Regulation of endothelial permeability and transendothelial migration of cancer cells by tropomyosin-1 phosphorylation

Abstract

BackgroundLoss of endothelial cell integrity and selective permeability barrier is an early event in the sequence of oxidant-mediated injury and may result in atherosclerosis, hypertension and facilitation of transendothelial migration of cancer cells during metastasis. We already reported that endothelial cell integrity is tightly regulated by the balanced co-activation of p38 and ERK pathways. In particular, we showed that phosphorylation of tropomyosin-1 (tropomyosin alpha-1 chain = Tm1) at Ser283 by DAP kinase, downstream of the ERK pathway might be a key event required to maintain the integrity and normal functions of the endothelium in response to oxidative stress.MethodsEndothelial permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of HUVECs grown to confluence in Boyden chambers. Actin and Tm1 dynamics and distribution were evaluated by immunofluorescence. We modulated the expression of Tm1 by siRNA and lentiviral-mediated expression of wild type and mutated forms of Tm1 insensitive to the siRNA. Transendothelial migration of HT-29 colon cancer cells was monitored in Boyden chambers similarly as for permeability.ResultsWe provide evidence indicating that Tm1 phosphorylation at Ser283 is essential to regulate endothelial permeability under oxidative stress by modulating actin dynamics. Moreover, the transendothelial migration of colon cancer cells is also regulated by the phosphorylation of Tm1 at Ser283.ConclusionOur finding strongly support the role for the phosphorylation of endothelial Tm1 at Ser283 to prevent endothelial barrier dysfunction associated with oxidative stress injury.

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis‐associated colon cancer

“…35,36 Furthermore, we showed that endothelial cell permeability of ICG in vitro was promoted by both PGE 2 , a downstream product of COX-2, 33 and H 2 O 2 , an upstream reactive oxygen species of iNOS. 33,37 Prostaglandin E 2 is known to increase local blood flow, resulting in vascular hyperpermeability. 32 In the present study, intradermal injection of PGE 2 induced ICG permeability and accumulation in normal ear tissues, suggesting that enhanced permeability induced by PGE 2 was likely to be one of the mechanisms of tumor accumulation of ICG.…”

Carbonic Anhydrase Activation Is Associated With Worsened Pathological Remodeling in Human Ischemic Diabetic Cardiomyopathy

“…Human recombinant CA‐I protein (Sigma) was added at 100 μmol/L to hCEC culture in vitro. Permeability across endothelial cell monolayers was measured using gelatin‐coated Transwell units (Boyden chambers) (6.5‐mm diameter, 0.4‐μm‐pore polycarbonate filter; Corning Costar), as previously described . Briefly, hCECs were plated at a density of 0.6×10 5 cells (or 1×10 5 in the case of transduced cells) per well (upper chamber) and were cultured for 3 days until a tight monolayer formed.…”

“…Permeability across endothelial cell monolayers was measured using gelatincoated Transwell units (Boyden chambers) (6.5-mm diameter, 0.4-lm-pore polycarbonate filter; Corning Costar), as previously described. 23 Briefly, hCECs were plated at a density of 0.6910 5 cells (or 1910 5 in the case of transduced cells) per well (upper chamber) and were cultured for 3 days until a tight monolayer formed. Endothelial permeability was determined by measuring the passage of FITC-labeled dextran (1 mg/mL of fluorescein isothiocyanate-dextran, molecular mass 40 kDa; Sigma-Aldrich) through the hCEC monolayer.…”

SASH1, a new potential link between smoking and atherosclerosis

“…This protein is involved in the polymerization of the actin cytoskeleton and protects endothelial cell-cell junctions from the oxidative stress triggered by aqueous cigarette smoke extract 237 . TPM1 phosphorylation state regulates endothelial permeability to cancerous cells 238 , and TPM1 was also demonstrated to be a tumor suppressor in various cancers, including breast cancer 239,240 , where it resensitizes cancer cells to anoikis (programmed cell death triggered by detachment from the extracellular matrix). As SASH1 binds TPM1, it could be envisioned that it preserves endothelial integrity through the stabilization of the actin cytoskeleton.…”