| Cell therapy, including endothelial progenitor cells (EPCs) transplantation, is appliedin the treatment of ischemic injury and cardiovascular diseases. Bone marrow-derivedmononuclear cells, blood-derived progenitor cells, mesenchymal stem cells andembryonic stem cells can be used as seed cells for the treatment of related diseases.Recently, more and more attentions are paied to embryonic stem cells (ESCs) whichcan be used in the research of key signals and regulators in embryogenesis. Currently,there are three main strategies used in the induction of endothelial cells (ECs) fromhuman embryonic stem cells (hESCs), including growth factors stimulation, hypoxiaand laminar shear stress (LSS) treatment. The addition of rhVEGF, rhbFGF and thetreatment of HIF1α lead to ECs commitment. But little is known about the effect ofLSS on ECs and the mechanism underline. The common understanding is LSS ismainly refers to the impact force generated by the blood flow in vessels in vivo. ECsare the basal lamina of vessels and could response to the stimulation of blood flowwhich results in modulation of vascular function, including contraction and relaxation.What’s more, LSS plays an important role in the differentiation and functionmodulation of ECs. Based on these knowledges, we would like to focus on thefunction of LSS on ECs differentiation and function.Human atonal homologue6(Hath6), an endothelial-selective gene expressed invascularized human tissues, was first identified as a novel shear-responsive basichelix-loop-helix (bHLH) transcription factor by transcriptional profile analysis ofHUVECs exposed to sustained laminar shear stress. The members of bHLH familyused to play important role in the regulation of differentiation and tissue formation inembryogenesis. On the other hand, LSS is a key stimuli in ECs differentiation andfunction modulation and Hath6is a LSS responsive transcriptional factor in ECs. All these suggest that Hath6may play a key role in human endothelial differentiation andfunction regulation.To address this hypothesis, gain-of-function and loss-of-function studies wereperformed using the human embryonic stem cell-endothelial cell (hESC-EC)induction model and endothelial cell lines. Since Hath6is a responser to LSS, theoverxepression of it might mimick the treatment of LSS.Firstly, to study Hath6‘s effects on ECs differentiation, we established the hESCs-ECsstepwise induction system. At the differentiation of stage2, we detected theexpression of endothelial markers including CD31, KDR and VE-cadherin up to16%、60%and10%respectively. The induced cells showed tubular structureformation capacity and the ability to cohere with fluorescein griffonia (bandeiraea)simplicifolia lectin Ⅱ. The cells were expanded with further induction ofdifferentiation and the resulting cells showed improved CD31expression up to30%with Dil-Ac-LDL incorporation. Taken together, these results suggested theestablishment of2D, serum-free and defined component induction system of ECsfrom hESCs, which is the basis of our research.Next, in consist with our hypothesis, the results of Q-PCR showed the endogenousexpression of Hath6was up-regulated during the formation of embryoid bodies (EBs)and the specific3staged-depended endothelial differentiation of hESCs. Then, weestablished the Hath6-hESCs and sh-Hath6-hESCs lines and induced them alongendothelial lineage to verify the role of Hath6in the process. Q-PCR indicated thatoverexpression of Hath6improved endothelial differentiation of hESCs as shown byan increased3times expression of endothelial-specific gene expression, improvedCD31+, KDR+, CD31+/KDR+populations detected by flow cytometry (1.5times ofcontrol, p<0.05)， a higher capacity for tubular structure formation and improvedDil-Ac-LDL uptake. All these indicated that the up-regulation of Hath6improved ECsdifferentiation. In contrast, Hath6gene knock-down with shRNA in hESCs decreasedendothelial differentiation. Taken together, these results suggested Hath6controlsendothelial lineage commitment.To further investigate the role of Hath6in endothelial phenotypic modulation, weperformed Hath6gain-and loss-of-function studies in ECs. The over-expression ofHath6facilitated the maturation of endothelial cells regarding endothelial geneexpression and cell migration. But Hath6did not improve the tubular structure formation and showed an inhibition on cell proliferation. The down-expression ofHath6showed reversed results including the down-regulation of endothelial geneexpression, decreased ability of tube formation (p<0.05), promoted cell proliferationand inhibited cell migration. All the results suggested that Hath6modulates ECsfunction.After confirming that Hath6could regulate ECs differentiation and function, webegan to consider by which means Hath6related to its biologic functions. Hath6is aLSS-responsive transcription factor which could regulate gene expression followed bythe ECs function modulation after the treatment of LSS. We suspected that its targetgene could also response to LSS and play roles in EC differentiation and functionmodulation. Hath6belongs to the bHLH transcriptional factor family whose membersare featured of the recognized and bound specifically with the sequence of E-box.Based on the above three features, we choose eNOS as our target gene. LSS couldupregulate the expression of eNOS followed with the change of NO production,which plays an important role in ECs differentiation and function modulation. Moreimportantly, bioinformatic analysis indicates the existence of some E-boxes in eNOSpromoter region. Therefore, in this part of study, we focused on eNOS to uncover themechanism of EC regulation exerted by Hath6. We first proved the interactionbetween Hath6and eNOS using LSS treatment in HUVECs and hESCs culturesystem. The upregulation of Hath6in HUVECs after treatment with LSS was up to6.2fold (p<0.001) and eNOS was increased to2.8fold (p<0.01). In hESCs, there wasmore than3.6times (p<0.05) expression of Hath6after treating with LSS at8h and6.1times (p<0.05) at20h, indicating a time depended manner of Hath6expressionupon LSS treatment. In addition, Western blot validated the correlative expression ofHath6and eNOS in ECs and in the hESC-EC differentiation process. Secondly, theaddition of eNOS inhibitor——L-NAME in the process of Hath6-hESCs-ECsdifferentiation showed decreased ECs induction efficiency initially induced by theoverexpression of Hath6. Finally, we detected the correlated down-expression ofeNOS (p<0.001) in the sh-Hath6-hESCs treated with LSS, which suggested thatHath6is the upstream signal of eNOS after LSS treatment. Taken together, theseresults suggested that Hath6, as a ECs transcriptional factor, activate eNOS signalingpathway to fulfill its role in the regulation of ECs differentiation and functionmodulation. In a word, the over-expression of Hath6prompts endothelial lineage commitment andendothelial differentiation is seriously impaired by Hath6knock-down. In addition,Hath6modulates endothelial proliferation, migration, and tube-like structureformation. Mechanism research indicates that as a transcriptional factor in ECs, Hath6regulated the expression of eNOS followed by the activation of signal pathways topromote ECs differentiation and function modulation. Currently, the mechanism ofLSS induced endothelial differentiation and function regulation is still unclear. Ourresearch on the role of Hath6might provide a new signal pathway for LSS. The studyalso provides new evidence for this gene family (the ATOH8family) on the regulationof embryonic development and may further enable the application of hESCs derivedendothelial cells in ischemia repair, the treatment of blood diseases and so on. Takentogether, all these might offer theoretical guidance and far-reaching impact onregenerative medicine and clinical application. |
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