The Function And Mechanism Of CREG Regulating Vascular Endothelial Cell Cycle And Its Effect On Mice Lower Limb Ischemia Model

Object：Endothelial injury is one of the initiating causes of atherosclerosis, myocardialinfarction, intrastent restenosis after percutaneous coronary intervention, peripheral arterialdisease (PAD) and shock. Promoting integrity of the endothelial cells (EC) and maintainingtheir normal function will provide a new way to the prevention and treatment for the abovediseases. The aim of this study is to investigate the function and underlying mechanism ofcellular repressor of E1A-stimulated genes (CREG), a newly found transcription regulatinggene, on cell cycle of human umbilical vein endothelial cell (HUVEC). We further explorethe role of CREG in regulating angiogenesis in a mice lower limb ischemia model.Method：(1) HUVEC were infected with adenovirus carrying either green fluorescence protein(GFP) or CREG to establish GFP control group and CREG over-expression group. Cellcounting analysis, BrdU incorporation assay, and flow cytometry (FCM) were performed toassess role of CREG in regulating HUVEC proliferation. Then Western Blot was used todetect expression of CREG, signaling molecules, cyclins and cyclin-dependent kinases(CDKs). Reverse transcription-polymerase chain reaction (RT-PCR) was applied to detectdifference in transcriptional expression of cyclins.(2) Stable HUVEC clones with CREGsilenced down or expressing scrambled negative control short hairpin RNA (shRNA)sequence were established by puromycin selection after retroviral infection. Cell countinganalysis, BrdU incorporation assay, and FCM were performed to evaluate the function ofCREG on HUVEC proliferation from perspective of loss of function.(3) According to theresults of Western blot, the signaling pathways affected by CREG significantly wereblocked with corresponding blockers. HUVEC proliferation was assessed by cell counting analysis, BrdU incorporation assay and FCM to determine the signaling pathway throughwhich CREG exerted its function. To identify the CREG related signaling pathways andCyclins, the expressions of Cyclins and signaling pathway protein were detected byWestern blot.(4) After blocking VEGF signaling pathway with anti-VEGF165neutralizingantibody in HUVEC with CREG over-expression or adding human recombinant VEGF165(rhVEGF165) in HUVEC with CREG knock down, cell proliferation was detected by cellcounting analysis, BrdU incorporation assay, FCM analysis, and the expressions of Cyclinsand signaling pathway protein was detected by Western blot at the same time to determinewhether VEGF is involved in regulation of CREG on HUVEC proliferation and Cyclins.(5)Using the normal HUVEC and GFP adenovirus infected HUVEC as control; the effect ofCREG over expression on angiogenesis was detected by the matrigel angiogenesis assay invitro and in vivo in mice. After establishment of mice lower limb ischemia model, withPBS and GFP-carrying adenovirus as control, the adenovirus carrying CREG was used toinfected local ischemia muscle tissue. To evaluate CREG function on the treatment of micelower limb ischemia and the effect on angiogenesis, the autologous amputation rate and theratio of trouble limb temperature that of normal limb were recorded, the pathologicalchange of skeletal muscle fiber was detected by H&E staining, and the blood perfusionratio of trouble limb to the contralateral was measured by blood flow perfusion imagingdetection.Results：(1) Compared with control groups, HUVEC with CREG over-expression showedhigher cell numbers by cell counting, increased BrdU positive cells percentage by BrdUincorporation experiment and increased percentage of S+G2phase by FCM analysis. Incontrast, HUVEC with CREG silenced down showed lower cell numbers by cell counting,decreased BrdU positive cells percentage by BrdU incorporation experiment and declinedpercentage of S+G2phase by FCM analysis.(2) Western blot analysis showed that CREGcould up-regulate expression of Cyclin E and activate the ERK, PI3K/Akt signalingpathway compared with control groups. RT-PCR found CREG could regulate theexpression of Cyclin E at transcription level.(3) After addition of signaling pathwayblockers, BrdU incorporation experiment and FCM analysis revealed that the promotioneffect of CREG on HUVEC proliferation was specifically mediated by ERK pathway, but not by PI3K/Akt pathway, although the two signaling pathway can both affect HUVECproliferation. As the same, it’s ERK but not PI3K/Akt signal pathway that mediates theregulation of CREG on CyclinE expression.(4) In vitro and in vivo matrigel angiogenesisexperiments found that CREG over-expression could significantly increase the capillarydensity. In the mice lower limb ischemia models, compared with the PBS and GFPadenovirus control groups, application of CREG adenovirus to infect ischemic muscletissue showed high temperature ratio of trouble limb to healthy limb, lower rate ofautologous amputation, significantly alleviated atrophy of muscle fibers and improvedperfusion ratio of trouble limb to healthy limb.Conclusion：(1) CREG can promote the proliferation of HUVEC cultured in vitro;(2) The functionof CREG regulation on HUVEC proliferation was mediated by ERK/Cyclin E pathway;(3)CREG over-expression can promote angiogenesis in mice lower limbs ischemic models.This study provides a new target of CREG for the treatment of PAD, and also a new prooffor CREG in the prevention and treatment of ischemic heart disease, intrastent restenosisafter percutaneous coronary intervention and late-onset thrombosis.