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PARP1 deficiency protects against hyperglycemia-induced neointimal hyperplasia by upregulating TFPI2 activity in diabetic mice

Diabetes mellitus (DM) promotes neointimal hyperplasia, characterized by dysregulated proliferation and accumulation of vascular smooth muscle cells (VSMCs), leading to occlusive disorders, such as atherosclerosis and stenosis. Poly (ADP-ribose) polymerase 1 (PARP1), reported as a crucial mediator in tumor proliferation and transformation, has a pivotal role in DM. Nonetheless, the function and potential mechanism of PARP1 in diabetic neointimal hyperplasia remain unclear. In this study, we constructed PARP1 conventional knockout (PARP1−/−) mice, and ligation of the left common carotid artery was performed to induce neointimal hyperplasia in Type I diabetes mellitus (T1DM) mouse models. PARP1 expression in the aorta arteries of T1DM mice increased significantly and genetic deletion of PARP1 showed an inhibitory effect on the neointimal hyperplasia. Furthermore, our results revealed that PARP1 enhanced diabetic neointimal hyperplasia via downregulating tissue factor pathway inhibitor (TFPI2), a suppressor of vascular smooth muscle cell proliferation and migration, in which PARP1 acts as a negative transcription factor augmenting TFPI2 promoter DNA methylation. In conclusion, these results suggested that PARP1 accelerates the process of hyperglycemia-induced neointimal hyperplasia via promoting VSMCs proliferation and migration in a TFPI2 dependent manner.


Introduction
Accelerated intimal thickening in diabetes mellitus is a critical cause of atherosclerosis and in-stent restenosis [1,2]. Hyperglycemia caused aberrant vascular smooth muscle cells (VSMCs) proliferation and migration promote the occurrence of arterial neointimal hyperplasia which is an inevitable stage in the development of vascular remodeling [3][4][5]. Sustained hyperglycemia will shorten the diameter of blood vessels and vessel calcification hindering percutaneous coronary intervention efficacy [6,7]. Increasing evidence has demonstrated that inflammation, proliferation, advanced glycation end products, and oxidative stress caused by diabetes play a pivotal role in intimal hyperplasia, in which suppressing the abnormal proliferation and migration of VSMCs is reported as an effective method to restrain neointimal formation and vascular lumen loss in diabetes [8,9].
Poly (ADP-ribose) polymerase 1(PARP1) is a nuclear protein that performs DNA repair mainly by catalyzing its own poly ADPribosylation and other chromatin-related proteins [10]. In addition, PARP1 is also involved in a variety of cellular functions, including transcriptional regulation, cell cycle progression, cell migration, inflammation, and energy metabolism [11][12][13]. Reactive oxygen/nitrogen species are effective activators of PARP1 due to the ability to damage DNA [14,15]. Recent studies have proven the increasing levels of oxidation in atherosclerotic plaques of DM mice, and carotid arteries after balloon injury in animal models [16][17][18][19]. However, to the best of our knowledge, no study has been reported regarding the role of PARP1 on neointimal hyperplasia caused by diabetes.
Tissue factor pathway inhibitor 2 (TFPI2) is a Kunitz-type serine proteinase repressor that is synthesized in large quantities of normal human tissue cells and secreted into the extracellular medium [20]. It has been reported that TFPI2 could inhibit plasmin and trypsin-mediated matrix metalloproteinase (MMP) activation [21], as well as directly inhibit the activity of MMPs [22][23][24][25]. Additionally, TFPI2 could suppress VSMCs proliferation by regulating cell cycle progression, which together with cell migration is an essential event in the process of neointimal formation [23,26]. Several studies have demonstrated that TFPI2 expression is largely diminished in atherosclerosis plaques, indicating that TFPI2 downregulation may contribute to neointimal hyperplasia and atherosclerosis progression [27,28]. Therefore, we speculated that PARP1 influences the process of neointimal hyperplasia dependent on TFPI2 expression under the condition of T1DM.
In the present study, we used human samples and T1DM murine models with a genetic deficiency in PARP1 to address the relationship between PARP1 and neointimal hyperplasia under the condition of diabetes. The result showed that hyperglycemia stimulated PARP1 abundance and genetic deficient in PARP1 attenuated the aorta remodeling process in STZ-treated mice. Furthermore, we proved the protective role of PARP1dificiency is dependent on the upregulation of TFPI2, which inhibits VSMCs proliferation and migration during the formation of neointima in diabetes mellitus.

