Proteomics Analysis Of Effect Of Endogeneous Digitalis-like Substance On Human Endothelial Cells

BackgroundDigitalis has been used in the treatment of chronic heart failure for more than 200 years since William Withering codified their use in his classic monograph on the efficacy of the leaves of the common foxglove plant. The search for an endogenous digitalis-like substance (EDLS) was aided in the last few decades when it became apparent that volume-expanded forms of hypertension may lead to the release of a natriuretic hormone. The Dahl-deWardener-Blaustein concept of a natriuretic hormone proposes that an enhanced production of endogenous inhibito(s) of the sodium pump occurs with the adaptive function of decreasing the volume of circulating fluid by means of inhibition of the Na⁺/K⁺-ATPase (NKA)in renal tubules in 1961. The increased production of endogenous digitalis-like compounds would also contribute to hypertension by means of inhibition of NKA in cardiovascular tissues. For some time it was not possible to establish that an endogenous digitalis actually exists. Hamlyn et al.were the first to demonstrate that the concentration of a circulating factor in blood plasma inhibiting purified NKA correlated with the blood pressure of the donors. This observation paved the way for the identification of endogenous digitalis as a group of cardenolides and bufadienolides whose physiological and pathophysiological function is only starting to be understood. Hence, it was demonstrated beyond doubt that the inhibitor from bovine hypothalamus is ouabain.Ouabain plays an imported role in essential hypertension,heart failure and Q-T long syndrome. The mechanisms of action of ouabain have been under extensive investigation for nearly 50 years, yielding one of the most specific mechanisms thus far defined for any agent so extensively used. Its ability to bind to and inhibit the NKA has been well established, as has the resulting increase in cellular [Ca²⁺] responsible for its positive inotropic action and its toxicity as well.However, recent observations suggest that additional actions of ouabian may modulate a number of other cell-signaling processes, with a potential for both short- and long-term changes in the way that have no affect on activity of NKA. reports have published that ouabain exerts dual effects (proliferation and death) on cell growth at different concentration in different cell lines, Some researchers proposed the mechanism for ouabain's proliferative inductive effect is its interaction with NKA; this interaction occurred at concentrations that did not induce enzyme activity inhibition and that transformed it into a signal transducer system. However, experimental evidence has been put forward showing that the effect of ouabain on cell survival and proliferation may be either independent of or dependent on the inhibition of cation transport. All of these data indicate that there maybe exist other target sites of digitalis in different cell lines besides NKA, and these target proteins play important roles in the regulation of cell growth and transformation with or without interaction with NKA.Vascular endothelial cells (VEC) , which are in direct contact with plasma and cellular components of blood, are targets of many endogenous molecules and xenobiotics. In particular, a number of anticancer drugs are toxic toward the vascular endothelium leading to clinical impairments such as pulmonary fibrosis or cardiac disorders. Vascular endothelialcells, whose functional integrity is crucial for the maintenance of blood flow and the antithrombotic activity, could be a target for endogenous ouabain.Genomics' aims are to characterise all the genes of an organism, including their sequences, polymorphism, structures, regulation, interaction and products. Correspondingly, proteomics is the biologists'search for an analogy of the encyclopedia of reactions known to chemistry, the characterisation and identification of all proteins expressed within any cell at any time. It is a field in which the mere scale is enlarging at an astounding rate. A few decades ago, it was thought that one gene was responsible for one polypeptide. It is now known that single genes may be responsible for a number of proteins, chaperonins may fold and activate proteins, time and place may affect protein synthesis and functional groups may be changed after synthesis, for example by phosphorylation and glycosylation .Correspondingly, the number of proteins estimated to exist in the human body has risen from the order of 100 000, when one gene was thought to produce one protein, to present estimates of between 1 and up to 100 million. Together, genomics and proteomics promise to revolutionise biological understanding, providing us with a molecular description of a biological entity and the effect of environment, disease and chemicals (drugs) upon it.With the discovery of most