In vitro large-wound re-endothelialization. Inhibition of centrosome redistribution by transient inhibition of transcription after wounding prevents rapid repair.

Abstract

Rapid, efficient re-endothelialization of large wounds is characterized by a specific sequence of cytoskeletal events that occur after wounding. Wounds 1.5 mm wide were created down the middle of confluent porcine aortic endothelial monolayers to study regulation of repair. The wounded cultures were incubated for short periods with cycloheximide or actinomycin D to test the hypothesis that transient inhibition of translation and transcription at the time of wounding disrupts rapid repair by interfering with centrosome redistribution to the front of the cell, an early event associated with cell migration. Although centrosome reorientation did not occur when protein synthesis was inhibited with 20 micrograms/mL cycloheximide for 1 hour before and for up to 4 hours after wounding, reorientation did occur by 2 hours after cycloheximide was washed out. The times taken for the wound to close for cycloheximide-treated and control cells did not differ (60 +/- 1.1 vs 60 +/- 0.8 hours). When transcription was inhibited with 0.25 micrograms/mL actinomycin D for 1 hour before and for 1 hour after wounding, re-endothelialization was dramatically reduced. The time taken for the wound to close was almost five times longer (288 +/- 5.3 hours) than for control cells. The cells moved very slowly, maintaining a flattened, spread-out shape, as opposed to being elongated. The centrosomes did not reorient to the front of the cell throughout the entire period. However, addition of actinomycin D for 2 hours when centrosomes had already moved to the front of the cells (4 hours after wounding) did not reduce subsequent wound repair (60 +/- 1.3 hours). This study supports our hypothesis that centrosome redistribution is essential for efficient wound repair and suggests that redistribution is regulated by transcription of essential gene(s) that is induced immediately after wounding by an unknown short-lived signal. Two possible signals are the loss of cell contact and/or a soluble substance released from the cells at the time of wounding. When the signal is unable to induce transcription, dysfunctional repair occurs by a very slow centrosome-independent process.

Inhibition of cytoskeletal reorganization stimulates actin and tubulin syntheses during injury-induced cell migration in the corneal endothelium

“…Similar results are observed in vascular endothelium, both in vitro [Gotlieb et al, 1984] and in the organ cultured system [Rogers et al, 1989]. Mechanisms underlying MTOC translocation are not fully understood, although it has been shown to depend on transcriptional activity occurring shortly after wounding [Ettenson and Gotlieb, 1993a]. The relocation of the MTOC appears to correlate with directed cell migration and interfering with this process retards wound repair [Ettenson and Gotlieb, 1993a].…”

“…Mechanisms underlying MTOC translocation are not fully understood, although it has been shown to depend on transcriptional activity occurring shortly after wounding [Ettenson and Gotlieb, 1993a]. The relocation of the MTOC appears to correlate with directed cell migration and interfering with this process retards wound repair [Ettenson and Gotlieb, 1993a]. Similarly, in the corneal endothelium, MTOC translocation may be coupled not only with directed migration but also to the polarized secretion of extracellular matrix protein.…”

Possible molecular mechanisms regulating endothelial repair in unstable fibroinflammatory atheroma

“…We showed that ECs at the edge of a wounded monolayer reposition their centrosome to a position between the cell nucleus and the leading edge of the cell [3,26]. Failure to reposition their centrosome leads to impaired EC migration at wound edges and markedly delays repair of the endothelial monolayer [26].…”

“…MTs play a role in maintaining cell shape and directional migration of ECs [3,26]. MTs are formed by self-assembly of alpha -beta (ah) tubulin heterodimers.…”

“…Cdc 42, dynein, and dynactin regulate MT organizing center reorientation independent of Rho-regulated MT stabilization [31]. Disruption of actin MFs resulted in a long delay in the reorientation of centrosomes in cells at the wound edge [26], suggesting that cooperation between cytoskeletal networks is important. In the presence of normal hemodynamic shear stress, distribution of MTs and centrosomes are similar in vivo and in vitro.…”