Diabetic mouse model and carotid artery ligation
All experimental procedures were under the institutional animal care guidelines and approved by the Shandong University Animal Ethics Committee.
PARP1 +/− mice were purchased as heterozygous breeding pairs from Jackson Laboratories (Bar Harbour, ME, USA). The wild type (PARP1 +/ + ) and PARP1 deficiency (PARP1 − /− ) offspring were obtained from PARP1 +/− mice were bred again. Mice were housed in a pathogen-free animal care facility at a constant temperature (24 • C) and a conventional light/dark cycle (12/12 h) under free conditions. The mice were randomized into the following three groups: (i) wild-type non-diabetic group (non-DM), (ii) wild-type diabetic group (DM), and (iii) PARP1− / − diabetic group (DM + PARP1− /− , n = 20, respectively). Type I diabetes was induced with 5 daily intraperitoneal injections of 50 mg/kg STZ in citrate buffer (0.05 mol/L; pH 4.5); control mice received an equivalent amount of citrate buffer solvents. Two weeks after the initial administration of STZ, mice with a blood glucose level of 16.7 mM (300 mg/dl) and above were considered diabetic and were included in the diabetic cohorts. Ligation of the left common carotid artery was performed just proximal to their bifurcations according to a procedure described previously 2 weeks after treatment with STZ or citrate solvent [29]. Mice were anesthetized. Postoperative analgesia was assured with 5 mg/kg SC carprofen. Animals were anesthetized at 3 weeks after surgery and sacrificed. Mice were fixed and perfused for 5 min with 4% formaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.3). The left and right carotid arteries were immersion-fixed in 70% ethanol. The length of common carotid arteries was 9 mm, of which the proximal and distal 2 mm were discarded and the remaining portion (5 mm) was cut in half. The two segments were embedded in paraffin, and serial sections (5 μm thick) were cut for analysis by immunostaining and hematoxylin-eosin staining for morphometry. Five or more sections spanning most of the vessel segment were analyzed for morphometry.

Human coronary artery samples
The human coronary arteries were obtained from both specimens with extensive diabetic atherosclerotic disease as well as healthy controls (n = 5). The human specimens were donated from the Shandong Red Cross Society (ethics approval No. KYLL-2018(KS)-233).

Histology and immunohistochemistry
Coronary artery morphologies were examined in 5 μm thick sections stained with hematoxylin. For immunohistochemical staining, paraffinembedded tissue sections were dewaxed and stained with an antibody, followed by a biotin-conjugated secondary antibody (1:1000), and then with horseradish peroxidase-conjugated streptavidin (Dianova, Rodeo, CA). Histopathological features were analyzed using Image Pro-Plus 6.0 (Media Cybernetics, MD, USA). The area of positive staining for TFPI2 was expressed as the percentage of the stained area divided by the total intima area in at least 15 high amplification fields (400 × ). For immunofluorescence, the cells were first stained with specific antibodies and then staining with fluorescently labeled secondary antibodies. Nuclei were counterstained with 4 ′ -6-diamidino-2-phenylindole (DAPI; Invitrogen). The images were analyzed by use of a LSM 710 laser confocal microscope (EC Plan-Neofluar 40x/1.30 Oil Objective) equipped with ZEN 2009 Light Edition software (Zeiss, Germany).

Generation of TFPI2 expression constructs
The pcDNA3.1/V5-His TOPO Expression Kit (Invitrogen) was used to express TFPI2 (Invitrogen). The cDNA, containing the entire coding region was generated without a stop codon by RT-PCR and subcloned into pcDNA3.1/V5-His TOPO vector (pcDNA3.1/TFPI2) and pcDNA3.1/ CT-green fluorescent protein (GFP), was used as a negative control for gene transfer into HASMCs.

Cell proliferation and cell cycle assay
Proliferation analysis was performed using the Cell-Light™ EDU assay kit (RiboBio, China), according to the manufacturer's instructions. The cells were collected 72 h after treatment, fixed with 20 • C with 70% ethanol, and then treated with ribonuclease (25 μg/mL) at 37 • C for 30 min, followed by staining with propidium iodide (PI, 50 μg/mL) in the dark at 4 • C for 30 min. The ratios of cells in G1, S, and G2/M phases were determined by FACS-Calibur flow cytometry (Becton Dickinson, USA).

Cell migration assay
HASMC migration assays were performed using transwell chambers (8-μm pore size; Corning Inc., Shanghai, China). Serum-starved HASMCs were seeded in the upper chamber, and the bottom chamber of the migration apparatus was filled with smooth muscle cell medium (SMCM) containing HG or OC. The cells were fixed with 4% formaldehyde and stained with crystal violet after culturing for 12 h. Cells were extracted on the surface with a cotton swab. Results are expressed as the average number of migrating cells per transwell membrane in 5 random fields of view (200 × ).

