Targeting Research Of Peptide NTSTH Specially Binding To Vascular Endothelial Cells Stimulated With Endotoxin

1. Objective and significanceSepsis is a series of clinical sydroms concerning systemic inflammatory response, prothrombotic diathesis and fibrinolytic disorders caused by invasion of microorganisms or other pathogens into human body, primarily representing as disfunction of immunologic and clotting system. It is considered as one of most serious complications of clinical acute and severe patients with trauma, burning, shock or inflammation, and an improtant cause to induce septic shock and multiple organ dysfunction syndrome (MODS). As a common factor causing acute and severe diseases and badly enfluencing the prognosis of the patients, sepsis is always highlighted in the basic and clinical researches of critical diseases. Sepsis is considered as a disease with high incidence rate, high death rate and high treatment cost. In America the incidence rate is increased by about 8.7％annually, while in the 750,000 new onsets of sepsis, there are 200,000 cases died of sepsis every year. Currently the number of sepsis cases is presented in a climbing tendency with the increase of the number of immunosuppressed patient, the use of invasive treatment method and test method, the increase of patients with microorganism resistance, the increase of the old population as well as the increase of the acknolodgement and diagnosis of sepsis. Therefore, it’s a great challenge confronted with the basic researches that how to decrease the incidence rate and death rate in this field.Generally, the pathomechanism of sepsis is closely associated with infection of gram-negative bacteria and endotoxin (ET) it produces. The main component of ET is lipopolysaccharide (LPS). It is considered as the main factor causing the generation of cytokine (CK) under the state of bacteria infection, trumma, burning and shock. Currently it is accepted that ET exert its effect mainly via the compound of soluble LPS and LPS binding protein. Then the compound is carried on the surface of myeloid cells and binds with CD 14 receptor, thus the cell response is activated to generate proinflammatory cytokine (PIC) and finally resulting in sepsis. At the same time some anti-inflammatory cytokines (AICs) is released to regulate inflammatory response. Provided such a regulation of early pro-inflammatory response failed, SIR will occur immediately. Sepsis is also usually accompanied with disorders of coagulation cascade and fibrinolysis system, with D-dimer of fibrinogen degration product (FDP) up-regulated and activated protein C (APC) decreased. As we all know that APC functions as maintaining homeostasis, but under the state of SIRS, the function of APC is inhibited therefore causing imbance of internal environment due to the effect of coagllen regulation factor and C receptor of endothelial cell, consumption of APC and injury of endothelism. Finally the fibrinolytic system is inhibited during the developping of sepsis and therefore cause prothrombotic diathesis and fibrinolytic disorders.Endothelism plays an important role in the development of sepsis. The following pathological changes may be caused by various of pro-inflammatory factors, activated complement system, oxygen radicals and vaso-excitor materials like AngⅡas well as the changes of the drive force of blood stream and blood vessel stress:(1) The function of coating and screening of blood vessel will be decreased while vascular permeability is elevated, causing the occurrence of edema; at the same time the blood is re-distributed both in and out of blood vessels, causing hypoperfusion of some vital organs and the occurrence of MODS. (2) During the progression of sepsis a large amount of pro-inflammatory factors is generated by vascular endothelial cells via various signal pathways like MAPK pathway and NFκB pathway, to form complicated cytokine network and finally the inflammation is therefore amplified. (3) Site-specific changes may also occur in the injuried vascular endothelial cells, causing the imbanlance of endothelium-derived vasoconstriction factors/endothelium-derived relaxing factors, aggravating sepsis and resulting in the occurrence of MODS. (4) Structures under the endothelial layer is exposed and collagenase is released, and in this way intrinsic coagulation pathway is started. Further more, tissue factors generated by vascular endothelial cells will induce extrinsic coagulation thus accelerating the generation of thrombase.In summary, the injury of endothelium is considered as the centre pathological process in the onset and progress of