Probing the crosslinking independent roles of tissue transglutaminase in vascular aging using a novel transamidation‐deficient TGM2‐C277S mouse model

Introduction Tissue transglutaminase (TG2) is a multifunctional protein of the transglutaminase family with putative transamidation‐independent functions in aging associated vascular stiffening and dysfunction. The development of preclinical models is critical to fully understand the physiological relevance of transamidation independent functions of TG2 and to identify the specific function of TG2 for therapeutic targeting. Therefore, In this study we harnessed the CRISPR‐Cas9 gene editing technology to introduce a mutation at the active site cysteine (C277) in the mouse TGM2 gene. Methods and results We investigated the contributions of the transamidation‐dependent vs. transamidaiton‐independent functions of TG2 in vascular aging using TGM2‐C277S mice, littermate WT mice, and age‐matched TG2−/− mice. Heterozygous and homozygous TGM2‐C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein abundance was similar to wild‐type (WT) in the TGM2‐C277S mutant mice in the liver, lung, heart, aorta, and kidney. GTP binding, and interactions with fibronectin and integrin beta1 were preserved in the TG2‐C277S mouse protein. Transglutaminase function was absent in the TG2 protein from TGM2‐C277S mice when compared with wild‐type (WT) littermates. Loss of transamidation function resulted in a marked loss in serotonin induced vasoconstriction and in vitro remodeling of the carotid artery in response to chronic vasoconstriction. On the other hand, cell adhesion and proliferation were comparable to that of WT in the vascular smooth muscle cells from TGM2‐C277S but not in those from TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse wave velocity, tensile testing, and pressure myography. TGM2‐C277S mice were markedly protected from age‐associated vascular stiffening, but not to the same extent as TG2−/− mice. Conclusions Together, these studies show that the TGM2‐C277S mice ubiquitously express a TG2 protein that lacks transamidation function and retains its other functions and can be used a tool for in vivo studies to examine the transamidation‐independent roles of TG2 in physiology and pathophysiology. While TG2‐mediated ECM crosslinking and VSMC dysfunction, both propagate vascular stiffening in aging, the transamidation function is the primary shareholder in aging associated mechanical decline of the large vessels. Support or Funding Information This work was funded by a StAAR Investigator Award (to LS) and a MedImmune Research Funds Award (to LS).