Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
  • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
  • G01N2333/8121—Serpins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
  • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders

Definitions

• TIA transient ischemic attack
• ACS acute ischemic coronary syndromes
• the invention relates in particular to a method of predicting the risk of a subject developing a cardiovascular event .
• the present invention further relates to the diagnosis of acute ischemic coronary syndromes (ACS) .
• ACS acute ischemic coronary syndromes
• the invention also relates to kits and biomarkers for use in the methods .
• prognostic biomarkers would be of major added value in recognizing patients who are at risk of suffering future cardiovascular events and who could then be targeted for aggressive preventive measures.
• prognostic value of biomarkers is very limited since these biomarkers only moderately add to standard risk factors.
• prognostic biomarkers are non-existent.
• the ideal approach in the search for biomarkers is an unbiased approach. Novel molecular techniques such as proteomics opened new possibilities for this purpose.
• plaque material can only be obtained through invasive procedures. It is therefore a first object of the present invention to provide an alternative method for predicting the risk of a subject developing a cardiovascular event .
• Chest-pain can result from many causes: gastric discomfort (e.g.
• indigestion indigestion
• pulmonary distress pulmonary embolism
• dyspnea pulmonary embolism
• dyspnea pulmonary embolism
• musculoskeletal pain pulled muscles, bruises
• ACS acute ischemic coronary syndrome
• Acute coronary syndrome is usually one of three diseases involving the coronary arteries: ST elevation myocardial infarction (30%) , non ST elevation myocardial infarction (25%) , or unstable angina (38%) . These types are named according to the appearance of the electrocardiogram (ECG/EKG) as non-ST segment elevation myocardial infarction (NSTEMI) and ST segment elevation myocardial infarction
• the physician has to decide if the patient is having a life threatening ischemic ACS or not.
• rapid treatment by opening up the occluded coronary artery is essential to prevent further loss of myocardial tissue.
• cardiac biomarkers have become an essential tool to define if a patient has a myocardial necrosis related to myocardial infarction.
• Favorable features of biomarkers of necrosis are high concentrations in the myocardium and absence in non- myocardial tissue, release into the blood within a
• ischemia is further defined by the addition of at least 1 of the following criteria: ischemic ST and T-wave changes, new left bundle-branch block, new Q waves, PCI-related marker elevation, or imaging showing a new loss of
• troponins can be detected in blood as early as 2 to 4 h after the onset of symptoms, elevation can be delayed for up to 8 to 12 h. This timing of elevation is similar to that of Creatine Kinase-MB but persists longer, for up to 5 to 14 days.
• proteomic analyses were performed on human plaque and plasma samples.
• the procedure was hampered, however, by the presence of high-abundant plasma proteins such as albumin and immune- globulins, which complicated the detection of potentially interesting low-abundant proteins. Therefore sub- fractions of plasma were investigated for the presence of proteins that may have predictive value for cardiovascular events.
• Protein secretion out of the cells can occur directly after production (constitutive pathway) or is first stored in the cell and released after a trigger (regulatory pathway) . Secretion, however, not only occurs with
• vesicles containing a large number of proteins and RNA. These vesicles are formed with a selection of lipids, protein and RNA from the secreting cell and are released as an intact vesicle.
• Vesicles in the size of 50-100 nm are called exosomes and the release of exosomes has been described for various cell types, including reticulocytes, B- and T- lymphocytes, dendritic cells, mast cells, platelets, macrophages and alveolar lung cells.
• reticulocytes including reticulocytes, B- and T- lymphocytes, dendritic cells, mast cells, platelets, macrophages and alveolar lung cells.
• dendritic cells dendritic cells
• mast cells reticulocytes, B- and T- lymphocytes, dendritic cells, mast cells, platelets, macrophages and alveolar lung cells.
• T cells, platelets, dendritic cells and mast cells secretion of exosomes is regulated by specific stimuli.
• Exosomes have a wide range of biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins.
• exosomes since exosomes express an array of proteins that reflect the originating host cell, they contain valuable information regarding ongoing (patho) physiologic processes in the human body including information of future cardiovascular events.