sepsis, while the difference molecules generated by inflamed vascular endothelial cells in comparison with normal cells interact with various of inflammatory factors, causing activation of various of signal pathways and amplification of inflammatory responses, is considered as direct pathway causing the occurence and progress of sepsis. It can be supposed that if there exist some small bioactive peptides with high affinity to difference molecules generated by endothelium under inflammatory conditons, there is possibility of these peptides to bind to some inflammatory mediators at its function site and competitively inhibit the binding of these inflammatory mediators to differece molecules on the surface of endothelium. In this way, the biological effect of these inflammatory mediators, such as releasing pro-inflammatory factors, mediating cell adhesion, etc., may be blocked or neutralized and then systemic inflammatory reactions may be inhibited or relieved. At the same time, such a peptide with targeted binding can also be used as leader peptide of some drugs, strengthing the targeted binding to endothelium and therefore treating, repairing and protecting blood vessels more efficiently.Characterized motif NTSTH is obtained via screening a phage 7-Mer cyclic peptide library by LPS-induced human umbilical vein endothelial cells (HUVEC), then sequencing 7-Mer cyclic peptide with targeted binding and bioinformatics analysis. The characteristics of motif NTSTH are as follows:(1) being from biosynthesized radom phage 7-Mer cyclic peptide library. Characterized motif NTSTH may actually exist in nature, while it also may do not exist in nature at all, but there is possibility to find its mimic functional protein in human protein library, which is associated with sepsis in function, anyway. And characterized motif NTSTH is considered as the mimotope of the target function protein which it mimics. (2) being possible of having the targeted binding to endothelium induced by LPS. Characterized motif NTSTH is considered as the shared epitope of the function proteins which are speically targeted to HUVEC induced by LPS, therefore, characterized motif may have relative strong targeted binding to HUVEC induced by LPS. (3) being homologous in more than one species. (4) being functionally related to endothelium under inflammatory conditions.According to the characteristics mentioned above, in this study we explored the potential biological information of characterized motif NTSTH firstly, try to find related functional targeted protein in human protein database. After searching with online tool and review of relavent researches, we find a homologous motif, NTTTH in protein Scube 1, which may be functionally associated with sepsis. The similarity between the two motifs is high up to 80%, suggesting the high homology. It should be noted that the protein Scube 1 maybe closely associated with the pathological process of sepsis. As the mimic epitope of targeted functional protein Scube 1, characterized motif NTSTH is studied concerning its targeted binding to endothelium induced by LPS both in vivo and in vitro.2. Methods and procedures2.1 Bio-information exploring of characterized motif NTSTHBasic local alignment search tool (BLAST) is applied to search the mimic protein of characterized motif NTSTH online, with the purpose to find the target function protein with similar function to NTSTH. And then, all the homologous proteins are screened on the base of large amount of review of related researches. The target function protein should have the following characters:being membrane protein, transmembrane protein or secreted protein; being mutiple homologous; being related to inflammatory responses. Bio-information exploring study is purposed to lay theory basis of further research on the targeted binding of characterized motif NTSTH.2.2 Construction of expression plasmid of NTSTH and the expression of fusion proteinOnline Blast searching results indicated that the target functional protein Scube 1 is mimicked by motif NTSTH. The protein Scube 1 is a secreted protein and closely associated with inflammation and blood coagulation. So it is very possible that NTSTH may be a function site where Scube 1 interact with endothelium induced by LPS, suggesting the possibility of targeted binding of NTSTH to endothelium. Because motif NTSTH is displayed by 2 phage clones, which are CNTSTHPHC and CNTSTHSLC respectively, we insert these two cyclic 7 peptides into the C terminal of expression plasmid pET14b-his-EGFP. In this way motif NTSTH is marked by his-EGFP and two expression plasmids of recombination protein, pET14b-his-EGFP-CNTSTHPHC and pET14b-his-EGFP-CNTSTHSLC are constructed respectively. At the same time, for the purpose of evaluating the targeted binding of another homologous motif, NTTTH, we added amino-acid residues RC at its C terminal and C at its N terminal, thus the third expression plasmid of cyclic 7 peptide CYNTTTHRC is contructed. The target proteins are expressed and purified for further use in the study. 