The essential role of transglutaminase 2 in the cytoskeletal responses of vascular smooth muscle cells to mechanical stretching

The hardening of the vasculature, otherwise known as arterial stiffening, often precedes the onset of hypertension. Indeed, vascular stiffening is an early and independent predictor of hypertension and a risk factor for life-threatening cardiovascular events. Notably, arterial stiffening increases pulsatile pressure and modulates circumferential stress-strain relationships in vascular smooth muscle cells (VSMC). This triggers a cascade of events that leads to structural changes, including accumulating VSMC fibrillar (F)-actin, as shown in the arteries of hypertensive humans and animal models. We and others have also demonstrated that tissue transglutaminase 2 (TG2) activity promotes arterial stiffening and inward remodeling. TG2 is a crosslinking enzyme that works as a G-protein and cellular receptor scaffold. However, it remains unclear how TG2 is activated in the arterial wall. Due to its structural conformation and location at the cell membrane, it has been suggested that mechanical forces activate TG2. Accordingly, we hypothesized that VSMC cytoskeletal responses to mechanical stretching require TG2 activity. We tested our hypothesis in cultured human aortic smooth muscle cells (HAoSMC) that were either left unstretched or exposed to cyclic uniaxial stretch using a Flexcell tension system with 11% elongation at 1 Hz for 6 hours simulating conduit artery pulsatility. TG2 activity was inhibited by treating HAoSMC with the nitric oxide (NO) donor s-nitrosoglutathione (400μmol/L for 6 hours) or cystamine (200μmol/L for 6 hours) under stretched conditions (n=5-12/condition). Additionally, TGM2 knockdown was performed in cultured HAoSMC using siRNA alongside scrambled siRNA negative controls under static and stretched conditions (n=5-6/condition). We also assessed ex vivo TG2 activity in abdominal aortic rings under unstretched or stretched (12mN for 1 hour) conditions (n=3/condition). All differences reported herein are significant at p<0.05. Our results show that in vitro mechanical stretching increases VSMC TG2 activity (presence of N-epsilon gamma glutamyl Lysine links, 1.3-fold), F-actin stress fibers (phalloidin staining, 1.3-fold), and ex vivo TG2 activity (cadaverine incorporation, 1.4-fold) relative to unstretched conditions. Notably, the increase in TG2 activity and F-actin content occurring in response to stretching was inhibited by the NO donor, cystamine, or TG2 knockdown. Accordingly, VSMC stiffening (1.3-fold) induced by mechanical stretching was significantly prevented by TG2 inhibition as determined using atomic force microscopy. These findings demonstrate that stretch-induced VSMC F-actin formation and stiffening require TG2 activity. They also offer valuable insights into the potential targetable mechanisms by which TG2 activation in response to mechanical stimuli contributes to arterial stiffening and remodeling. National Institutes of Health grant: R01HL153264 to LM-L and JP This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.