A Histone Deacetylase 7-derived 7 Amino Acid Peptide Acts As A Phosphorylation Carrier And Promotes Angiogenesis

Background and aims:Intimal injury is a common pathological basis in the process of cardiovascular diseases(CVD),and the recovery of the injured endothelium is essential for the protection of CVD.Dysfunction of endothelial cells(ECs)is thought to play a pivotal role in the injured endothelium,and EC health was suggested to be maintained by the consistent supply of supporting grow factors and activation of local stem/progenitor cells which have the ability to differentiate towards ECs.It is well documented that vascular progenitor cells(VPCs),which are mainly located in the adventitial,also contribute to the repair of the injured endothelium via migration toward endothelium and differentiation into EC lineage.It is well established that in eukaryotic species a single gene can produce multiple messenger RNA(mRNA)molecules through the alternative splicing process,giving rise to different protein variants with different even opposite functions.Recent studies have shown that the translation of a peptide/protein can be initiated from different ATG or CTG(take up to 15%)codon within a single mRNA molecule.It means a single mRNA molecule may produce different peptides or proteins,which makes the genetic information stored in the gene sequence even more vast.Histone deacetylases(HDACs)are a family of enzymes that remove acetyl groups from N-acetylated lysine residues on histones,involved in gene transcriptional regulation through modulating chromatin structures.HDAC7,a member of the class II HDACs,is specifically expressed in the vascular endothelium and contributes to the maintenance of vascular integrity during early embryogenesis.Due to alternative splicing,there are 4 and 8 transcript variants in human and mouse HDAC7 mRNA respectively.In this study,we demonstrated that a short open reading frame(sORF)within one of the mouse HDAC7 transcript variant could be translated,giving rise to a 7 amino acid(aa)-peptide.This peptide could modulate vascular progenitor cell behaviour through functioning as a phosphorylation carrier.Methods1.The effect of VEGF on HDAC7 mRNA alternative translationAs a peptide with only seven amino acids is not easy to be detected by routine methods,we created an artificial construct containing the whole 5'UTR and 225aa of the main ORF,designated as pShuttle2-HD7FH.In this plasmid,a Flag tag was inserted upstream of the stop codon in the sORF and an HA tag was fused to the 225aa of the main ORF.The expression of FLAG and HA can reflect the translation of the short and main ORFs respectively.Double immunofluorescence staining detect FLAG and HA.ELISA with anti-FLAG and anti-HA antibodies detect FLAG and HA ratio.2.VEGF and 7A 7aa-peptide phosphorylation2.1 To whether the serine residue of 7A could be phosphorylated,we firstly performed IP assay with anti-FLAG beads,followed by ELISA with anti-phospho-serine antibody.Next,we performed a special biotin-labelled peptide pulldown assay,to verify whether phosphorylation signal derived from the FLAG-tagged 7A or its associated proteins.2.2 To identify the potential upstream kinase for 7A phosphorylation,we performed a pilot proteomics analysis of the peptides-associated proteins,especially on their phosphorylation change in response to VEGF treatment.Then we verify the results by Western Blot assay and detect phospho-MEKK1S393 in the presence of 7S,7Aa,7A and PBS.Finally,we knockdown MEKK1 by SiRNA and detect 7A phosphorylation.3.The role of phosphorylated 7A on 14-3-3? protein To test whether 7A was directly involved in 14-3-3? phosphorylation,the phosphorylated bio-7A peptide was purified by pulldown assay and incubated with commercially available recombinant 14-3-3? protein,followed by ELISA detection of the phosphorylated 7A with anti-phospho-Ser antibody and Western blot analysis of phosphorylated 14-3-3? with anti-phospho-Ser and anti-phospho-Thr antibodies respectively.Then we incubate 7S,7Sp,7A,7Ap and PBS with recombinant 14-3-3?in cell-free buffer system and detect phosphor-14-3-3?.Next,we explore the effect of 14-3-3? in VPCs by immunofluorescence staining.4.The effect of 7aa-peptide on VPCs migration We performed single cell colony formation assay in 96-well plates with normal growth medium to explore 7A role on cell proliferation.Then transwell migration assays and wound healing model to observe 7A effect on VPCs migration with or without VEGF.5.The role of 7aa-peptide on VPCs differentiation To test whether 7A be involved in cell differentiation process,VPCs were cultured in differentiation medium in the presence of 7A and/or VEGF,followed by quantitative RT-PCR analysis of EC and SMC marker expression.We also performed tube formation assay to test differentiated cell function.6.The role of 7aa-peptide on VEGF-induced VPCs migration and differentiation.To test whether 7A bridged between MEKK1 and 14-3-3?.We knockdown of MEKK1 by siRNA,and detect VEGF-induced 14-3-3?Thr145 phosphorylation level.Then to explore whether MEKK1 and 14-3-3y involved in 7A induced VPCs migration and differentiation,we knockdown of MEKK1 or 14-3-3? respectively in VPCs,and performed transwell assay and differentiation assay.7.The effect of 7aa-peptide in vivo.7.1 Matrigel plug assays were performed in C57BL/6J mice via subcutaneous implantation of peptide-containing Matrigell5,followed by observation of CD31 positive cells in plug section harvested at day 7 and 14 post-implantation7.2 A wire-guided femoral artery injury model was then introduced in ApoE-/-micel6.Pluronic-127 gel containing 10ng/ml of peptides or PBS was applied surrounding the adventitia of the injured vessels,which were harvested 4 weeks post-surgery.The HE staining of cryo-section analysis neointima formation.7.3 A hindlimb ischemia model was introduced in C57BL/6J mice,in which Pluronic-127 gel containing 10ng/ml peptides was applied surrounding the injured vessels17.Foot blood perfusion was measured by a Doppler Scanner at day 7 and day 14 post-surgery.Immunofluorescence staining on the skeletal muscle sections with anti-CD31 and anti-Scal antibodies.Results1.VEGF induced HDAC7 mRNA underwent alternative translation.Double immunofluorescence staining detected FLAG and HA within a single cell in pShuttle2-HD7FH-transfected VPCs,indicating that the sORF indeed can be translated.As plasmid transfection efficiency was very low,we created Ad-HD7FH viral particles.ELISA with anti-FLAG and anti-HA antibodies confirmed that both FLAG and HA were expressed in Ad-HD7FH infected VPCs as compared to that in the control virus-infected cells.In differentiated VPCs,the HA expression was significantly increased due to an increased virus infection efficiency.However,the ratio of FLAG to HA was decreased in differentiated VPCs as compared to that in VPCs,which was restored by VEGF treatment.2.VEGF induced 7aa-peptide phosphorylation via MEKK1Serine phosphorylation was detected in Ad-HD7FH-infected VPCs as compared to that in the control virus infected cells,which was significantly increased by VEGF treatment.Peptide pulldown assay results demonstrated that the serine residue in 7A was phosphorylated and the phosphorylation could be enhanced by VEGF treatment,and that this phosphorylation was sequence specific as the serine residue in the scrabble 7aa-peptide(7S,MPHASGD)was not phosphorylated.We noticed that the phosphorylation of the peptide RSSRIK from MEKK1 was elevated in Bio-7Aa but reduced in Bio-7A-associated samples,which was confirmed by Western blot analysis.The presence of 7A increased the de-phosphorylation of the phosphorylated MEKK1,while the addition of 7Aa could retain MEKK1 phosphorylation.These observations imply that MEKK1 may function as an upstream kinase for 7A phosphorylation.Indeed,knockdown of MEKK1 by siRNA significantly attenuated 7A phosphorylation.These results suggest that VEGF induces the sORF translation and the derived peptide phosphorylation via MEKK1.3.The phosphorylated 7A directly phosphorylated 14-3-3? proteinFrom the pilot proteomics study,we noticed that the phosphorylation of a peptide(RATVVESSEK)from 14-3-3? protein was increased in Bio-7Ap(the serine residue was synthetically phosphorylated)and VEGF-treated Bio-7A associated samples.The physical binding of the bio-7A and Bio-7Ap to 14-3-3? protein was confirmed by Western blot analysis.The phosphorylation signal in bio-7A was increased by VEGF but significantly reduced by further incubation with recombinant 14-3-3?.No phosphor-Ser bands were detected,but a phosphor-Thr positive band was detected corresponding to 14-3-3? protein by Western blot.Direct incubation of 7Ap with recombinant 14-3-3? protein in buffer system could increase threonine phosphorylation in 14-3-3? protein.Immunofluorescence staining revealed that the phosphorylated 14-3-3? was mainly located in nucleus and upregulated by VEGF treatment,which