大鼠2/3肝切除72 h后再生肝脏质膜比较蛋白质组学研究

大鼠2/3肝切除模型为研究肝细胞增殖和生理性血管生成提供了一个很好的活体内模型.为了揭示肝再生过程中与肝细胞增殖终止相关及与血管生成启动相关的质膜蛋白质,本研究对大鼠肝2/3部分切除72 h后的肝脏质膜进行了研究：利用两步蔗糖密度梯度离心法对切除组和假手术组的肝脏质膜进行纯化;然后通过双向电泳和质谱技术对肝切除样品进行了比较分析并对几个关键蛋白程序性凋亡相关蛋白-6和丝蛋白-A进行了免疫印迹验证.相对于假手术对照组(Sham组),21种蛋白质在切除后72 h的肝脏中上调,15种蛋白质下调.所鉴定的差异表达蛋白参与了血管生成、细胞分裂增殖和凋亡、细胞分化调控、肝脏组织重新构建、代谢及应急反应.本研究为肝脏再生及其血管生成的研究提供了理论依据.     

大鼠肝再生过程中肝细胞质膜微区差异蛋白质鉴定与分析

动物肝是具有极强再生能力的器官,研究并阐明肝再生的机制可为肝移植等与肝损伤相关的疾病治疗提供理论依据.质膜包括“脂筏(lipid rafts)”和“质膜微囊(caveolae)”的微区,具有参与胞吞胞饮、信号转导、运输胆固醇等重要功能.肝再生过程中,肝质膜微区脂筏蛋白质受到内部调控的影响会发生改变. 捕获脂筏微区信号蛋白分布的变化,对于理解和阐明肝再生过程中信号通路途径有重要意义.本研究应用成熟的大鼠2/3肝切除模型结合蔗糖密度梯度离心法,提取假手术组与肝再生组大鼠肝细胞质膜,并进一步纯化获得质膜微区蛋白质.通过SDS-PAGE分离以及ESI-Q-TOF质谱鉴定,对获得的质膜微区蛋白质进行差异分析. 结果显示,有30个微区蛋白质差异表达,其中13个上调、17个下调.生物信息学分析表明,所鉴定到的蛋白质主要参与细胞增殖、程序性死亡、细胞凋亡等调控,同时涉及到与肝再生密切相关的血管生成等信号通路.本文为质膜微区蛋白质的研究提供了方法上的参考以及相关基础数据,为后续临床肝再生的研究奠定了一定的基础.     

Human peripheral blood eosinophils induce angiogenesis

Eosinophils play a crucial role in allergic reactions and asthma. They are also involved in responses against parasites, in autoimmune and neoplastic diseases, and in fibroses. There is increasing evidence that angiogenesis plays an important role in these processes. Since eosinophils are known to produce angiogenic mediators, we have hypothesized a direct contribution of these cells to angiogenesis. The effect of human peripheral blood eosinophil sonicates on rat aortic endothelial cell proliferation (in vitro), rat aorta sprouting (ex vivo) and angiogenesis in the chick embryo chorioallantoic membrane (in vivo) have been investigated. To determine whether eosinophil-derived vascular endothelial growth factor influences the eosinophil pro-angiogenic activity, eosinophil sonicates were incubated with anti-vascular endothelial growth factor antibodies and then added to the chorioallantoic membrane. Vascular endothelial growth factor mRNA expression and vascular endothelial growth factor receptor density on the endothelial cells were also evaluated. Eosinophils were found to enhance endothelial cell proliferation and to induce a strong angiogenic response both in the aorta rings and in the chorioallantoic membrane assays. Pre-incubation of eosinophil sonicates with anti-vascular endothelial growth factor antibodies partially reduced the angiogenic response of these cells in the chorioallantoic membrane. Eosinophils also increased vascular endothelial growth factor mRNA production on endothelial cells. Eosinophils are able to induce angiogenesis and this effect is partially mediated by their pre-formed vascular endothelial growth factor. This strongly suggests an important role of eosinophils in angiogenesis-associated diseases such as asthma.     

A high-throughput approach for subcellular proteome: identification of rat liver proteins using subcellular fractionation coupled with two-dimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis

Four fractions from rat liver (a crude mitochondria (CM) and cytosol (C) fraction obtained with differential centrifugation, a purified mitochondrial (PM) fraction obtained with nycodenz density gradient centrifugation, and a total liver (TL) fraction) were analyzed with two-dimensional liquid chromatography tandem mass spectrometry analysis. A total of 564 rat proteins were identified and were bioinformatically annotated according to their physicochemical characteristics and functions. While most extreme alkaline ribosomal proteins were identified in the TL fraction, the C fraction mainly included neutral enzymes and the PM fraction enriched alkaline proteins and proteins with electron transfer activity or oxygen binding activity. Such characteristics were more apparent in proteins identified only in the TL, C, or PM fraction. The Swiss-Prot annotation and the bioinformatic prediction results proved that the C and PM fractions had enriched cytoplasmic or mitochondrial proteins, respectively. Combination usage of subcellular fractionation with two-dimensional liquid chromatography tandem mass spectrometry was proved to be a high-throughput, sensitive, and effective analytical approach for subcellular proteomics research. Using such a strategy, we have constructed the largest proteome database to date for rat liver (564 rat proteins) and its cytosol (222 rat proteins) and mitochondrial fractions (227 rat proteins). Moreover, the 352 proteins with Swiss-Prot subcellular location annotation in the 564 identified proteins were used as an actual subcellular proteome dataset to evaluate the widely used bioinformatics tools such as PSORT, TargetP, TMHMM, and GRAVY.     

Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.     

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.     

SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.     

Systemic analysis of tyrosine phosphorylated proteins in angiopoietin-1 induced signaling pathway of endothelial cells

Angiogenesis is an essential process in physiological and pathological processes and is well-regulated to maintain the cellular homeostasis by balancing the endothelial cells in proliferation and apoptosis. Angiopoietin-1 (Ang1) regulates angiogenesis as a ligand of Tie 2 receptor tyrosine kinase. However, the regulation pathways are not well-understood. To date, only a few of the signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. In this study, we systematically identified tyrosine-phosphorylated proteins in Ang1-induced signaling cascade in human umbilical vein endothelial cells (HUVECs), employing proteomic analyses combining two-dimensional gel electrophoresis, Western analysis using phosphotyrosine antibody and mass spectrometry (MALDI-TOF MS and nanoLC-ESI-q-TOF tandem MS). We report here the identification, semiquantitative analysis, and kinetic changes of tyrosine-phosphorylated proteins in response to Ang1 in HUVECs and identified 66 proteins among 69 protein spots showing significant changes. Of these, p54nrb was validated as a molecule involved in cell migration. These results suggest that Ang1 induces stabilization of neo-vessel network by regulating the phosphorylations of metabolic and structural proteins.     

Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy

Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1alpha (hypoxia inducible factor-1alpha) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1alpha and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1alpha was assessed by quantitative RT-PCR and protein levels of HIF-1alpha and VEGF were assessed by western blot. HIF-1alpha DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48 h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1alpha and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1alpha and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1alpha activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1alpha and VEGF induced by partial hepatectomy alone.     

Leptin-leptin receptor are involved in angiogenesis in human hepatocellular carcinoma

Recent evidence in vitro and in vivo indicates that leptin, an adipose tissue-secreted hormone which is involved in the regulation of satiety, metabolic rate and thermogenesis, is implicated in angiogenesis. However, the role of leptin-mediated angiogenesis in hepatic carcinogenesis has not yet been completely elucidated. In this study, we have correlated microvascular density and leptin/leptin receptor (Ob-R) expression in endothelial and tumor cells with the histopathological type in human hepatocellular carcinoma (HCC). For this purpose, specimens of 40 primary HCC were submitted to immunohistochemical investigation using anti-CD31, anti-leptin and anti-Ob-R antibodies. Poorly-differentiated HCC had a higher degree of vascularization than other stages and leptin/Ob-R expression in both tumor and endothelial cells increased in parallel with the grade of malignancy and was highly correlated with the degree of angiogenesis. In the chick embryo chorioallantoic membrane in vivo assay, HCC biopsy specimens induced a strong angiogenic response, which was counteracted by an anti-leptin antibody. Taken together, these findings indicate that leptin/Ob-R correlate with angiogenesis and tumor progression in patients with HCC and that an anti-leptin antibody exerts an angiostatic activity in HCC.     

Arachidonic acid and calcium signals in human breast tumor-derived endothelial cells: a proteomic study

Intracellular calcium signals activated by growth factors in endothelial cells during angiogenesis regulate cytosolic and nuclear events involved in survival, proliferation and motility. Among the intracellular messengers released upon proangiogenic stimulation, arachidonic acid (AA) and its metabolites play a key role, and their effects are strictly related to calcium homeostasis. In human breast tumor-derived endothelial cells (B-TECs) AA stimulates proliferation and tubulogenesis in a calcium-dependent way. Here, to characterize the proteins whose expression is regulated by AA-induced calcium entry, we used a proteomic approach (two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, 2-DE and MALDI-MS) and we compared the proteomes of B-TECs stimulated with AA in presence or in absence of calcium entry (with addition to the culture medium of the calcium chelator EGTA, which completely prevents calcium fluxes throughout the plasma membrane). We found that six proteins increased their levels of expression, all higher when AA-induced calcium entry was abolished. These proteins have been identified by mass spectrometry and database search, and their potential roles in AA-stimulated pathway and in angiogenesis are discussed.     