• 'plasma exosome sample' can refer both to a sample of isolated exosomes and a sample of body fluid, in particular serum or plasma, comprising exosomes.
• any biomarker with prognostic value may be used.
• specific markers were identified in or on plasma exosomes that have predictive value for secondary
• the invention thus provides a method of predicting the risk of a subject developing a cardiovascular event comprising detecting a biomarker in an exosome sample or other micro-vesicles of smaller or larger size from said subject, wherein said biomarker comprises at least one protein selected from the group of 6 proteins consisting of :
• Serpin F2 (IPI : IPIO0879231, SWISSPROT :A2AP_HUMA ) ,
• CD14 (IPI: IPI00029260, SWISSPROT : CD14_HUMAN) ,
• Cystatin C (IPI: IPI00032293, SWISSPROT: CYTC_HU A ) ,
• Plasminogen (IPI : IPI00019580 , SWISSPROT: PLMN_HUMAN)
• Nidogen 2 (IPI:IPI00028908,SWISSPROT:NID2_HUMAN)
• SerpinGl ( IPI : IPIO0291866 ; SWISSPROT: IC1_HUMAN) .
• a biomarker comprises one protein or a set of multiple proteins
• a biomarker is also identified herein as a profile or protein profile.
• a profile may comprise 1, 2, or more ..than 2 such as 3, 4, 5, 6 of the proteins:
• Serpin F2 (IPI : IPIO0879231, SWISSPROT :A2AP_HUMAN) ,
• CD14 (IPI:IPI00029260, SWISSPROT : CD14_HUMA ) ,
• Cystatin C (IPI : IPI00032293 , SWISSPROT: CYTC_HUMA ) ,
• Plasminogen (IPI : IPI00019580 , SWISSPROT: PLMN_HUMAN) ,
• Nidogen 2 (IPI : IPIO0028908 , SWISSPROT :NID2_HUMAN) ,
• a profile may be used that comprises any number and any combination of these proteins.
• the biomarker protein or a peptide fragment thereof is detected in exosomes or other vesicles somewhat larger or smaller in size that are preferably found in body fluids like serum, plasma or blood.
• exosomes or such other vesicles from other body fluids such as urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, breast milk, saliva can be used.
• the biomarker protein or a peptide fragment thereof is detected in serum or plasma.
• biomarkers that are attached to, anchored in or adhered to exosomes are detected.
• cardiovascular event to be predicted is preferably selected from the following conditions: vascular death or sudden death, fatal or non fatal stroke, fatal or non fatal myocardial infarction, fatal or non fatal rupture of an abdominal aortic aneurysm, rupture of abdominal aortic aneurysm confirmed by laparatomy, vascular intervention, coronary artery disease, transient ischemic attack (TIA) , peripheral arterial disease, acute coronary syndrome, heart failure or re-stenosis of carotid, coronary, femoral or other arteries.
• vascular death or sudden death fatal or non fatal stroke
• fatal or non fatal myocardial infarction fatal or non fatal rupture of an abdominal aortic aneurysm
• rupture of abdominal aortic aneurysm confirmed by laparatomy, vascular intervention, coronary artery disease, transient ischemic attack (TIA) , peripheral arterial disease, acute coronary syndrome, heart failure or re-stenosis of carotid, coronary, femoral or
• the method of the present invention may suitably be used for risk stratification and/or patient selection (such as for clinical trials) , for monitoring of disease, and the markers may be used as clinical biomarkers for safety and efficacy studies (e.g. as surrogate endpoint markers) .
• the invention also relates to a biomarker for use in the prognosis of the risk of a subject developing a
• cardiovascular event comprising a protein selected from Vitronectin, Serpin F2, CD14 , Cystatin C, Plasminogen, Nidogen 2, Serpin Gl .
• the biomarker comprises a combination of two or more proteins selected from Vitronectin, Serpin F2, CD14, Cystatin C, Plasminogen, Nidogen 2, Serpin Gl .
