Rho and Rac but not Cdc42 regulate endothelial cell permeability

Endothelial cell permeability is a key factor in vascular physiology and disease. Prior research has shown that thrombin and histamine can increase permeability through actin reorganization and junctional disassembly. However, the specific roles of Rho family GTPases in this process remain unclear. No prior work had resolved how Rho, Rac, and Cdc42 differentially regulate endothelial barrier function. This gap motivated an investigation into their individual contributions. Understanding these mechanisms could clarify how endothelial permeability is controlled. The study sought to address whether Rho, Rac, or Cdc42 are essential for permeability changes. The uncertainty around their roles in junctional dynamics and actin remodeling drove the experimental design. This work builds on prior knowledge of GTPase signaling in cell structure.

The aim of this study was to determine how Rho, Rac, and Cdc42 influence endothelial permeability. Specifically, the researchers wanted to assess whether these GTPases regulate permeability through effects on actin and junctional structures. The study focused on thrombin- and histamine-induced permeability changes in endothelial cells. The motivation came from gaps in understanding how each GTPase contributes to barrier function. The researchers hypothesized that Rho and Rac might have distinct roles in this process. They also wanted to clarify whether stress fibre formation is essential for permeability. The study aimed to test whether dominant-negative constructs could alter permeability outcomes. This approach allowed for direct comparison of each GTPase’s impact on endothelial function.

The researchers used adenovirus-mediated gene transfer to express RhoA, Rac1, and Cdc42 in human umbilical vein endothelial cells. They tested the effects of dominant-negative constructs and a Rho kinase inhibitor, Y-27632. Permeability was measured using transendothelial resistance and intercellular gap formation. The study included thrombin and histamine stimulation to induce permeability changes. Actin stress fibre assembly was analyzed using fluorescent markers. Adherens and tight junctions were assessed for structural integrity. The methods allowed for direct comparison of each GTPase’s role in permeability. This approach provided insights into how each protein affects endothelial barrier function.

Dominant-negative RhoA and Y-27632 inhibited thrombin- and histamine-induced permeability and junctional disassembly. These treatments also prevented stress fibre formation and contractility. In contrast, dominant-negative Rac1 increased permeability in unstimulated cells and enhanced thrombin-induced permeability. However, it inhibited stress fibre assembly, suggesting that stress fibres are not essential for permeability. Dominant-negative Cdc42 reduced thrombin-induced stress fibre formation but had no effect on permeability or histamine responses. The results showed that Rho and Rac regulate permeability through distinct mechanisms. Rho inhibition preserved junctional integrity and reduced permeability. These findings clarify the differential roles of Rho family members in endothelial function.

The authors concluded that Rho and Rac regulate endothelial permeability through different mechanisms. Rho inhibition prevents permeability by preserving junctional integrity and reducing stress fibre formation. In contrast, Rac inhibition increases permeability despite reducing stress fibres, indicating that stress fibres are not essential for permeability. Cdc42 does not significantly affect permeability or junctional responses. These findings suggest that Rho and Rac have distinct roles in barrier function. The results provide a mechanistic explanation for their effects on permeability. The study supports the idea that Rho and Rac are key regulators of endothelial permeability. However, Cdc42 appears to play a minimal role in this process. These conclusions align with the observed effects of dominant-negative constructs and inhibitors.