2.3 In vivo study on targeted binding of motif NTSTHThe cultured HMVEC in petri-dishes were added with 600 nmol/L his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC respectively 12 h following the stimulation of 1 ug/ml LPS. EGFP was taken as control. After incubation at 37℃in the cell culture system for 30 min, the cells were washed with PBS, fixated and dyed with DAPI. The cells were observed under Zeiss fluorescence microscope. Image analysis software Image-pro plus (IPP) was applied for semi-quantitation of mean stained area (MSA) and mean integrated optical density (MIOD) in different groups.2.4 Distribution of his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC in septic-shock miceSeptic-shock mice were intravenously injected with 0.7 nmol his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC via tail vein respectively. Then the mice were perfused via left ventricle with PBS for 10 min and fixated with 4％paraformaldehyde. Various of tissues from liver, heart, spleen, lung, kidney, brain, small intestine, skin and muscle were sampled and exposed under Kodak in-vivo Imaging System to observe the distribution of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC in various of tissues in septic shock mice. EGFP was taken as control. At the same time the perfused tissues were homogenated and the supernate was used for fluorescent quantitation and western-blotting.2.5:Localization of his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC in tissues of septic-shock miceSeptic-shock mice were intravenously injected with 0.7 nmol his-EGFP-CNTSTHPHC, his-EGFP-CNTSTHSLC and his-EGFP-CYNTTTHRC via tail vein respectively. Then the mice were perfused via left ventricle with PBS for 10 min and fixated with 4％paraformaldehyde. Tissues of liver, heart, spleen, lung, kidney, brain, small intestine, skin and muscle were sampled and cryosections were made to observe the binding site of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC in septic-shock mice under Ziess fluorescence microscope. EGFP was taken as control. 2.6 Specificity evaluation for targeted binding of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC in septic-shock mice12 mice were processed into his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC groups with 4 mice for each.4 different processing methods are for 4 mice in each group:(1) septic model plus injection with recombination protein; (2) septic model plus injection with his-EGFP; (3) non-treated mice plus injection with recombination protein; (4) non-treated mice plus injection with his-EGFP. After the injection the fresh tissues were sampled and homogenated and the supernate was used for fluorescent quantitation and western-blotting analysis.3 Results3.1 Bio-information exploring of characterized motif NTSTHCharacterized motif NTSTH mimics a new protein, Scube 1 very well. According to relevant researches, characteristics of scube 1 are as follows:(1) being conservative in 2 more species like mice, monkey, zebrafish and human; (2) membrane protein and secreted protein; (3) being originally found in cDNA library of HUVEC and strongly expressing in human blood vessels; (4) being closely associated with blood coagulation; (5) being closely associated with inflammation and indicating dynamic response to the stimulation of LPS. Motif NTSTH is highly homologous to NTTTH, a motif from a cysteine-rich domain in scube 1, which is much possible to be a function site for scube 1 to bind to active endothelium. Therefore it can be inferred that motif NTSTH not only has the possiblity to bind to endothelium under inflammatory conditions, but also be a mimic epitope of scube 1.3.2 Construction of expression plasmid of NTSTH and the expression of fusion protein3 expression plasmids of his-EGFP-CNTSTHPHC/CNTSTHSLC/ CYNTTTHRC were constructed and the protein of his-EGFP-CNTSTHPHC/ CNTSTHSLC/CYNTTTHRC were expressed and purified respectively. his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC as cyclic peptides were all marked by his-EGFP.3.3 In vivo study on targeted binding of his-EGFP-CNTSTHPHC/ CNTSTHSLC/CYNTTTHRCAfter incubation with LPS-induced HMVEC for 30 min, obvious binding of protein