Role And Mechanism Of PAK1in Embryonic Vascular Development

p21-activated kinase1plays as an intracellular serine/threonine protein kinase with awide range of functions in organisms. In the past decades, researches of PAK1werefocused on its roles and mechanisms in cancer. Recent years, an increasing number ofstudies were performed to investigate its effects in organic development. However, fewstudy demonstrated the roles of PAK1in embryonic vascular development. Although itseemed that PAK1was associated with tumor angiogenesis and pathological increasedvascular permeability, its effects in physiological angiogenesis was not yet clear. It hadbeen found that PAK1was one of the target genes of endothelial-specific microRNA-126,which was related to embryonic vascular integrity, suggesting that PAK1might have someeffects on embryonic vascular development. However, its molecular mechanisms of actionhad not been disclosed.Vascular development is a complex process involving various signal pathways likeVEGF, PDGF, Notch, et al. Notch is one of the most important pathways in angiogenesis,regulating the differentiation of angioblasts, maturation of endothelial cells, arterial-venouscell fate decision and sprouting. Studies have shown that in many tumor cells, PAK1canbe activated by growth factors such as VEGF and PDFG which were associated with thevascular development, and it was proved that activation of PAK1in human embryonickidney cells could inhibit Notch pathway by phosphorylation of its substrate proteinSHARP, suggesting that PAK1may play important roles in vascular development.Objective: To investigate the effects of p21-activated kinase1(PAK1) on embryonicvascular development in zebrafish and its impact on vascular endothelial cells, confirmingits roles and mechanisms in embryonic angiogenesis through Notch pathway.Methods: We cloned the coding sequence of pak1by PCR and constructed its expressionplasmid by inserting the CDS into PCS2vector. The PCS2-pak1plasmid DNA waslinearized by Not I digestion. In vitro transcription reactions were performed with thelinearized templates and SP6RNA polymerase. Microinjection method was performed tooverexpress pak1mRNA in single-cell stage embryos of wild-type zebrafish. Phenotype ofthese embryos was observed under an inverted microscope. The incidence of phenotypicabnormalities, the spatial and temporal characteristics of the phenotype and the outcomesof the injected embryos were recorded in different developmental stage. Furthermore, weused endothelium-specific Tg (flk1: mcherry-ras) transgenic zebrafish to investigate theshape, number and distribution of vessels by confocal microscopy. Embryos were treated with2%paraformaldehyde and glutaraldehyde, embedded and sectioned, stained withuranyl acetate and lead, and the ultrastructure of the vessel wall was observed bytransmission electron microscope. Transfection of pBSKI2-caPAK1in cultured HUVECwas performed by using Lipofectamine LTX with its PLUS reagent. Control cells weretreated with the same concentration of liposomes. Morphological changes of cells wereobserved under an inverted optical microscope and expression level of PAK1was verifiedby quantitative real-time PCR after transfection. MTT based assay was used to detect cellviability. The absorbance was measured with a microplate reader at a wavelength of490nm. HUVECs were treated with anti-PCNA and DAPI to detect cell proliferation afterPAK1overexpression. The results were showed by the confocal microscopy and analyzedby SPSS statistical software. Apoptosis of HUVEC was demonstrated by flow cytometrywith Annexin V/7-AAD double-staining. Expression of cell-cell junctions were detectedby quantitative PCR and the distribution of vascular endothelium-specific adhesionmolecule CDH5was illustrated by confocal microscopy with immunofluorescence. Wedetected the activation of Notch pathway when the expression of PAK1changed in vivoand in vitro. Overexpression and knockdown of PAK1in cultured HUVEC was performedby transfection of pBSKI2-caPAK1and specific siRNA of human PAK1, respectively.Overexpression of pak1in zebrafish was performed by mRNA microinjection in thesingle-cell embryos，while knockdown of pak1was achieved by injection of morpholino.The expression level of Notch pathway molecules and its target genes were all detected byquantitative real-time