpx1 and Gpx4, resp.) [32].
To ascertain the role of miR-181a in ROS-mediated H9c2 cells apoptosis, miR-181a expression was modulated via a miR-181a inhibitor and miR-181a mimic. ROS cause damage to intracellular macromolecules, including DNA breakage and lipid membrane peroxidation, both of which can be detected morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation and extracellular exposure of phosphatidylserine). We found that the downregulation of miR-181a expression protected against the H 2 O 2 -induced injury of H9c2 cells by restoring the alterations of H9c2 morphology and nuclear condensation, inhibiting the production of ROS and blocking LDH release and MDA production, two indicators of oxidative stressinduced injury [33].
The pivotal role of mitochondria in cell death and cell survival has been well established: mitochondrial dysfunction constitutes a critical event in the apoptotic process [34,35]. Excessive levels of ROS damage mitochondria, induce the translocation of Bax and Bad, open the permeability transition pore (PTP), and, thus, lead to mitochondrial depolarization and outer membrane rupture, accompanied by the mitochondrial release of cytochrome c and late activation of caspase-3, finally causing cell apoptosis or death [19]. Our data demonstrate that the anti-miR-181a blocked H 2 O 2induced H9c2 apoptosis by regulating mitochondria-related apoptotic pathways. H 2 O 2 induced a decrease in the mitochondrial membrane potential, suggesting an impairment of mitochondrial function. Transduction of the anti-miR-181a, however, restored the mitochondrial membrane potential. These data demonstrate that the anti-miR-181a protected H9c2 cells from H 2 O 2 -induced apoptosis by maintaining mitochondrial membrane integrity and cardiomyocyte function. Moreover, previous studies indicated that the Bcl-2 family is upregulated during the opening of the PTP [36]. Critical interactions between Bcl-2 family proteins cause permeabilization of the outer mitochondrial membrane, a common decision point early in the intrinsic apoptotic pathway that irreversibly commits the cell to death [37,38]. Our results demonstrate that miR-181a regulates the expression of the Bcl-2 family. The anti-miR-181a significantly increased Bcl-2 levels while decreasing Bax protein levels, attenuating the alterations caused by H 2 O 2 -induced injury. One recent study demonstrated that cardiac mitochondria from GPx1 −/− mice lost their resistance to hypoxia/reoxygenation damage [39]. Previous studies have indicated that Bcl-2 knockout mice have reduced glutathione levels and glutathione peroxidase activity in brain tissue [40]. Other works have shown that GPx1 may modify the ratio of Bax to Bcl-2 to create a more antiapoptotic environment [41]. Therefore, it is reasonable to conclude that miR-181a regulates the expression of Gpx1 and that Gpx1 directly influences the expression of Bcl-2. However, the direct regulation of Bcl-2 by miR-181a has been confirmed by many investigations, mainly in tumor cells [42][43][44]. In the future, luciferase reporter assays and western blot analyses should be used to validate Bcl-2 as a direct target of miR-181a in H9c2 cells.
In addition to cis-regulation (direct targeting of mRNAs to induce degradation or inhibit protein translation), miR-NAs may alter the expression of transcription factors or other regulatory genes that can affect the regulation of the target gene via trans-regulatory mechanisms [45]. Prior studies have identified several targets for miR-181a, including GRP78, a major endoplasmic reticulum chaperone and signaling regulator [46,47], and sirtuin-1, an NAD-dependent protein deacetylase [48]. We assume that these targets may participate in the trans-regulation of Gpx1. Furthermore, other studies have demonstrated that miR-181a represses important antiapoptotic targets such as X-linked inhibitor of apoptosis (XIAP), a function that could help explain the proapoptotic effects of this miRNA [32].
In other studies, miRNAs have been shown to be directly linked to the regulation of antioxidant enzymes, including catalase [49], SOD 2 [50], and NADPH oxidase [51]. Our study illustrates the role of miR-181a as a proapoptotic modulator in the regulation of Gpx1. Future studies, especially in ischemia/reperfusion animal models, are necessary to validate the possible therapeutic use of miR-181a regulation.
In summary, we have demonstrated that miR-181a expression is upregulated in H 2 O 2 -treated H9c2 cells and that inhibition of miR-181a confers cardiac protection against oxidative stress-induced H9c2 cell apoptosis through the direct inhibition of Gpx1 expression and ROS generation, which are important for the maintenance of mitochondrial membrane integrity and the inhibition of mitochondrial apoptotic pathway under oxidative stress conditions. These novel findings may have extensive diagnostic and therapeutic implications for a variety of cardiovascular diseases related to ROS, including atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy.