The Role Of Nucleolin In Angiogenesis And Its Underlying Mechanisms

Abstract:Angiogenesis is a complex biological process and its underlying mechanisms are not well documented. Nucleolin is a multifunctional nucleolar phosphoprotein, which is highly conserved in eucaryon. It is an important RNA-binding protein, which has also been described as a shuttling molecule among nucleus, cytosol and cell surface. It has been reported that nucleolin localized on the cell surface is involved in the regulation of angiogenesis. However, it is unclear about the role of the RNA binding potential of intracellular nucleolin in the regulation of angiogenesis. This study was divided into two parts:(1) the effect of nucleolin on angiogenesis which was investigated in vivo and in vitro;(2) the relationship between the RNA binding ability of nucleolin and angiogenesis was further investigated via in vitro experiments.Main results of our study are as follows:1. Increased nucleolin expression and angiogenesis in ischemic myocardium in mice.Myocardial ischemia model was prepared by permanent occlusion of the left anterior descending coronary artery (LAD) and confirmed by visual observation (e.g. pale) and continuous ECG (electrocardiograph) monitoring. Heart tissues from ischemic area were collected at15days after occlusion. Then the samples were treated for isolation the total protein and RNA, or for preparation the tissue sections respectively depending on the demands of the followed experiments.(1) Assays of immunohistochemistry and immunofluorescence showed that the density of micro-vessel in the ischemic heart were higher than that in sham group. The expression of VE-cadherin was also increased in ischemic hearts.(2) RT-PCR and immunoblotting assays revealed that the expression of angiogenesis-related genes (such as MMP9, COX43, ENG, VEGF and VE-cadherin) was increased in ischemic heart which was accompanied by an increase of nucleolin expression. (3) In heart of transgenic mice with cardiac-targeted over expression of nucleolin, the density of blood vessel and the expression of angiogenesis-related genes are higher than in the wild type mice.These findings in vivo implied that nucleolin maybe contribute to angiogenesis.2. VEGF induced the expression of nucleolin which is involved in VEGF-mediated tube formation in HUVECs (human umbilical vein endothelial cells).To further understanding the role of nucleolin in angiogenesis, a cellular model of angiogenesis in vitro was developed by using VEGF-mediated tube formation of HUVECs. Gene-gain and-loss function assays were carried out by delivery of expression plasmids or antisense olignucleotides of nucleolin gene into cells. Gene expression was analyzed at protein and mRNA levels by using RT-PCR and immunoblotting. Tube formation on Matrigel and Wound-healing assays were used to assess the potential of angiogenesis in vitro.(1) RT-PCR and immunoblotting assays showed that the expression of nucleolin is increased in a dose and time-dependent manner in HUVEC after exposure to VEGF.(2) Knockdown of nucleolin inhibited VEGF-mediated tube formation and migration of HUVECs, vice versa, overexpression of nucleolin promoted VEGF-mediated tube formation and migration of HUVEC.(3) As indicated by RT-PCR, knockdown of nucleolin inhibited the expression of angiogenesis-associated genes (VE-cadherin, Tie-2, MMP9, Endoglin, COX43) induced by VEGF in HUVECs.These findings suggested that nucleolin promoted angiogenesis.3. Potential mechanisms by which nucleolin regulated angiogenesis.Due to its ability of RNA binding, nucleolin serves as an important regulator of mRNA stability contributing to posttranscriptional regulation of specific genes. So, we postulated that nucleolin regulates angiogenesis via its RNA binding domain-mediated regulation of gene expression at posttranscriptional level. We screened the complete mRNA sequence of some angiogenesis-related genes with binding motif of nucleolin " CS-RBD (consensus sequence type RNA binding domain)" by using bioinformatics. We predicted13genes which maybe the target mRNA with one or more binding sites of nucleolin within the3’-UTR (untranslation region) or5’-UTR or coding region. Further protein-RNA immunoprecipitation and RT-PCR assays showed that nucleolin interacted with the mRNA of angiogenesis-related genes MMP9, ENG and integrin αV.Although we did not provide more powerful evidences, our findings revealed still a clue that mRNA of MMP9, ENG and integrin aV are the possible targets of nucleolin, and their stability may be regulated by nucleolin at the posttranscriptional level, which endows the ability of nucleolin to promote angiogenesis. Considered the complexity of angiogenesis process, nucleolin-mediated posttranscriptional regulation of gene expression would provide a novel insight into the underlying mechanisms of angiogenesis regulation, and nucleolin may be a novel target for developing drugs or chemicals to control angiogenesis in ischemic heart.