Microarray analysis
The microarray data in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/index.cgi; GSE141193).
HASMCs were treated with PARP1 inhibitor PJ34 or PBS, using RNeasy Mini Kit (Qiagen, Hilden, Germany) purified total RNA. Concentration and purity were detected using a NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Biotin-labeled cRNA was synthesized using the GeneChip 3 IVT PLUS Reagent Kit (Thermo Fisher Scientific). Each sample was hybridized to a GeneChip™ Human Transcriptome 2.0 Array (Thermo Fisher Scientific) after fragmentation.
The procedure was carried out according to the manufacturer's

Detection promoter DNA hypermethylation of TFPI2
Genomic DNA (1 g) purified from HASMCs using a QIAamp DNA Minikit (Qiagen, Valencia, CA) was modified with sodium bisulfate using the EZ DNA methylation toolkit (Zymo Research, Orange, CA). The MassARRAY platform (Sequenom, San Diego, CA) was used to determine DNA methylation levels as described previously [30]. The detailed primers were designed using the online software Epidesigner (at http://www.epidesigner.com/). For each reverse primer, an additional T7 promoter tag was added for in vivo transcription, while a 10-m tag was used on the forward primer to adjust the melting temperature difference. The following primers based on the reverse complementary strands of the TFPI2-promoter were used: (5 ′ -AGGAAGAGAGGGAATTTATTTTTTGAAGGTATGAA-3 ′ and 3 ′ -CAGTAATACGACTACCTATAGGGAGAAGGCTCCTCCCAAAA-TACTAAAATTACAAA-5 ′ ). Analysis of methylation data for individual units (1-3 CpG sites per unit) using EpiTyper software (Sequenom).  statistical differences were evaluated with Student t-test for two groups and one-way ANOVA for three or more groups (Tukey's post-test). P < 0.05 was considered statistically significant.

PARP1 deficiency protects against hyperglycemia-induced augmented neointimal hyperplasia by inhibition of HASMC proliferation
To prove the involvement of PARP1 in hyperglycemia augmented neointimal hyperplasia, we firstly used STZ injection to induce DM, where the blood glucose levels of DM group increased to >23 mM versus 5.78 mM in control group (Table 1; n = 20; P < 0.05). Carotid arteries were ligated 2 weeks after STZ or citrate treatment and collected for analysis 3 weeks after surgery. Using immunochemical and western-blot analysis, we found the expression of PARP1 elevated apparently in DM group ( Fig. 1A and B). Meanwhile, PARP1− /− diabetic mice displayed dramatically decreased neointimal thickening compared to the wildtype group (Fig. 1C). According to immunochemical staining and Westernblot, we analyze the proliferation of neointimal VSMCs using PCNA (proliferating cell nuclear antigen), an indicator of cell proliferation. An elevated PCNA protein level was seen in diabetic mice while decreased PCNA protein expression in PARP1 deficient diabetic mice (Fig. 1D and E).
To further determine the role that PARP1 plays in regulating HGinduced VSMCs proliferation, 5 μM PJ34 treatment was used to inhibit PARP1 expression. HG treatment significantly increased the PARP1 and PAR (an enzymatic product of PARP1) by approximately two times, while PJ34 treatment reduced its protein level (p < 0.05, Fig. 1F).
Moreover, cell proliferation was demonstrated using the EDU incorporation assay. Compared with the control group, HG increased EDU incorporation, and PJ34 treatment reversed the increase in EDU incorporation (Fig. 1G). In order to explore the potential mechanism of PARP1 inhibition in the proliferation of VSMCs, we performed cell cycle analysis on high glucose-stimulated VSMCs by measuring the DNA content of cells stained with sodium iodide. There was a decline in cells corresponding to the G1 phase and an increase in cells corresponding to the S phase in the high glucose-stimulated VSMCs compared with the control, and PARP1 inhibition reduced the high glucose mediated transition from the G1 to the S phase in VSMCs (Fig. 1H).

PARP1 deficiency inhibits glycemia-induced VSMCs migration and attenuates the enzymatic activity and expression of MMPs
To better illustrate the effect of PARP1 on glycemia-induced VSMCs Migration, we used the transwell migration test finding that genetic inhibition of PARP1 significantly suppressed high glucose-stimulated cell migration (Fig. 2A). Moreover, we observed that PJ34 treatment markedly attenuated the high glucose-induced increase in MMP2 and MMP9 protein expression, consistent with the changes in gelatin zymography of MMP activity ( Fig. 2B and C). Furthermore, the immunostaining and gelatin zymography analysis revealed that the expression and activity levels of MMP2 and MMP9 were significantly increased in the neointima of diabetic mice compared with the controls, whereas PARP1 deletion markedly decreased their intensity (Fig. 2D and E). Meanwhile, consistent results were found using immunochemical analysis (Fig. 2F).

