Abstract: FR-PO625

Endogenous Klotho May Play Direct Roles in the Pathogenesis of Diabetic Vascular Disease

Session Information

Category: Diabetes

  • 501 Diabetes Mellitus and Obesity: Basic - Experimental

Authors

  • Zhu, Langjing, The First Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong, China
  • Liu, Qinghua, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Ding, Yan, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Chang, Yu-Chun, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Hsiao, Li-Li, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
Background

Diabetes is a known risk factor for cardiovascular disease (CVD) and chronic kidney disease. Dysfunction of vascular smooth muscle cells (VSMCs) plays a key role in the pathogenesis of diabetic vascular disease, which requires the migration and proliferation of VSMCs as well as the production of biological mediators such as TGF-β1, Fractalkine, and matrix metalloproteinases (MMPs). We previously showed that endogenous Klotho is a master regulator of CVD by reducing vascular calcification in the VSMC layer. This study aims to explore the functional roles of endogenous Klotho in the pathogenesis of diabetic vascular disease.

Methods

Diabetic model is created using Human Aortic-SMCs (HA-SMCs) with D-glucose under dose-response (5.5mM, 25mM, 35mM, and 50mM) and time-dependent fashion (0 day to 10 days); mannitol (35mM and 50mM) is used to assess the osmotic effects. Expression of endogenous Klotho, TGF-β1, Fractalkine, MMP2, and MMP9 are assessed by Western blot. The direct effects of endogenous Klotho are achieved by Klotho-siRNA and Klotho-plasmid transfection. MTS assay is used to assess the cell proliferation, flow cytometry with propidium iodide staining is used to study cell cycle, and the wound-healing migration assay is for the migratory activity.

Results

The Klotho expression in HA-SMCs is upregulated by high glucose (HG) in a dose-dependent manner; and the expression peaks at day 3 with 35mM D-glucose treatment. However, the Klotho expression is not affected by mannitol, indicating that differential expression of klotho is not caused by hyperosmolarity. HG (35mM D-glucose) also upregulates the expression of TGF-β1, Fractalkine, MMP2, and MMP9, which are further enhanced under the Klotho deficiency state using Klotho-siRNA transfection, and mitigated by Klotho over-expression using Klotho-plasmid transfection. Neither HG nor Klotho has effects on the migration or proliferation of HA-SMCs.

Conclusion

HG regulates the expression of endogenous Klotho and in an unique diphase pattern. Endogenous Klotho may play direct roles in the pathogenesis of diabetic vascular disease using the knock-down and knock-in Klotho system, which suggest that Klotho may serve as a potential treatment target in diabetic vascular disease.

Funding

  • Government Support - Non-U.S.