his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC on cell membrane. However, no affinity of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC can be observed on normal cells without stimulation of LPS, and, no affinity of his-EGFP was observed on either LPS-induced or non-induced cells. Semi-quantitation analysis of fluorescence indicated that MSA of his-EGFP-CNTSTHPHC (F= 47.761, P< 0.001), his-EGFP-CNTSTHSLC (F= 36.802, P< 0.001), his-EGFP-CYNTTTHRC (F= 19.099, P< 0.001) and MIOD of his-EGFP-CNTSTHPHC (F= 69.974, P< 0.001), his-EGFP-CNTSTHSLC (F= 32.031, P< 0.001), his-EGFP-CYNTTTHRC (F= 30.640, P< 0.001) were significantly higher in LPS-induced group vs control. These results suggest the specific affinity of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC on HMVEC.3.4 Distrbution of his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC in septic-shock miceObservation under Kodak In-Vivo Imaging System indicated that the distribution of his-EGFP-CNTSTHPHC is primarily in tissues of liver, kidney, brain and small intestine, and distribution of his-EGFP-CNTSTHPHC is also observed in heart and lung tissues; CYNTTTHRC is obviously distributed in liver and kidney; as for CNTSTHSLC, beside the distribution in liver, kidney and brain, it also can be observed to distribute in lung and heart tissues. All the recombination proteins are obviously distributed in vascularized tissues like kidney, liver, lung, brain and small intestine, however, little distribution was observed in non-vascularized tissues, such as muscles and skin. Fluorescent quantitation and western-blotting analysis of tissue homogenate also indicated similar results, suggesting the targeted binding of recombination proteins to blood vessels in various tissues.3.5 Localization of his-EGFP-CNTSTHPHC/CNTSTHSLC/CYNTTTHRC in tissues of septic-shock miceUnder fluorescence microscope it was observed on cryo-sections of various tissues that the recombination proteins his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC are all localized in vascular endothelium, while little fluorescence was observed in adjacent tissues, tissue spaces and adipose tissues. The recombination proteins were also observed to distribute in thrombus in some tissues from liver, kidney and brain, suggesting the recombination protein may be associated with blood coagulation.3.6 Specificity evaluation for targeted binding of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC in septic-shock miceFluorescent quantitation and western-blotting analysis indicated strong targeted binding to blood vessels in liver and kidney, but very poor binding of these recombination proteins is observed in non-treated mice. As a control, EGFP was observed to exert no influence on the targeted binding of the three recombination proteins to blood vessels without any fluorescence detected both in fluorescent quantitation and western-blotting analysis, suggesting the specificity of targeted binding of his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC to blood vessels in septic-shock mice. As a shared motif of these three proteins, the characterized motif NTTTH can be inferred to be the special binding site to blood vessels in septic-shock mice.4. Conclusion4.1 Recombination proteins his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC can target to vascularized organs and tissues. The targeted binding is mediated by LPS but not associated with their marker, his-EGFP.4.2 The localization of recombination proteins his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC in various tissues is primarily in vascular endothelium of septic-shock mice.4.3 The targeted binding of the recombination proteins his-EGFP-CNTSTHPHC, CNTSTHSLC and CYNTTTHRC is specific to vascular endothelium of septic-shock mice.4.4 The shared motif NTSTH/NTTTH of cyclic 7 peptides CNTSTHPHC, CNTSTHSLC and CYNTTTHRC is also the function site of the targeted binding. The binding activity of these recombination proteins is not associated with the amino acid residues besides the shared motif.4.5 The characterized motif NTSTH/NTTTH can specially target to vascular endothelium stimulated by LPS.4.6 The targeted binding of NTSTH/NTTTH, which is marked by large molecule EGFP, suggesting the potentiality of NTSTH/NTTTH as a leader peptide of some macromolecular drugs.4.7 It can inferred that the characterized motif NTSTH is the mimic epitope of Scube 1, while NTTTH is possibly one of the function sites of Scube 1.4.8 The high affinity of NTSTH/NTTTH to vascular endothelium induced by LPS suggests its potential competitive inhibition to its mimicked protein, Scube 1, but further evidence is needed.