T2DM, a disease of carbohydrate metabolism, should be considered a vascular disease because diabetic patients have higher incidence of atherosclerosis . Metabolic and hematologic abnormalities exist in T2DM, including hyperglycemia, insulin resistance, dyslipidemia, inflammation, and thrombophilia . Diabetes itself is an independent risk factor for cardiovascular events. T2DM increases the risk for CAD by 2 to 4 times that of the overall population . CAD is more ubiquitous and has a worse prognosis in adults with diabetes mellitus than non-diabetic patients . Restenosis in patients undergoing PCI results in a lowered long term survival rate and increased rates of repeat revascularization . Moreover, the likelihood of target vessel revascularization in patients with diabetes mellitus after PCI is increased . A serial intravascular ultrasound study showed that neointimal formation was closely related to diabetes mellitus restenosis after vascular injury .
The tagging of newly synthesized DNA in cells or tissues to identify proliferation is an important experimental technique. 3H]Thymidine has been quite useful for studying DNA replication and assessing cell proliferation. However, the procedure is burdensome. BrdU staining was believed to be the “gold standard” method for detecting DNA synthesis. However, detection of BrdU requires DNA denaturation of the specimen which makes the staining process difficult and penetration of BrdU-antibody through fixed tissue is comparatively difficult. Recently, the use of EdU has been described to detect DNA replication in vivo and in vitro in animals. The application of EdU was also reported in plants and fission yeast [36, 37]. Grenier et al. determined the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin, and function of micronuclei in cleavage stage rat embryos using EdU incorporation to monitor DNA synthesis . In addition, Škalamera et al. transferred protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection into lentiviral expression vector using the highly efficient Gateway recombination cloning and labeled transduced cells with EdU to detect cells progressing through S phase . The full potential of EdU in biomedical research remains to be explored.
Carotid artery injury was induced by balloon de-endothelialization in our previous study. Cell proliferation in obese Zucker rats was higher than in lean Zucker rats at day 7 after injury, and the neointimal area of obese Zucker rats was also broader than that of lean Zucker rats at day 7 after injury . Since Zucker rats and GK rats performed the same in this research. GK rats were used for some of the following research to replace Zucker rats. Time course of neointimal formation in our study was in agreement with experiments performed in our previous study . We have evaluated the effect of rosiglitazone on VSMCs proliferation in Zucker obese and lean rats after carotid artery injury with the use of BrdU incorporation to assess DNA synthesis in vivo. The rats received intraperitoneal injections of 50 mg/kg BrdU at hours 18, 12 and 2 before euthanasia. Following this procedure, the number of BrdU-labeled positive cells in the intima and media was counted . In the present day, GK and Wistar rats on the 7th day after injury were chosen to detect EdU-labeled DNA synthesis of neointimal formation and for making morphological comparisons. In the current study, we report the use of EdU to conveniently and quickly detect DNA synthesis of neointimal formation in rats after injury. EdU was injected into model rats at different doses and different injection frequencies to reach an optimal injection method for staining. Better staining results were obtained with multiple EdU injections of 100 mg/kg. This may be related to the fact that more EdU can be incorporated into proliferating cells during DNA synthesis of neointimal formation than a single injection or low doses. Attempts to obtain higher fluorescence intensity and more positive cells by injecting EdU at 200 mg/kg were unsuccessful. Along with enhancement in fluorescence intensity of EdU positive cells, the brightness of the background was simultaneously increased; however, the actual percentage of EdU positive cells remained almost the same.
PI3K/Akt signaling plays a key role in essential cellular functions such as cell growth and survival [40, 41]. Aberrant regulation of the PI3K/Akt pathway has been discovered in insulin resistant T2DM, leading to enhanced Akt activity . Activation of Akt increases translation of cell cycle-associated genes, such as cyclins and cyclin-dependent kinases. The entry of vascular cells into cell cycle plays an important role in the pathogenesis of post-angioplasty restenosis . Immediately after injury, VSMCs leave their resting state and enter the cell cycle. Arterial injury results in the proliferation and migration of VSMCs into the intimal layer of the arterial wall. The contribution of vascular proliferation to the postangioplasty restenosis is particularly important. In addition, the proliferative response is also reflected by PCNA activation, which is a well-accepted marker of cell proliferation and assists in DNA replication. In a study of rat aortic VMSCs, Akt signaling is highly activated following stimulation with platelet-derived growth factor (PDGF) . Therefore, we established an in vivo rat carotid artery injury model and determined whether neointimal SMCs exhibit activated Akt signaling. Cell proliferation involves changes at the levels of gene transcription and protein translation. In our study, cell proliferation was assessed by using immunohistochemistry staining for PCNA expression and localization to identify the actively cycling cells within the media and intima. The results showed that there were significantly more PCNA-positive VSMCs in diabetic rats than in non-diabetic rats. Using Western blot analysis we found that the protein levels of PCNA and p-Akt were both significantly increased in injured arteries, and were much higher in diabetic rats. qRT-PCR detection of PCNA mRNA levels revealed similar results, which was consistent with the detection of EdU-labeled DNA synthesis. Therefore, we indirectly and strongly confirmed the accuracy of the EdU incorporation and EdU staining by detecting PCNA expression with three distinct molecular biological methods.
To summarize, carotid artery balloon catheter injury led to cell proliferation, which was validated at the levels of PCNA and p-Akt protein as well as PCNA mRNA. And we concluded that EdU incorporation and staining was useful means for detecting DNA synthesis in the vascular neointima quickly and efficiently. Additionally, it only takes less than 2.5 hours for EdU staining, compared to 5 hours and overnight incubation for BrdU antibody detection method. In this work, we also compared the percentages of EdU-positive cells in GK and Wistar rats. We discovered that at day 7 after catheter balloon injury, DNA synthesis in the vascular neointima could be more readily observed in GK rats than in Wistar rats by intraperitoneal injections of EdU at a dose of 100 mg/kg three times. Therefore, it is recommended that a saturated dose and multiple injections can be used to obtain reliable and accurate results. In conclusion, we demonstrate the suitability of EdU incorporation and staining in GK and Wistar rats, and further probe the pathological features of diabetes mellitus by observing a higher amount of DNA synthesis in GK rats.