PARP1 downregulates TFPI2 gene expression
Genome microarray assays of HASMCs with or without PARP1 inhibition were performed to profile DEGs that might be involved in PARP1 regulation. A heat map was used to illustrate the expression profile of DEGs (>2-fold) in untreated and PJ34-treated HASMCs (Fig. 3A). Among these genes, TFPI2 was the one exhibiting the greater down-regulation change in expression (>3.65-fold). Some studies have reported that TFPI2 closely corresponds to the process of VSMCs proliferation and migration by regulating MMP2 and MMP9 [22][23][24]，so we chose TFPI2 as a target regulated by PARP1 in the present study. To verify the microarray analysis data, we examined TFPI2 expression by western blotting. What's more, PJ34 treatment evidently reversed the reduced TFPI2 expression due to high glucose (Fig. 3B). Immuno-analysis also showed that TFPI2 levels in the neointima of the carotid artery and tunica media of the aorta were markedly lower in diabetic mice than in control mice, which were reversed in PARP1− /− diabetic mice (Fig. 3C and D). In addition, lower TFPI2 expression was observed in the neointima of stenotic coronary arteries of diabetic patients than in that of healthy controls (Fig. 3E).

TFPI2 mediates HASMC proliferation and migration
Furthermore, we evaluated the role of TFPI2 in the proliferation and migration of HASMCs. HASMCs cultured in DMEM were transfected with pcDNA3.1/TFPI2 or siRNA for TFPI2.
TFPI2 overexpression significantly inhibited PCNA, MMP2, and MMP9 expression (Fig. 4A). Further validation by TFPI2 knockdown showed increased expression of PCNA, MMP2, and MMP9 protein (Fig. 4B). The gelatin zymography of MMP2 and MMP9 activity showed results that were consistent with the above conclusions ( Fig. 4C and D). TFPI2 overexpression was associated with decreased EDU incorporation, and the TFPI2 knockdown obviously increased the incorporation as compared with siNC transfected cells (Fig. 4E and F). Additionally, overexpressed TFPI2 led to waning migration of HASMCs, whereas TFPI2 silencing promoted the migration than the siNC control ( Fig. 4G and H).

TFPI2 overexpression inhibits HG-induced HASMC proliferation and migration
We then investigated the mechanism of TFPI2 expression on neointimal formation in diabetes. The pcDNA3.1/TFPI2 transfected into HASMCs can significantly inhibit the proliferation of hyperglycemia- induced HASMCs compared with negative control (pcDNA3.1/CTgreen; Fig. 5A). Flow cytometry showed that increased G1 phase and decreased S phase were found in high-glucose-stimulated HASMCs compared to the controls, while TFPI2 overexpression reduced high glucose-mediated transition from G1 to S phase in HASMCs (Fig. 5B). Transwell migration assays revealed that TFPI2 overexpression reduced the high glucose-stimulated HASMC migration (Fig. 5C). Moreover, TFPI2 overexpression markedly attenuated the high glucose-induced increase in MMP2 and MMP9 protein expression, which is consistent with the changes in the gelatin zymography of MMP activity (Fig. 5D and E).

PARP1 mediated HG-induced TFPI2 promoter DNA methylation
Studies have shown that the TFPI2 promoter contains some of the CpG islands participating in transcriptional silencing [31][32][33]. To determine whether PARP1 mediates hyperglycemia-induced TFPI2 promoter DNA methylation thus decreasing TFPI2 activity, the methylation levels of CpG islands in the TFPI2 promoter were examined using the MassARRAY assay (Sequenom).
One region in the TFPI2 promoter (− 372 bp to − 57 bp, nucleotide positions relative to the translation initiation sites) was analyzed, wherein all 10 CpG sites were divided into 8 CpG units (Fig. 6A). We found that the methylation status at the CpG_3 site was much higher in the high-glucose-stimulated HASMCs than in the control groups, whereas PARP1 inhibition significantly decreased the high glucosestimulated methylation levels at the CpG_3 and CpG_4 sites ( Fig. 6B and C). To assess whether hyperglycemia-induced DNA methylation was responsible for TFPI2 suppression, we treated HAECs transfected with 5-M 5-ara-dC, a methyltransferase inhibitor, for 36 h. TFPI2 suppression was reversed by 5-ara-dC treatment as shown by western blotting (Fig. 6D). Meanwhile, 5-ara-dc also significantly reduced the expression of PCNA and MMP2/MMP9 in HASMCs induced by hyperglycemia ( Fig. 6E and F).

