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SUMMARY Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease.

Aims In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. Methods and results Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor α stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. Conclusion Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response. [ABSTRACT FROM AUTHOR]