Peri/epicellular protein disulfide isomerase PDI acts as an organizer of cytoskeletal mechanoadaptation in vascular smooth muscle cells

Abstract

Mechanobiologycal and redox processes sinergize to regulate physiological or pathological vascular conditions. More specifically, adaptations to cyclic stretch on vascular smooth muscle cells (VSMC) have been shown to be redox‐regulated. However, mechanisms connecting mechanoadaptation to oxidant generation are still unclear. Previously, we reported that protein disulfide isomerase A1 (PDI), a redox chaperone from endoplasmic reticulum, is targeted to peri/epicellular (pec) space and supports an anti‐constrictive remodeling effect in balloon‐injured arteries by means of cytoskeleton and extracellular matrix architecture organization. We hypothesized that pec PDI acts as a global redox adaptor by connecting oxidant generation with responses to extra‐ or intracellular forces. First, pecPDI inhibition through specific neutralizing antibody (PDI Ab) incubation, prevented stress fiber assembly in response to prolonged exposure to equibiaxial stretch (10–12% at 1 Hz for 24 h). In addition, uniaxial stretch promotes cell repositioning perpendicularly to stretch orientation. Such response was also regulated by pecPDI, as PDI Ab treatment attenuated cell alignment in stretched VSMC at 4h. Through biotinylation experiments followed by western detection, we showed that pecPDI sustains a pro‐oxidant effect on beta1 integrin thiols. To address whether pecPDI organizes intracellular force distribution in such events, we used traction force microscopy, which revealed a significantly decreased net contractile moment in PDGF‐exposed VSMC after pecPDI neutralization (0.70 ±0.08 AU vs control=0.9 ±0.09, p<0.05). Thus, pecPDI involvement in mechanoresponse is not only limited to external forces. The balance between intracellular traction forces and cell adhesion governs cell migration. Indeed, in a model of single VSMC migration, pecPDI impaired migration persistence without affecting total distance or velocity. Since the Rho GTPase RhoA is known to act as a master mechanoregulator, we addressed if downstream pecPDI‐related mechanisms involve RhoA. Neither RhoA expression nor total activity were affected by pecPDI inhibition. However, the polarized distribution of RhoA or caveolin‐3 clusters promoted by cyclic stretch was disrupted by pecPDI inhibition, which promoted a non‐polarized pattern of RhoA/caveolin‐3 cluster colocalization. Moreover, we searched for pecPDI effects on localized RhoA activity using a FRET biosensor. The higher local RhoA activity at cell protrusions compared to perinuclear regions was disrupted by pecPDI neutralization, confirming its regulation of localized RhoA activation. In conclusion, pecPDI acts as a redox organizer able to restrict the noise of cytoskeletal repositioning during mechanoresponses in VSMC. This effect may have several implications, including the role of redox processes and pecPDI on vascular remodeling.Support or Funding InformationSupported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CEPID‐Redoxoma grant 2013/07937‐8 and scholarship grant 2013/17115‐5This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.