• the cardiovascular event may be a primary event in a subject that has not yet suffered a cardiovascular event but is in particular a secondary event occurring in a subject already having suffered such an event before. According to the invention it is possible to discriminate between patients that already had a cardiovascular event and are at risk of suffering an additional event and patients who had such an event and do not have an increased risk of suffering a further event .
• the prognosis is made by using exosomes as the sample and preferably the biomarker comprising
• the prognosis is made by using serum comprising the exosomes as the sample and preferably the biomarker comprising Vitronectin, Serpin F2, CD14, Cystatin C, Plasminogen, Nidogen 2 or any combination thereof as the protein (s) to be detected in or on the exosomes .
• ischemic event immediately activates endothelial cells that attract
• vesicles are formed with a selection of lipids, protein and NA from the secreting cell and are released as an intact vesicle. They are generally called microvesicles and have a size between 20 and 1000 nm. From these microvesicles, exosomes are the best described
• particles having a size between 50 and 100 nm having a size between 50 and 100 nm.
• ACS ACS
• microvesicles are secreted from several cells and tissues.
• the most obvious tissue is the myocardium.
• Apoptosis of cardiomyocytes occurs almost instantly after occluding the coronary artery and subsequent ischemia.
• the apoptopic cardiomyocytes secrete vesicles in the blood that are called_apoptopic bodies .
• proteomic analyses were performed on human plasma samples.
• the procedure was hampered, however, by the presence of high-abundant plasma proteins such as albumin and immune-globulins, which complicated the detection of potentially interesting low-abundant proteins. Therefore sub- fractions of plasma were investigated for the presence of proteins that may have diagnostic value for the
• exosome is thus intended to include other vesicles that are smaller than about 50 nm or larger than 100 nm but still fall within the range of about 20 to about 500 nm.
• T cells T cells
• platelets dendritic cells
• mast cells secretion of exosomes is regulated by specific stimuli. While early studies focused on their secretion from diverse cell types in vitro,
• exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, broncho-alveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have a wide range of biological functions,
• exosomes express an array of proteins that reflect the originating host cell and that they contain valuable information regarding ongoing (patho) physiologic processes in the human body including information on the occurrence of ACS .
• biomarker with diagnostic value may be used.
• specific markers were identified in/on plasma exosomes that have diagnostic value for ACS.
• the invention thus provides a method for the diagnosis of ACS comprising detecting a biomarker in an exosome sample or micro-vesicles of smaller or larger size from said subject, wherein said sample comprises at least one protein selected from the group of 3 proteins consisting of: Serpin F2 (IPI : IPI00879231 ,
• index numbers databases accessions
• the referenced index numbers include reference to fragments, isoforms and modifications thereof, hence the present invention foresees the use of fragments of the proteins as well as modifications and derivatives of the proteins disclosed herein as biomarkers in the context of the various aspects of the present invention.
• a biomarker comprises one protein or a set of multiple proteins. Such a biomarker is also identified herein as a profile or protein profile.
• a profile may comprise 1, 2 or 3 of the proteins Serpin F2
• biomarker protein instead of detecting the complete biomarker protein, one may detect peptide fragments of said biomarker proteins which are derived from the biomarker proteins by fragmentation
• peptide fragment refers to peptides having between 5 and 50 amino acids. These peptide fragments preferably provide a unique amino acid sequence of the protein, and are associated with the cardiovascular events as disclosed herein.
• proteins and/or peptide fragment may optionally be detected as chemically modified proteins and/or peptides, such chemical modification may for instance be selected from the group consisting of glycosylation, oxidation,
• biomarkers may also be part of the membrane.
• biomarkers that are attached, anchored or adhered to the exosome can also be detected in samples of body fluid, in particular in serum.
• the biomarker protein or a peptide fragment thereof is detected in, on or attached to exosomes that are preferably found in body fluids like serum, plasma or blood.
• the invention further relates to a kit for
• the kit comprises means for detecting the presence of a biomarker as defined above.
• the means for detecting the presence of the biomarker are preferably antibodies, antibody fragments or antibody derivates or via mass
• detection means optionally comprise a detectable label.