was significantly enhanced by the presence of 7A.As expected,the exogenous 7Ap could increase 14-3-3? phosphorylation in the absence of VEGF treatment4.The 7aa-peptide enhanced VPCs self-renewal and VEGF-induced VPCs migrationIn a limited dilution cologenic assay performed in 96-well plates with normal growth medium,the presence of 7A especially 7Ap significantly increased the colony numbers and cell numbers within a colony.Besides,in clonogenic assay performed in 6-well plates,7A especially 7Ap dramatically increased the cells numbers per colony and the average area occupied by per cell.These results suggest that 7A especially 7Ap increase VPCs self-renewal and may also increase VPCs migration.Indeed,the transwell migration assays and wound healing model showed that 7A increased and significantly enhanced VEGF-induced VPCs migration and that 7Ap alone dramatically stimulated VPCs migration,which was comparable to that observed in VEGF plus 7A5.The 7aa-peptide enhanced VEGF-induced VPCs differentiation toward EC lineage in vitro7A increased CD31 and CD 144 mRNA levels,which was significantly enhanced by VEGF although VEGF alone only had slight effect.The effect of 7Ap alone on CD31 and CD 144 expression was comparable to the combined effect of 7A and VEGF.7A alone had no effect on SM22 expression but significantly decreased SM22 expression in the presence of VEGF,although VEGF had slight increasing effect.7Ap alone significantly decreased SM22 expression.These results suggest that 7A especially 7Ap favours VPCs differentiation toward EC lineage while suppresses SMC differentiation.The EC differentiation was further confirmed by the tube formation assays.6.The MEKKl-7Ap-14-3-3y signal pathway mediated VEGF-induced VPCs migration and differentiation.Knockdown of MEKK1 by siRNA decreased the basal and abolished VEGF-induced 14-3-3?Thr145 phosphorylation,indicating that MEKK1 is an upstream kinase for 14-3-3? phosphorylation.In MEKK1 knockdown VPCs,the addition of 7Ap alone could induce 14-3-3?Thr145 phosphorylation.In the transwell migration assays,knockdown of MEKK1 or 14-3-3? decreased the basal level and abolished VEGF-induced VPCs migration,suggesting that both MEKK1 and 14-3-3y are essential for VPCs migration.In MEKK1 or 14-3-3y knockdown cells,7A had no stimulatory effect any more,suggesting that MEKK1 and 14-3-3? are also essential for 7A-mediated VPCs migration.However,7Ap alone could induce VPCs migration in MEKK1 but not in 14-3-3y knockdown cells,indicating that 7Ap is downstream of MEKK1 but upstream of 14-3-3y.7.The 7A increased vascular injury repair and angiogenesis in ischemic tissues in vivo.At day 7 post-implantation,few CD31 positive cells could be observed in PBS group,and only a few CD31 positive cells but no capillary structures were present in 7S group.In contrast,several capillary structures could be observed in 7A group.At day 14 post-implantation,only a few CD31 positive cells and capillary structures could be observed in both PBS and 7S groups,but multiple capillary structures were observed in 7A group.For wire-guided femoral artery injury model,the HE staining of cryo-sections revealed that 7A or 7Ap administration significantly reduced the neointima formation.For hindlimb ischemia model,7Ap can promote the foot blood perfusion recovery.Immunofluorescence staining on the skeletal muscle sections with anti-CD31 and anti-Scal antibodies revealed that there were more double positive cells in 7Ap group.Importantly,there were many Scal+ cell clusters in 7Ap group.These results suggest that 7A especially 7Ap stimulate VPCs migration,proliferation and differentiation toward EC lineage in vivo,contributing to vascular injury repair and angiogenesis in ischemic tissues.Conclusion:1.VEGF induces HDAC7 mRNA to undergo translation shift from the main ORF to the short one in VPCs,giving rise to a 7aa-peptide(7A).2.VEGF activates MEKK1 via phosphorylation at the Ser393 residue.The activated MEKK1 transfers the phosphate group from Ser393 to the serine residue within the 7aa-peptide.The phosphorylated 7aa-peptide(7Ap)can transfers the phosphate group to the Thr145 residue within 14-3-3y protein,leading to cadherin-plakoglobin-catenin-14-3-3y complex disruption and 14-3-3y nuclear translocation.3.7aa-peptide promote angiogenesis via MEKK-7A-14-3-3?.4.7aa-peptide may play an important role in cellular processes like proliferation,migration and differentiation via acting as a phosphorylation carrier.