Cellular localization and characterization of proteins that bind high density lipoprotein.

High density lipoprotein (HDL) stimulates excretion of excess intracellular cholesterol from cells, presumably by interacting with a cell-surface receptor. A 110 kDa membrane protein that is a candidate for the HDL receptor has been identified by ligand blot analysis. In this study we determined the cellular localization of this and other HDL-binding proteins and characterized their properties. The plasma membranes (PM) of cultured bovine aortic endothelial cells were labeled with trace amounts of [3H]cholesterol, and cell homogenates were fractionated on sucrose and Percoll gradients. Ligand blot analysis of homogenates of cultured bovine aortic endothelial cells demonstrated that cells contain multiple proteins that bind HDL3, including a major membrane protein with an apparent M(r) of 110 kDa and two minor ones with M(r) of 105 and 130 kDa. The gradient distribution of the 105, 110, and 130 kDa HDL-binding proteins mirrored that of labeled cholesterol and 5'-nucleotidase, both PM markers. Treatment of intact cells with the water-soluble cross-linker bis(sulfosuccinimidyl)suberate abolished the HDL binding activity of the 110 and 130 kDa proteins but not that of the 105 kDa protein. These findings suggest that the 105, 110, and 130 kDa HDL-binding proteins are localized to the PM and that at least two of these proteins are exposed to the extracellular fluid. Solubilized 110 and 130 kDa proteins were retained on wheat-germ agglutinin and abrin lectin columns, showing that they are glycoproteins. The cellular localization and physical properties of the 110 and 130 kDa proteins suggest that they may play a role in binding of HDL to the cell surface.     

Subproteomic analysis of the mitochondrial proteins in rats 24 h after partial hepatectomy

A 70% partial hepatectomy (70%PHx) induces cell proliferation until the original mass of the liver is restored. Mitochondria are involved directly in the process of liver regeneration (LR); however, their role in the early phase of LR is not clear. In an attempt to identify mitochondrial proteins that are correlated with the early phase of LR, we obtained a mitochondrial fraction via Nycodenz(R) density gradient centrifugation and subcellular proteomic analysis was performed. The mitochondrial proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Compared to the sham-operation control group, 3 proteins were up-regulated and 22 proteins were down-regulated at 24 h after 70%PHx. We identified 22 differentially expressed proteins that were associated with carbohydrate metabolism, lipid metabolism, the respiratory chain and oxidation-phosphorylation, biotransformation and other metabolic pathways. Prohibitin, a proliferation-regulating protein that was down-regulated at 24 h after PHx, was analyzed by applying RNAi (PHBi) with BRL-3A. This demonstrated a decreased mitochondrial membrane potential, implying a potential role in maintaining mitochondrial integrity. These results indicated that hepatic mitochondrial adaptations to LR after 70%PHx affect various cellular metabolic pathways, which advances our knowledge of the role of mitochondria in the early phase of LR.     

Absence of Direct Delivery for Single Transmembrane Apical Proteins or Their “Secretory” Forms in Polarized Hepatic Cells

The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.     

Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.     

Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis

Mesenchymal stem cells (MSCs) accelerate regeneration of ischemic or injured tissues by stimulation of angiogenesis through a paracrine mechanism. Tumor necrosis factor-α (TNF-α)-activated MSCs secrete pro-angiogenic cytokines, including IL-6 and IL-8. In the present study, using an ischemic hindlimb animal model, we explored the role of IL-6 and IL-8 in the paracrine stimulation of angiogenesis and tissue regeneration by TNF-α-activated MSCs. Intramuscular injection of conditioned medium derived from TNF-α-treated MSCs (TNF-α CM) into the ischemic hindlimb resulted in attenuated severe limb loss and stimulated blood perfusion and angiogenesis in the ischemic limb. Immunodepletion of IL-6 and IL-8 resulted in attenuated TNF-α CM-stimulated tissue repair, blood perfusion, and angiogenesis. In addition, TNF-α CM induced migration of human cord blood-derived endothelial progenitor cells (EPCs) through IL-6- and IL-8-dependent mechanisms in vitro. Intramuscular injection of TNF-α CM into the ischemic limb led to augmented homing of tail vein-injected EPCs into the ischemic limb in vivo and immunodepletion of IL-6 or IL-8 from TNF-α CM attenuated TNF-α CM-stimulated homing of EPCs. In addition, intramuscular injection of recombinant IL-6 and IL-8 proteins resulted in increased homing of intravenously transplanted EPCs into the ischemic limb and improved blood perfusion in vivo. These results suggest that TNF-α CM stimulates angiogenesis and tissue repair through an increase in homing of EPCs through paracrine mechanisms involving IL-6 and IL-8.