• the kit of the invention is intended for use in a method of predicting the risk of a subject developing a cardiovascular disease by determining the presence of a biomarker in or on exosomes of the subject or for diagnosing ACS or for risk prediction for coronary heart disease in females.
• the kit may further comprise reagents and/or instructions for using the means for detecting a biomarker in any such method.
• Figure 1 the graph shows two ROC analyses for CD1 .
• the solid grey line is the reference line and represents an AUC of 0.5 (that is, no discrimination).
• Figure 2 the graph shows two ROC analyses for
• the solid grey line is the reference line and represents an AUC of 0.5 (that is, no
• the solid grey line is the reference line and represents an AUC of 0.5 (that is, no
• the solid grey line is the reference line and represents an AUC of 0.5 (that is, no discrimination) .
• Figure 5 the graph shows two ROC analyses for
• the solid grey line is the reference line and represents an AUC of 0.5 (that is, no discrimination) .
• Figure 6 the graph shows two ROC analyses for Nidogen 2.
• Figure 7 a published ROC curve.
• Figure 8 Typical CD9 western blot showing CD9 levels in original serum (serum 3) and in exosome pellet after lx (pellet 1) 2x (pellet 2) and 3x (pellet 3) exosome precipitation using ExoquickTM.
• Serum 1 is loading control.
• Sup is Serum (Supernatant) after lx (Sup 1) , 2x (Sup 2) and 3x (Sup 3) precipitation.
• Figure 9 Nanosight Sample Report of an exosome pellet after resuspension and dilution.
• Figure 10 Area under the curve analysis for Troponin (Trop, solid curve) measured in blood taken at intake of the patient with chest pain and Troponin plus Serpin F2 (SerpinF2_CP, dashed curve) .
• FIG. 11 Schematic representation of flotation experiment (left) and subsequent SDS PAGE for protein separation (right) . Microvesicles preparation from
• plasma/serum samples was performed by ultracentrifugation on a linear sucrose gradient of 2.0-0.4 M. Microvesicles will float on a different sucrose gradient density based on their different buoyancy and density.
• Figure 12 Western Blot analysis on microvesicles obtained from the flotation experiment.
• CD9 protein was used as the microvesicle marker protein. All four biomarkers were found to be present in collected fractions of microvesicles: fractions densities of 1.176 g ml "1 to 1.216 g ml "1 for CD14 ; 1.196 g ml “1 and 1.176 g ml “1 for Cystatin C 50 kD and 1.196 g ml "1 to 1.245 g ml “1 for Cystatin C 180 - 200 kD; 1.176 g ml "1 to 1.245 g ml “1 for Serpin F2 ; and 1.196 g ml "1 to 1.216 g ml “ 1 for Serpin Gl .
• Figure 13 Predictive biomarkers in microvesicles isolated with ExoQuick Precipitation Solution. Multiplex Luminex assay was used to validate the significant
• the Athero-Express is a longitudinal vascular biobank study, which includes biomaterials from patients undergoing carotid and femoral end-arterectomy in two Dutch hospitals (UMC Utrecht and St. Antonius Hospital).
• Exosomes were isolated from frozen human plasma by filter separation followed by ultracentrifugation (cf .
• exosomal proteins such as CD9 and CD81 were detected in the exosome pellet using western blotting. FACS analysis with beads of defined sizes demonstrated that the pellet contains mostly particles of 50-100 nm which is in accordance with the size of exosomes.
• the exosome pellets collected in the Athero-Express biobank plasma were after ultracentrifugation dissolved in 40 ⁇ 6% SDS in HPLC pure water. Plaque protein was, after grinding the plaque material without any blood remains to powder, also extracted with 6% SDS. Digestion and subsequent labeling, HPLC separation and mass spectrometry analysis was identical for plaque and exosome proteins .
• the protein content was determined by 2-D Quant Kits. After protein reduction and alkylation, the protein mixture was diluted 20 times with 50 mM triethylammonium bicarbonate (TEAB) and protein digestion was initiated by adding trypsin in a 1:40 trypsin-to-protein ratio. The protein digests were desalted using a Sep-Pak C18 cartridge and dried in a Speedvac.