Discussion
PARP1, a nuclear protein participating in DNA repair, showed an elevated expression in diabetes due to increased DNA damage caused by hyperglycemia [16,17]. PARP-1 activation plays a key role in the pathogenesis of various diabetic complications such as vasculopathy, cardiomyopathy, retinopathy and neuropathy [8,34]. However, its specific role in neointimal hyperplasia in diabetes and precise molecular mechanisms have not been clarified. Here, we confirmed a higher PARP1 abundance in the pathogenesis of neointimal hyperplasia in both humans and mice. Recently, emerging evidence shows that PARP1 is involved in various cellular processes, such as inflammatory response, vascular function, cell proliferation and migration, independent of its classic function of DNA repair [10][11][12][13][35][36][37]. A previous study showed that minocycline suppressed VSMCs proliferation and plaque formation by inhibiting PARP1 and inducing CDK inhibitor p27 expression [38]. It has also been reported that PARP1 inhibition could promote the transmigration of VSMCs to the intima of atherosclerotic plaques by increasing metalloproteinase 2, a tissue inhibitor, and thus enhancing the plaque stability [18]. Using PARP1 knockdown mice, our study is the first to prove that genetic inhibition of PARP1 significantly alleviated neointimal thickening in diabetic mice by inhibiting glucose-stimulated VSMCs proliferation dependent on the G1-S phase transition, as well as impeding VSMCs migration by suppressing MMP2/9 expression and activity in vitro and vivo.
TFPI-2, as a Kunitz-type serine proteinase inhibitor, is mainly synthesized by VSMCs and endothelial cells [39]. It has been reported that TFPI2 is expressed in a variety of human tissue cells such as the heart, skeletal, muscle, liver, pancreas, and kidney tissues, wherein the protein is secreted into the extracellular matrix (ECM) and prevents ECM hydrolysis by inhibiting the activation of plasmin-mediated MMPs [24,40]. In addition, TFPI2 has been discovered to be widely distributed in the cytoplasm and nucleus. Interaction of the nuclear-localized TFPI2 with a transcription factor AP-2α results in inhibition of cell migration by negatively regulating the transcription of MMP2 mRNA in breast cancer cells [41]. Moreover, as a direct inhibitor of MMP-2 and MMP-9 and potent inhibitor of both matrix-bound and cell-associated plasmin, TFPI-2 was reported to inhibit the progression of atherosclerosis and promote atherosclerotic plaque stability [42,43]. Our study found TFPI2 overexpressed under the condition of PARP1 inhibition using transcriptome analysis, and TFPI2 could efficiently inhibit the hyperglycemia-induced VSMCs proliferation and migration.
DNA methylation, a biological process where methyl groups are added to the DNA molecule, typically represses gene transcription via inhibiting the binding of transcription factor(s) to DNA [44]. PARP1 has been reported to play an important role in gene regulatory pathways including basal transcription machinery, histone modifications, chromatin remodeling, and DNA methylation [11]. Moreover, promoter region analysis of TFPI-2 gene 5 ′ -flanking region revealed a high GC-rich content with masses of CpG sites. Transcription of TFPI2 is often silenced by aberrant promoter hypermethylation in tumor tissues [45].Furthermore, it has also been demonstrated that TFPI2 promoter methylation is linked to low activity in carotid atherosclerotic plaques [28]. In this article, we used the MassARRAY platform validating that high glucose-stimulated PARP1 upregulation promoted TFPI2 hypermethylation, which also accounts for its low expression in vascular neointima.
In conclusion, PARP1 overexpression in diabetes caused excessive VSMC proliferation, migration, and intimal hyperplasia via silencing TFPI2 through DNA hypermethylation (Fig. 7). Understanding this pathway provides important molecular insights into PARP1 for diabetic neointimal hyperplasia treatment. Further studies are needed to elucidate the underlying mechanism by which PARP1 regulates downstream molecules.

Funding
This work was supported by the National Natural Science Foundation of China (#81100206, #82000417 and #81970251), the Taishan Scholar Project of Shandong Province of China (No. ts20190972), 2020SFXGFY06 and Key Issues of Jinan Science and Technology Bureau (#20170409).

Declaration of competing interest
None.