human genes in 2000, it is now apparent that a 'factory approach' to address biological problems is desirable if we are to gain a comprehensive understanding of complex biological processes. Proteomics is the large-scale study of proteins, usually by biochemical methods. The word proteomics has been associated traditionally with displaying a large number of proteins from a given cell line or organism on two-dimensional polyacrylamide gels. The central tool for displaying the proteome is twodimensional gel electrophoresis, Analysis of gel images with specialised software 10 allows comparisons of multiple gels both within a laboratory and,via links, to comprehensive proteome databases on the internet. Thus, by a process of subtraction, differences (eg, presence, absence, or intensity of proteins or different forms) between healthy and diseased samples can be revealed. Proteins of interest can then be identified on the basis of knowledge of the isoelectric point and apparent molecular size determined from the two-dimensional gels, supplemented by a combination of methods, generally applied hierarchically. Increasingly these include highly sensitive mass-spectrometric methods, which require smaller amounts of material and have a higher throughput than conventional sequencing methods.With the accumulation of vast amounts of DNA sequences in databases, researchers are realizing that merely having complete sequences of genomes is not sufficient to elucidate biological function. A cell is normally dependent upon a multitude of metabolic and regulatory pathways for its survival. There is no strict linear relationship between genes and the protein complement or 'proteome' of a cell.Proteomics is complementary to genomics because it focuses on the gene products, which are the active agents in cells. For this reason, proteomics directly contributes to drug development as almost all drugs are directed against proteins. Many studies of cardiovascular disease and cancer have used two-dimensional electrophoretic and mass spectrometric techniques and, this is likely to remain a principal approach in the future. Nonetheless, proteomics has already revolutionized the discovery process for antimicrobial drugs by accelerating target identification and evaluation, assay development, mechanism of action studies, and follow-up support for medicinal chemistry programs. An important starting point for drug discovery is to supply a validated therapeutic target, and the proteomics study of ouabain on VEC. may yet provide insights into the mechanism of cardiovascular disease, and provide new starting points for therapeutic intervention.This article is combibed by four parts: the first part is to estimate the ouabain-triggered HUVEC model and observe the effect of ouabain on cell growth; the second part is to find the different expression proteins induced by ouabain treatment by 2-DE approaches; the third part is to identify the differently expressed proteins regulated by ouabain and the last part, is to confirmed the results of compare proteomics.The pathway is showed as follows:Objective:The aim of this work was to characterize the effect of ouabain on cell growth in endothelial cells, and, to do the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to ouabain and examine changes in protein expression, further to supply a validated therapeutic target and provide newavenues for the treatment of cardiovascular diseases.Methods:1 Effects of EDLS on VEC cell growthHuman umbilical vein endothelial cells (HUVEC) were cultured to the 2-4 passage and divided into 4 groups accorging to the time point at 12h,24h and 48h, each group was divided into control group,ouabain-treated group,digoxin-treated group and etacrynic acid -treated group. Cell viability was measured by microscopic examination of trypan blue dye exclusion. Cell survival was estimated with MTT colorimetric assay. To evaluate changes in morphology, cells under the effect of ouabain were visualized by transmission electron microscope (TEM), Apoptosis was confirmed byflow cytometry (FCM) and the activities of Caspase3 were determined by chromometry.2 The establish of 2-DE profiles of HUVEC and ouabain-treated cells Total protein of cultured HUVEC and ouabain-treated HUVEC were extracted.The proteins were separated differently using immobilized pH gradients 2-DE and visualized by silver staining. The digitized images which got with scaner were then analyzed with PDQuest software in order to establish the diferential expression profiles.3 Mass spectrometry identify the differently expressed proteinsThe diferential protein spots were cut from the gels using proteomework spot cutter and subjected to in-gel digestion with trypsin. The digested peptides' separation was conducted by a Finnigan LCQ MS coupled with a Surveyor HPLC system.LC M S/ MS was performed (Thermo, San Jose, CA, USA)using an LTQ linear mass spectrometer '[he system was fitted with a C18 RP column. Mobile phaseA (0.1% formic acid in water) and the mobile phaseB (0.1% formic acid in ACN) were selected. The LTQ linear mass spectrometer was set so that one full MS scanwas followed by ten MS/MSscans on the ten most intense ions from the MS spectrum with the exclusion duration. A singly charged peptide must tryptic, and the crosscorre.fati.on score (Xcorr) had to be at least be 1.9. Pryptic or partially tryptic peptides with a charge state of +2 have pro Xcorr of at least 2.2. Triply charged tryptic or partially tryptic peptides with a +3 charge state was accepted if the Xcorr was≥3.75. Generally, an Xcorr greater than 2.0 indicates a highly significant match, and a DeltaCn higher than 0.1 indicates a significant distinction between the best match and the second-best match.All identified proteinswere classified by their molecular function, cellular component, and biological process with the tools on www. geneontology. org.4 Prelimary study on the role of differently expressed proteins in ouabain's proliferative effectThree identified proteins (myosin regulation light chain2,profilin-land heat shock protein 60) were selected for further research. The expression of the three proteins in ouabain-treated HUVEC were observed using western blot analysis. The association between the profilin-1 expression and ouabain actions such as cell growth,mobility were analyzed by approaches as showned in part 1.Results:1 EDLS plays dual effects on HUVEC cell growth.MTT results showed that the ouabain increased HUVEC cell number when exposed to concentrations≤20nmol/l less than 48h, and induced cell apoptosis at higher concentrations. Using the trypan blue exclusion assay, an increase in cell number up to 40% of HUVEC treated with 10nM ouabain for 24h was observed when compared with the control group.Digoxin showed dual effects on HUVEC cell growth, it stimulated cell proliferation when exposed to concentrations≤=50nmol/l and inhibited cell growth at high concentrations. The inhibiting effect of digitalis compounds on cell growth is concentration and time dependent.FCM and TEM confirmed that ouabian's actions are proliferation,apoptosis and necrosis.The nonsteroid inhibitor of Na⁺/K⁺-ATPase, ethacrynic acid, which binds to the same enzymatic conformation on which digitalis compounds act: E₂-P, unable to induce proliferative growth on HUVEC; consequently, the EDLS mechanism for cell growth induction may be different to the simple interaction with NKA.The results obtained in this study suggest that EDLS stimulats cll growth without inhibiting NKA.2 2D gel images from ouabain-treated HUVEC and control cellsIn our experiments, we got the 2-DE of low-dose ouabain treated HUVEC and control HUVEC; at least three 2-D gels were run per sample. Visualized spots in control group were 2792±124 while ouabain-treated group were 2806±153. The match ratio ananlysis by PDQuest soft got that in control group was 86.5% and in ouabain-treated group was 87.5%, which means a good replication.Image analysis of silver-stained 2D gels by PDQuest software revealed that 39 protein spots showed diferential expression ( >2 folds,p<0.05)in ouabain-treated HUVEC compared to control cells, of which 8 spots showed more than 5 folds expression changes (p <0.01). Among these 32 protein spots, the upregulated preoteins were 18, while down regulated were 14;and that 5 vs 3 in the eight significantly changed protein spots.3 Mass spectrometry identify the differently expressed proteins Compared the protein profiles of cells exposed to ouabain, 32 diferentiallyexpressed protein spots were selected and identified with electrospray ionization tandem MS (ESI-MS-MS).Being analyzed by ESI-MS-MS and database searching.4 Differently expressed proteins can be canbidated target in cardiovascular disease interventionThe expression of MRLC2,profilin-land heat shock protein 60 in low-dose ouabain treated HUVEC meet the result of 2-DE (p<0.01) . Further study on the profilin-1 support that the ifferently expressed proteins gained by proteomics approaches can be canbidated target in cardiovascular disease intervention.Conclusions:1 EDLS plays dual effects on HUVEC cell growth in a time and concentration dependent way, it stimulats cll growth without inhibiting NKA.2 The identified proteins involved in various aspect of endothelial cellular function, including metabolism, cell motility and signal transduction as well. This indictes that ouabain plays its role in cardiovascular disease not only through its inhibiting NKA, but also have another potential target sites.3 Profilin-1 plays an important role in ouabain-triggered proliferation in HUVEC.4 This study provides an important knowledge of ouabain-treated HUVEC protein expression in vitro and should be helpful for the further elucidatation of the molecular mechanisms involved in EDLS-involved cardiovascular disease.