• TEAB triethylammonium bicarbonate
• digests were labeled with iTRAQ reagents according to the manufacturer's protocol. Briefly, digested proteins were reconstituted in 30 ⁇ of dissociation buffer and mixed with 70 ⁇ of ethanol-suspended iTRAQ reagents
• the dried fraction was reconstituted in 100 ⁇ of
• Quantitative proteomics were performed on exosomes from 50 patients that suffered a coronary event during follow up (Group 1) and from 50 matched control patients that did not suffer a secondary event during follow up
• Quantitative data were available from 2 pooled events samples (Group 1 in duplo) and 2 pooled control samples (Group 2 in duplo) . Based on pilots, it was determined that a ratio of 1.2 and above means that there is significantly higher level of the protein in the event while a ratio of 0.8 and lower is a significant lower level in the event. First selection was based on proteins with identical duplo' s (both below 0.8, both above 1.2 or both between 0.8 and 1.2) .
• Second selection was based on proteins with lower (events/controls ⁇ 0.8) or higher (events/controls >1.2) expression in group 1 vs. group 2. This revealed a list of 116 proteins.
• this group of 102 differentially expressed proteins was complemented with a selection of plaque material derived proteins and finally narrowed down to a combined set of 34 selected exosome- and plaque-derived proteins for further validation in exosome samples of individual patient samples.
• Athero-Express cohort 40 carotid end- arterectomy patients were selected of which 20 had a
• Quantitative proteomics was performed on plaque samples as for the exosome proteomics. However, since 40 individual plaques were analyzed, four plaque extracts were run simultaneously each differently labeled by the iTraq reagent (114, 115, 116, 117 resp.). Each run consisted of two plaque extracts of patients that suffered a
• plaque is the origin of atherosclerotic disease leading to cardiovascular events. For this, it is very likely that plaque proteins related to future cardiovascular events can also be found in, on, anchored or adhered to exosomes especially the plaque proteins that are related to the pathways over-represented in exosome proteins that differ between cardiovascular events and controls.
• the objective of this study was to identify in blood samples of individual patients which of those 17 biomarkers were differentially expressed between patients suffering from a secondary coronary event and healthy controls.
• a primary cerebral-vascular event i.e. a stroke or Transient Ischemic Attack (TIA) and were followed-up for three years.
• the 17 markers were measured in blood samples of patients who suffered from a secondary coronary event (29 samples) and age and sex matched controls (30 samples) .
• the secondary coronary events were defined as myocardial infarction (fatal and non-fatal) , cardiovascular death, sudden death, coronary angioplasty, and coronary artery bypass graft (CABG) .
• Exosomes were isolated from the plasma using the ultracentrifugation technique. Proteins extracted from the exosome samples were measured in multiplex Luminex bead assays .
• ROC analyses were performed to determine the ability of the marker, in conjunction with a risk score, to
• the risk score was based on 7 traditional cardiovascular risk factors (gender, age, cholesterol, systolic blood pressure, smoking status, history of peripheral artery disease, and history of coronary artery disease) .
• the objective of the present example is to evaluate whether a favourable biomarker profile can rule out the chance of a cardiac event in the following years.
• QICS study Quality of service
• exosome bound biomarkers predictive for secondary cardiovascular events were identified (Example 1 and 2) .
• serum of 240 patients of the QICS cohort was used to measure the expression of these three exosome-based biomarkers in serum samples with and without isolating the specific exosome fraction of the serum.
• Cystatin C, CD14 and Serpin F2 can be measured in serum samples without exosome isolation as well as in isolated exosomes using ExoquickTM and that in both samples (with or without exosome isolation) these three markers are predictive for secondary coronary events.
• Table 1 shows that indeed the serum levels of markers progressively decrease, almost disappear after three consecutive ExoquickTM extraction cycles, thus proving the fact that these markers are exosome bound.