PCR. To confirm that the bleeding phenotype caused by pak1overexpression was associated with Notch pathway, we used DAPT to inhibit Notchpathway by soaking the embryos. Phenotype changes were checked under an invertedmicroscope. The bleeding rate and spatial temporal distribution of hemorrhage in Notchsuppression embryos were compared with that induced by pak1overexpression. Thenincrease the pak1expression and Notch activation at the same time by co-injection of pak1mRNA and Notch intracellular domain, closely observing the changes of phenotype.Results: The coding sequences were successfully cloned and inserted into PCS2expression vector. PAK1mRNA was microinjected into wide-type zebrafish embryos. Themortality and malformation rate were quite similar between experimental and controlgroup. Intracerebral hemorrhage was observed in30-36hpf embryos injected with pak1mRNA. The incidence of bleeding was20-30%. The most severe phenotype and highestbleeding rate were observed in36-48hpf embryos. The hemorrhage was attenuated at about60hpf and most of the embryos recovered at72hpf. The survival embryos werecontinued to develop with no other abnormalities. There was no apparent hemorrhageobserved in control embryos. Tg (flk1: mcherry-ras) transgenic zebrafish embryos wereinjected with pak1mRNA. The morphology and distribution of vessels were observedusing confocal microscopy. The axial vessels and intersegmental vessels werewell-developed. However, abnormal vascular branching was noted in the bleeding sites inembryos overexpressing pak1. Transmission electron microscopic analysis showed integrallumen but damaged endothelial cell of the intracerebral vessel in the bleeding embryos.Expression level of intracellular PAK1detected by quantitative PCR was significantlyincreased48h after transfection. Cultured HUVEC presented morphological changes underoptical microscopy after overexpression of PAK1, with reduced number and smaller size.Some of the cells even rounded up and gradually detached from the cell culture substratum.MTT survival assay showed decreased cell viability in HUVEC overexpressing PAK1(P <0.01), while cell proliferation detected by anti-PCNA/DAPI double staining showed nostatistical significant difference in these cells compared with control (P>0.05). However,the flow cytometry data demonstrated a higher apoptosis rate in cells with PAK1overexpression (P <0.01). Expression level of endothelial cell-cell adhesion were changedsignificantly, with an obvious decrease both in CDH5and CDH2(P <0.05and P <0.01,respectively). However, the expression level of tight junctions such as ZO-1and CLDN5were not changed (P>0.05). Immunofluorescence showed CDH5depolymerization fromthe cell membrane after PAK1overexpression. Overexpression of PAK1in culturedHUVEC suppressed the expression of RBP-Jκ, a transcription factor of Notch, anddownstream target molecules HES1, HEY1and HEY2(all P <0.05). Opposite changeswere presented after PAK1was knockdown, with increased expression of RBP-Jκ, HEY1and HEY2(all P <0.05). However, FLK1, the important VEGF related molecule did notchange its expression level with PAK1overexpression or knockdown (P>0.05). In vivoresults were consistant with that from in vitro experiments. The expression level of efnb2which was related to arterial-venous specification and regulated by Notch pathway wasdecreased significantly when PAK1overexpressing in zebrafish embryos (P <0.05).Expression of Notch target genes hey1, hey2and her6were reduced as well (P <0.05).Opposite changes were presented after pak1was knockdown. Cerebral hemorrhage wasobserved in Notch suppressing embryos treated with DAPT, and the phenotype andbleeding rate were both similar with that occurred in pak1overexpressing embryos. However, activation of Notch pathway by NICD did not cause any abnormalitys.Activation of Notch pathway by NICD could rescue the phenotype induced byoverexpression of pak1, with a significant reduction in hemorrhage (20-30%vs.0-2%，P <0.01).Conclusion: PAK1could affect vascular integrity with its mainly influence on thefunctions of endothelial cells. PAK1was involved in the regulation of endothelial cellapoptosis as well as the formation and distribution of intercellular connections. PAK1played important roles in the regulation of embryonic vascular development through theNotch signaling pathway.