Photochemically induced increase in endothelial permeability regulated by RhoA activation

Area of Science:

• Cell Biology
• Biochemistry
• Medical Science

Background:

• Photodynamic therapy (PDT) involves photochemical reactions that can damage normal cells and tissues.
• This damage increases endothelial permeability, contributing to neointimal formation and restenosis.
• The precise mechanisms underlying PDT-induced endothelial permeability increase require further investigation.

Purpose of the Study:

• To investigate the in vitro mechanisms by which photochemical reactions increase endothelial permeability.
• To identify key molecular players involved in PDT-induced endothelial cell morphology changes and permeability.

Main Methods:

• Human umbilical vein endothelial cells were treated with delta-aminolevulinic acid and irradiated with a 646 nm LED.
• Endothelial permeability was assessed using Transwell assays.
• Cell morphology, VE-cadherin localization, and stress fiber formation were analyzed using fluorescence microscopy.
• Protein phosphorylation (GDP-RhoA to GTP-RhoA) was evaluated by Western blotting.
• Inhibitors of RhoA signaling (forskolin/rolipram, 8CPT-2'O-Me-cAMP) were used to block downstream effects.

Main Results:

• Photochemical reactions significantly increased endothelial permeability by 200%.
• PDT induced cell shrinkage, capillary-like structure destruction, VE-cadherin mislocalization, and stress fiber formation.
• Western blotting confirmed photochemical induction of GDP-RhoA to GTP-RhoA phosphorylation.
• Inhibition of RhoA signaling prevented stress fiber formation and VE-cadherin mislocalization, maintaining normal endothelial permeability.

Conclusions:

• Photochemically induced RhoA activation is a critical mechanism increasing endothelial permeability.
• RhoA activation alters endothelial cell morphology, leading to increased permeability.
• Targeting RhoA signaling may offer a therapeutic strategy to mitigate PDT-induced vascular damage.