• CD9 is a trans-membrane protein that is associated with the membrane of exosomes and is one of the most common exosome proteins and used as exosome marker. Again the same 4 serum samples were used as above. CD9 was measured by Western Blot analysis before ExoquickTM exosome precipitation and after 1, 2 and 3 times ExoquickTM precipitation.
• Nanosight measures the Brownian movement of vesicles by shattering light on the exosome pellet re-suspended in PBS. The less the vesicles move the bigger they are.
• An exosome pellet after ExoquickTM precipitation was re- suspended and diluted at least a 100.000 time and brought on the Nanosight machine resulting in Figure 9, which shows that the vesicles in the ExoquickTM pellet are a very
• ARTerial disease Study, with the objective to investigate brain changes on MRI in patients with symptomatic atherosclerotic disease.
• UMCU University Medical Center Utrecht
• peripheral arterial disease or an abdominal aortic aneurysm were included.
• MR magnetic resonance
• AUC Area under the curve
• the AUC reflects the overall added value of a model and does not directly indicate its clinical value, therefore the Net Reclassification Index of the biomarkers were assessed.
• the Net Reclassification Index is a tool to assess what effect a biomarker will have in classifying patients in pre-specified risk groups.
• the NRI analysis is a relatively new phenomenon in statistics .
• the American Heart Association recently published a new set of criteria for the evaluation of novel cardiovascular biomarkers, which
• Group 1 ( ⁇ 5% risk) consisted of 527 patients (451+ 74+2)
• Group 2 (5-10% risk) consisted of 389 patients (86+ 279 + 24)
• Group 3 (>10% risk) consisted of 136 patients (102 + 34) .
• SerpinGl is an important biomarker for the exosome panel .
• each of the markers individually achieves on top of the traditional risk factors a reclassification effect ranging from 6.0% for CystatinC to 9.8% for SerpinF2, and for CD14 and SerpinGl at 8.0% and 8.4%, respectively. These percentages already demonstrate the impact on patient reclassification of the individual markers .
• the combinations of 4 markers on top of the traditional risk factors yields the best and clinically very relevant NRI score of 15.5%. Table 3
• Plasma and tissue samples were obtained from all patients before (blood) or during end-arterectomy. All patients underwent clinical follow-up 1 year after surgical intervention and filled in postal
• Exosomes were isolated from frozen human plasma by filter separation followed by ultracentrifugation.
• exosomal proteins such as CD9 and CD81 were detected in the exosome pellet using western blotting. FACS analysis with beads of defined sizes demonstrated that the pellet contains mostly particles of 50-100 nm which is in accordance with the size of exosomes. Protein extraction and digestion
• the exosome pellets collected in the Athero-Express biobank plasma were after ultracentrifugation dissolved in 40 ⁇ 6% SDS in HPLC pure water. Plaque protein was, after grinding the plaque material without any blood remains to powder, also extracted with 6% SOS. Digestion and subsequent labeling, HPLC separation and mass spectrometry analysis was identical for plaque and exosome proteins. The protein content was determined by 2-D Quant Kits. After protein reduction and alkylation, the protein mixture was diluted 20 times with 50 mM triethylammonium bicarbonate (TEAB) and protein digestion was initiated by adding trypsin in a 1:40 trypsin-to-protein ratio. The protein digests were desalted using a Sep-Pak C18 cartridge and dried in a Speedvac.
• TEAB triethylammonium bicarbonate
• Quantitative data were available from 2 pooled events samples (Group 1 in duplo) and 2 pooled control samples (Group 2 in duplo) . Based on pilots, it was determined that a ratio of 1.2 and above means that there is significantly higher level of the protein in the event while a ratio of 0.8 and lower is a significant lower level in the event. First selection was based on proteins with identical duplo' s (both below 0.8, both above 1.2 or both between 0.8 and 1.2).
• expressed proteins was complemented with a selection of plaque material derived proteins and finally narrowed down to a combined set of 34 selected exosome and plaque derived proteins for further validation in exosome samples of individual patient samples.
