Oscillatory shear potentiates latent TGF-ß1 activation more than steady shear as demonstrated by a novel force generator.

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor ß1 ubiquitously in a latent form (LTGF-ß1). While complex shear stresses can activate LTGF-ß1, the mechanisms underlying LTGF-ß1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-ß1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-ß1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-ß1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-ß1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-ß1 activation. OSS-generated active TGF-ß1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-ß1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-ß1 to drive vascular remodeling.

Platelet TGF-ß1 deficiency decreases liver fibrosis in a mouse model of liver injury.

Transforming growth factor-ß1 (TGF-ß1) signaling in hepatic stellate cells (HSCs) plays a primary role in liver fibrosis, but the source of TGF-ß1 is unclear. Because platelets are rich in TGF-ß1, we examined the role of platelet TGF-ß1 in liver fibrosis by challenging wild-type (WT) mice and mice deficient in platelet TGF-ß1 (PF4CreTgfb1f/f) with carbon tetrachloride (CCl4), an inducer of acute hepatic injury and chronic fibrosis. CCl4 elicited equivalent hepatic injury in WT and PF4CreTgfb1f/f mice based on loss of cytochrome P450 (Cyp2e1) expression, observed at 6 hours and peaking at 3 days after CCl4 challenge; PF4CreTgfb1f/f mice exhibited less liver fibrosis than control mice. Activated platelets were observed during acute liver injury (6 hours), and WT mice with transient platelet depletion (thrombocytopenia) were partially protected from developing fibrosis compared with control mice (P = .01), suggesting an association between platelet activation and fibrosis. Transient increases in TGF-ß1 levels and Smad2 phosphorylation signaling were observed 6 hours and 3 days, respectively, after CCl4 challenge in WT, but not PF4CreTgfb1f/f , mice, suggesting that increased TGF-ß1 levels originated from platelet-released TGF-ß1 during the initial injury. Numbers of collagen-producing HSCs and myofibroblasts were higher at 3 days and 36 days, respectively, in WT vs PF4CreTgfb1f/f mice, suggesting that platelet TGF-ß1 may have stimulated HSC transdifferentiation into myofibroblasts. Thus, platelet TGF-ß1 partially contributes to liver fibrosis, most likely by initiating profibrotic signaling in HSCs and collagen synthesis. Further studies are required to evaluate whether blocking platelet and TGF-ß1 activation during acute liver injury prevents liver fibrosis.