Ectopic expression of KLF2 induces both functional and morphological changes in endothelial cells which mimic the effects of shear stress. KLF2 was shown to affect the expression of vascular tone regulating genes which enables the establishment of a functionally quiescent endothelium. ECs expressing KLF2 display anti-inflammatory, anti-thrombotic, anti-migratory, antifibrotic and anti-oxidant properties. A number of thrombotic and inflammatory mediators originate from EC-specific, elongated secretory organelles called WeibelPalade bodies. WPBs function as storage vesicle for von Willebrand Factor, a multimeric glycoprotein which plays a crucial role in platelet plug formation. In addition, these organelles also contain other bioactive compounds including P-selectin, lamp3, Ang2, IL-8, eotaxin-3, osteoprotegerin-1 and endothelin-1, the release of which enables the endothelium to actively participate in inflammatory responses, angiogenesis and regulation of vascular tone. WPBs fuse with the plasma membrane resulting in release of their contents in the circulation and exposure of P-selectin on the plasma membrane. However, a subset of WPBs is able to escape regulated exocytosis in response to cAMP-raising agonists and form a perinuclear cluster at the microtubule organizing center. The minus-end VE-821 directed transport of WPBs along the microtubules to the MTOC is mediated by the dynein/ dynactin complex and protein kinase A.
We have recently shown that expression of KLF2 modulates the thrombin-induced release of WPBs whereas the epinephrineinduced release of these organelles was not affected. In this work we have further characterized the atheroprotective phenotype induced by KLF2 with regard to the secretory pathway of ECs. We show that lentiviral expression of KLF2 leads to an altered morphology and composition of WPBs and results in impaired regulated secretion of Ang2 and IL-8. Strikingly, we found that KLF2 expressing cells no longer display perinuclear clustering of WPBs after stimulation with cAMPmediated agonists.
Our data indicate that the atheroprotective phenotype of KLF2 expressing ECs extends to their regulated secretory pathway and markedly alters the composition and regulation of their secretory response. KLF2 is responsible for the shear stress induced modulation of gene expression that limits inflammatory responses and thereby sustains endothelial quiescence. In this study we explored the effects of KLF2 on the dynamics and properties of WPBs. In agreement with previous observations we show that the number of secretable WPBs increases upon expression of KLF2. We anticipate that the increased number of WPBs results from the upregulation of VWF mRNA levels induced by KLF2. The increase in number of WPBs in KLF2-transduced cells suggests that generation of WPBs is mainly driven by the amount of VWF synthesized by endothelial cells. It has been well-established that the formation of WPBs is strictly dependent on the presence of VWF. Absence of WPBs affects the regulated release of other WPB constituents such as P-selectin.