Figure 2. Mechanisms of pulmonary endothelial barrier-disruption and pericyte apoptosis induced by ACE/Ang II/AT1R, IL6 and TNF-α-mediated pathways are illustrated (Red pathways): Endothelial barrier-disruption results from actin-myosin interaction after MLC-phosphorylation, which is regulated by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Activation of the actin myosin contractile apparatus disperses cortical actin and increases actin stress fibre formation, resulting in cell contraction and tensional force applied to adherens junction (AJ) proteins. RhoA acts via effector protein Rho-associated protein kinase (ROCK) to activate MLCK and inhibit MLCP. MLCK activation is modulated by Ca2+ which enters the cytosol from endoplasmic reticulum (ER) or extracellular space. Phosphorylation of specific tyrosine residues of cytoskeletal proteins and adhesion molecules including vascular endothelial cadherin (VE-cadherin) as well as microtubule disassembly are MLCK-independent mechanisms of barrier-disruption; Src mediated VE-cadherin phosphorylation leads to VE-cadherin internalisation. Nuclear factor κB (NF-κB) activation by Ang II, TNF-α and IL6 promotes a proinflammatory state resulting in degradation of the endothelial glycocalyx, which may expose leukocyte ligands. Ang II/AT1R activates non-specific cation channels and voltage dependent calcium channels resulting in increased cytosolic calcium in pericytes resulting in pericyte contraction and depolarization, this is coupled with opening of calcium dependent chloride channels enhancing depolarization pericyte contraction. Ang II regulates gene-expression of Ang 2 and antagonizes AngI/Tie2 pathway resulting in nuclear translocation of FOXO3A and pericyte apoptosis. TNFα increases caspase 3 activity and FOXO DNA-binding activity via p38MAPK and JNK pathways to induce pericyte apoptosis.Downregulation of ACE2 activity results in decreased barrier enhancement activity (Green pathways): RhoA activity is inhibited by the GTPases Rap1 and Rac1 as well as cyclic AMP (cAMP) induced protein kinase A (PKA) activation. cAMP levels increase in response Ang1-7-masR activity to induce activation of PKA (which inhibits RhoA) as well as the guanine exchange factor, exchange protein activated by cAMP (Epac). Epac (via Rap1) enhances VE-cadherin junctional integrity and actin reorganisation. Rap1 enhances barrier function via inhibition of Rho and activation of Cdc42 as well as a cooperative association with VE-cadherin. Cdc42 directly regulates cortical actin organisation and proteins including MLCK and neural-Wiskott Aldrich syndrome protein (N-WASP) that mediate cortical actin formation via interaction with focal adhesion kinase (FAK) and actin-related protein (ARP) thus strengthening AJ and tight junction (TJ) formation as well as cell adhesion to the extracellular matrix (ECM). FAK also signals via effector molecules to inhibit RhoA and activate Rac1. Ang1-7 acts on masR and increases the expression of Akt along with Akt phosphorylation resulting in raised Akt/phosphorylated Akt ratio leading to Rac 1 activation.