The endothelial monolayer partitioning underlying tissue from blood components in the vessel wall maintains tissue fluid balance and host defense through dynamically opening intercellular junctions. Edemagenic agonists disrupt endothelial barrier function by signaling the opening of the intercellular junctions leading to the formation of protein‐rich edema in the interstitial tissue, a hallmark of tissue inflammation that, if left untreated, causes fatal diseases, such as acute respiratory distress syndrome. In this review, we discuss how intercellular junctions are maintained under normal conditions and after stimulation of endothelium with edemagenic agonists. We have focused on reviewing the new concepts dealing with the alteration of adherens junctions after inflammatory stimulus.

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture–induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1β reduced cAMP generation and CREB-mediated transcription of VE-cadherin. Furthermore, endothelial cell–specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase–mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS.

Lung vascular endothelial barrier disruption and the accompanying inflammation are primary pathogenic features of acute lung injury (ALI); however, the basis for the development of both remains unclear. Studies have shown that activation of transient receptor potential canonical (TRPC) channels induces Ca²⁺ entry, which is essential for increased endothelial permeability. Here, we addressed the role of Toll-like receptor 4 (TLR4) intersection with TRPC6-dependent Ca²⁺ signaling in endothelial cells (ECs) in mediating lung vascular leakage and inflammation. We find that the endotoxin (lipopolysaccharide; LPS) induces Ca²⁺ entry in ECs in a TLR4-dependent manner. Moreover, deletion of TRPC6 renders mice resistant to endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. TRPC6 induces Ca²⁺ entry in ECs, which is secondary to the generation of diacylglycerol (DAG) induced by LPS. Ca²⁺ entry mediated by TRPC6, in turn, activates the nonmuscle myosin light chain kinase (MYLK), which not only increases lung vascular permeability but also serves as a scaffold to promote the interaction of myeloid differentiation factor 88 and IL-1R–associated kinase 4, which are required for NF-κB activation and lung inflammation. Our findings suggest that TRPC6-dependent Ca²⁺ entry into ECs, secondary to TLR4-induced DAG generation, participates in mediating both lung vascular barrier disruption and inflammation induced by endotoxin.

The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol 12-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-α. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-α with Rho. Activated PKC-α but not PKC-ε induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-α is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.

RhoA activation and increased intracellular Ca²⁺ concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Gα_q mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca²⁺ concentration by the inhibitor of Gα_q or phospholipase C and the Ca²⁺ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca²⁺ also inhibited RhoA activation, indicating the requirement of Ca²⁺ entry in the response. RhoA activation could not be ascribed to storeoperated Ca²⁺ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca²⁺ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Gα_q-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca²⁺ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca²⁺ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCα activation. Our results demonstrate that Gα_q activation of TRPC6 signals the activation of PKCα, and thereby induces RhoA activity and endothelial cell contraction.

We tested the hypothesis that RhoA, a monomeric GTP-binding protein, induces association of inositol trisphosphate receptor (IP₃R) with transient receptor potential channel (TRPC1), and thereby activates store depletion-induced Ca²⁺ entry in endothelial cells. We showed that RhoA upon activation with thrombin associated with both IP₃R and TRPC1. Thrombin also induced translocation of a complex consisting of Rho, IP₃R, and TRPC1 to the plasma membrane. IP₃R and TRPC1 translocation and association required Rho activation because the response was not seen in C3 transferase (C3)-treated cells. Rho function inhibition using Rho dominant-negative mutant or C3 dampened Ca²⁺ entry regardless of whether Ca²⁺ stores were emptied by thrombin, thapsigargin, or inositol trisphosphate. Rho-induced association of IP₃R with TRPC1 was dependent on actin filament polymerization because latrunculin (which inhibits actin polymerization) prevented both the association and Ca²⁺ entry. We also showed that thrombin produced a sustained Rho-dependent increase in cytosolic Ca²⁺ concentration [Ca²⁺]_i in endothelial cells overexpressing TRPC1. We further showed that Rho-activated Ca²⁺ entry via TRPC1 is important in the mechanism of the thrombin-induced increase in endothelial permeability. In summary, Rho activation signals interaction of IP₃R with TRPC1 at the plasma membrane of endothelial cells, and triggers Ca²⁺ entry following store depletion and the resultant increase in endothelial permeability.