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CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells via Activating Akt/eNOS Pathway and Promotes Ischemic Angiogenesis in Diabetic Limb Ischemia

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Abstract

Purpose

Endothelial progenitor cells (EPCs) play a critical role in repairing damaged vessels and triggering ischemic angiogenesis, but their number is reduced and function is impaired under diabetic conditions. Improving EPC function has been considered a promising strategy to ameliorate diabetic vascular complications. In the present study, we aim to investigate whether and how CXCR7 agonist TC14012 promotes the angiogenic function of diabetic EPCs.

Methods

High glucose (HG) treatment was used to mimic the hyperglycemia in diabetes. Tube formation, cell scratch recovery and transwell assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and cleaved-caspase3 expression were used to evaluate the angiogenic capability, cell migration, and apoptosis of EPCs, respectively. Hind limb ischemia (HLI) model was used to appraise the ability of TC14012 in promoting diabetic ischemic angiogenesis in vivo.

Results

HG treatment impaired EPC tube formation and migration, and induced EPC apoptosis and oxidative damage, while TC14012 rescued tube formation and migration, and prevented HG-induced apoptosis and oxidative damage of EPCs. Furthermore, these beneficial effects of TC14012 on EPCs were attenuated by specific siRNAs against CXCR7, validating that CXCR7 is a functional target of TC14012 in EPCs. Mechanistic studies demonstrated that HG treatment reduced CXCR7 expression in EPCs, and impaired Akt and endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production; similarly, these signal impairments in HG-exposed EPCs could be rescued by TC14012. However, the protective effects of TC14012 on tube formation and migration, Akt and eNOS phosphorylation, and NO production in HG-treated EPCs were almost completely abolished by siRNAs against CXCR7 or Akt specific inhibitor wortmannin. More importantly, in vivo study showed that TC14012 administration enhanced blood perfusion recovery and angiogenesis in the ischemic hind limb and increased the EPC number in peripheral circulation of db/db mice, demonstrating the capability of TC14012 in promoting EPC mobilization and ischemia angiogenic function.

Conclusion

TC14012 can prevent EPCs from HG-induced dysfunction and apoptosis, improve eNOS activity and NO production via CXCR7/Akt signal pathway, and promote EPC mobilization and diabetic ischemia angiogenesis.

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Data Availability

All data relevant to the study are included in the article or uploaded as supplementary information.

Code Availability

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Funding

This study was supported by a Junior Faculty Award (1–13-JF-53) from the American Diabetes Association, National Key Research and Development Program of China (2017YFA0506000), National Natural Science Foundation of China (81770305, 81873466, 82070834), Natural Science Foundation of Zhejiang Province (LY22H070005, LY22H020005), Foundation for Distinguished Young Scholars of Sichuan Province (2019JDJQ0042), Key Research and Development Support Plan of Chengdu (2019-YF05-00275-SN), Fund of Development and Regeneration Key Laboratory of Sichuan Province (SYS18-04), Grant from Science and Technology Bureau of Wenzhou (Y20210010), and Basic Scientific Research Foundation of Wenzhou Medical University (KYYW201907).

Author information

Author notes

  1. Kai Wang and Shiyue Sun contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China

    Kai Wang & Chaosheng Lu

  2. Pediatric Research Institute, Department of Pediatrics, University of Louisville School of Medicine, 570 South Preston Street, Baxter-I Building Suite 304E, Louisville, KY, 40202, USA

    Kai Wang, Qian Lin, Oscar Chen, Lu Cai & Yi Tan

  3. Chinese-American Research Institute for Diabetic Complications, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China

    Shiyue Sun, Guigui Zhang, Zixian Lu, Hui Chen, Xia Fan, Chunjie Gu, Xiaohong Pan & Xiaoqing Yan

  4. School of Biosciences and Technology, Chengdu Medical College, Chengdu, Sichuan, China

    Xiaozhen Dai

  5. Department of Obstetrics, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China

    Xiao Wang

Authors
  1. Kai Wang
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Contributions

Kai Wang, Xiaoqing Yan, and Yi Tan conceived and designed the study. Kai Wang, Shiyue Sun, Guigui Zhang, Zixian Lu, Hui Chen, Xia Fan, Chunjie Gu, Xiaohong Pan, Oscar Chen, Qian Lin, Xiao Wang, and Chaosheng Lu performed in vitro cell culture, cell biology, molecular biology experiments, and histology analysis. Kai Wang and Xiaozhen Dai performed in vivo therapeutic studies in mice. Kai Wang, Xiaoqing Yan, and Yi Tan wrote the manuscript. Kai Wang and Lu Cai edited the manuscript with important intellectual content. Xiaoqing Yan and Yi Tan supervised this study.

Corresponding authors

Correspondence to Xiaoqing Yan or Yi Tan.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the Declaration of Helsinki. All protocols were approved by the Institutional Review Board of the First Affiliated Hospital of Wenzhou Medical University.

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10557_2022_7337_MOESM1_ESM.pdf

Fig. S1. TC14012 protects EPCs from HG-induced impairment of migration. EPCs were exposed to high glucose (HG, 33 mmol/L) with or without TC14012 (TC, 5 μmol/L) treatment for 24 h, the equivalent concentration of mannitol (Man) was used as osmotic control. The migration of EPCs was determined by transwell assay (A) and the migrated cells were counted using ImageJ (B). Three independent experiments were performed. Data were expressed as Mean ± SD. *P<0.05 vs. Man; #P< 0.05 vs. HG. Fig. S2. TC14012 decreased MDA content of HG-treated EPCs. The MDA content in EPCs was detected using MDA detecting kit. Three independent experiments were performed. Data were normalized to cell number and expressed as Mean ± SD. *P<0.05 vs. Man; #P< 0.05 vs. HG. Fig. S3. CXCR7 knockdown abolishes the protective effect of TC14012 on migration of HG-treated EPCs. Three siRNAs against CXCR7 sequences were chosen and RT-PCR was used to detect knockdown efficiency (A). CXCR7-siRNA-441 with the highest transfection efficiency was selected, CXCR7 protein expression was evaluated by Western blot (B). The migration of EPCs was determined by transwell assay (C) and the migrated cells were counted using ImageJ (D). Three independent experiments were performed. Data were expressed as Mean ± SD. *P<0.05 vs. Ctrl-siRNA-Man; #P< 0.05 vs. HG; & P<0.05 vs. CXCR7-siRNA-Man. Fig. S4. Akt inhibitor wortmannin abolisheS the protective effect of TC14012 on migration of HG-treated EPCs. EPCs were exposed to high glucose (HG) with or without TC14012 (TC) treatment in the present of absent of wortmannin (Wort) for 24 h, the equivalent concentration of mannitol (Man) was used as osmotic control. The migration of EPCs was determined by transwell assay (A) and the migrated cells were counted using ImageJ (B). Three independent experiments were performed. Data were expressed as Mean ± SD. *P<0.05 vs. Man; #P< 0.05 vs. HG. Fig. S5. TC14012 did not affect blood glucose of db/db mice. Random blood glucose of mice was tested using glucose testing strips (Roche). n=6, 7 per group, respectively. (PDF 299 KB)

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Wang, K., Sun, S., Zhang, G. et al. CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells via Activating Akt/eNOS Pathway and Promotes Ischemic Angiogenesis in Diabetic Limb Ischemia. Cardiovasc Drugs Ther 37, 849–863 (2023). https://doi.org/10.1007/s10557-022-07337-9

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