Nat Commun. 2020 Jul 17;11(1):3595.
doi: 10.1038/s41467-020-17329-8.
Background
Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. Another situation is, following leukocyte crossing, microvascular integrity quickly recovers through re-annealing of inter-endothelial junctions. However, the molecular basis of these processes is poorly defined. Most mechanistic studies have been carried out in-vitro. In-vivo studies have used EC-contraction mediators such as thrombin and LPS which also affect other cellular functions and cells, particularly immune cells thus adding confounding factors which prevent the mechanistic study of EBF recovery.Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. The authors have investigated the molecular mechanism of EBF recovery in response to endothelial contraction and junction release-inducing stimuli using in-vitro and in-vivo models of MHC-induced vascular leakage in the context of inter-endothelial CD31 interactions.
Methods
1. Isolation and culture of primary microvascular endothelial cells, 2. Measurement of endothelial permeability in-vivo, 3. Antibody-mediated EC activation, 4. Measurement of trans-endothelial electrical resistance(TEER), 5. Actin cytoskeleton analysis, 6. Measurement of ECAR and OCR.
Results
1. CD31 is required for endothelial barrier recoveryPermeability of EC barrier function was monitored by trans-endothelial electrical resistance(TEER), and the authors found that MHC-I activation (by antibody) triggered quick and similar reduction in TEER by both WT and Cd31 KO EC, however, recovery of TEER of Cd31 KO EC after post stimulation is significantly prolonged.(Blue: WT EC,Orange: Cd31KO EC)
As endothelial contractility is associated with F-actin polymerization and stress fiber formation, the authors further analyzed EC cytoskeletal rearrangements following MHC-triggering.While CD31 triggering on its own did not elicit any effect, co-ligation with MHC molecules significantly increased F-actin polymerization above the levels induced by MHC-signals, suggesting that CD31 is required for efficient anchorage of actinfibers to the intercellular junctions during EC contraction.2. CD31 signal prevents b-catenin phosphorylationIntegrity of intercellular junctions is a major determinant of permeability of the endothelium, and de-phosphorylation of VE-cadherin-catenin complexes in adherens junctions mediate both anchorage and mechanical coupling of the cytoskeleton of adjacent EC including during leukocyte extravasation. The authors therefore analyzed β-catenin phosphorylation in response to MHC- and ICAM-1-mediated signals by CD31-expressing or -deficient EC, and they found that CD31 signals prevent VE-cadherin and β-catenin phosphorylation in response to MHC stimulation via ITIM 686-thyrosine phosphorylation.3. CD31 KO EC display impaired glycolysis and similar level of mitochondrial respiration upon stimulation.
The observations above do not explain the intense membrane-associated cytoskeleton reorganization and EC contraction induced by MHC and CD31 co-engagement. Although Src-induced phosphorylation of VE-cadherin prevents the binding of β-catenin, this is not sufficient to increase endothelial permeability as reported.The authors draw attention to EC metabolism, as it has recently emerged as animportant regulator of endothelial function.
Conclusions
MHC triggering induces RhoA and Erk activation and ECcontraction.MHC signals induce CD31 ITIM phosphorylation and SHP-2 recruitment. SHP-2 prevents the phosphorylation of b-catenin and VE-cadherin.Dephosphorylated b-catenin can also transfer to the nucleus where it induces cMyc transcription. In parallel, SHP-2 induces AKT activation which in turn inhibits FoxO1 nuclear translocation, thus preventing inhibition of cMyc transcription.cMyc transcription and FoxO1 translocation leads to enhanced transcription of glycolysis enzymes and enhanced glycolysis required for actin remodeling and maintenance of junctional anchorage.