• Athero-Express cohort 40 carotid end- arterectomy patients were selected of which 20 had a
• Quantitative proteomics was performed on plaque samples as for the exosome proteomics. However, since 40 individual plaques were analyzed, four plaque extracts were run simultaneously each differently labeled by the iTraq reagent (114, 115, 116, 117 resp.). Each run consisted of two plaque extracts of patients that suffered a
• the plaque is the origin of atherosclerotic disease leading to cardiovascular events. For this, it is very likely that plaque proteins related to future cardiovascular events can also be found in exosomes especially the plaque proteins that are related to the pathways over-represented in exosome proteins that differ between patients suffering from a cardiovascular events and healthy controls . Having established that 3 canonical pathways (acute phase,
• Selection was based on the presence of proteins that are related to the 3 atherosclerosis related canonical pathways and for which 2 antibodies and a recombinant protein were available.
• markers were selected based on over-representation of 3
• Luminex bead assay development From the selected plaque and exosome proteins for which antibodies and recombinant protein were available, 34 proteins were chosen for Luminex bead assay development. For 17 proteins out of those 34 proteins (including Cystatin C, Serpin F2 and CD14) , a reproducible and quantitative Luminex bead assay was set up that could be used for
• QICS Quick Identification of acute Chest pain Study
• EBT electron beam
• CT computed tomography
• MSCT contrast multi-slice CT
• Cystatin C, Serpin F2 and CD14 were measured using Luminex multiplex technology on/in or attached to exosomes that were isolated with ExoquickTM from 250 ul of serum of individual QICS patients.
• ROC analyses were performed to determine the ability of the marker, in conjunction with a risk score, to
• Serpin F2 (238 samples) showed a p-value of p ⁇ 0.001 between ACS and non-ACS while CD14 gave a p-value of 0.002 (238 samples)
• Serpin F2 The strongest marker Serpin F2 was analyzed to see if it had additional value, in diagnosing acute coronary syndrome on top of Troponin levels measured at the intake of the patient .
• ROC curves show that the area under the curve increases from 0.835 for Troponin alone to 0.881 for Troponin plus Serpin F2.
• Serpin F2 plus the maximum levels of troponin measured (Tropmax) is significantly different from Tropmax alone and Serpin F2 plus High sensitive (Hs) - Troponin is also significantly different from Hs-Troponin alone showing the added value of Serpin F2.
• Important biological component such as membrane- linked protein markers are often present in microvesicles found in body fluids such as urine, amniotic fluid,
• sucrose gradient centrifugation and ExoQuick isolation were used to isolate microvesicles from plasma samples .
• Sucrose gradient separation using ultra- centrifugation on plasma samples is based on the different buoyancy (density) of microvesicles compared to free protein and large protein complexes.
• sucrose gradient centrifugation large complexes will go to the bottom of the tube (highest density) while free proteins will go to the top of the gradient (lower density) .
• Microvesicles will float in the intermediate density layers that are also identified by microvesicle markers like CD9.
• ExoQuick solution manufactured by System Biosciences (SBI) was used to precipitate microvesicles/exosomes out of the plasma samples according to the manufacturer's protocol
• Citrate-anticoagulated human whole blood samples were collected and centrifuged at 1850 x g for 10 minutes at room "temperature to eliminate cell debris.
• the resulting clear solution on the very top layer known as blood citrate plasma was transferred into a 15 ml tube, snapped freeze in liquid nitrogen, and stored in -80°C for further use.
• the supernatant was aspirated using an aspirator leaving the pellet containing microvesicles/exosomes undisturbed at the bottom of the tube.
• the pellet was resuspended in 20 ⁇ lx PBS , and mixed with 1.5 ml of 2.5 M sucrose in a new ultracentrifuge tube.
• the plasma samples were centrifuged at 3000 x g for 15 minutes at room temperature prior to use.
• a pre-treated 0.45 ⁇ filter with 100 ⁇ of pre-heated MQ water at 37°C was prepared, centrifuged at 10000 x g for 2 minutes at room temperature, and transferred into a new empty filter tube.
• the plasma was added into the pre-treated filter followed by centrifugation at 12000 x g for 10 minutes at room
• the pellet was resuspended in Roche Complete Lysis-M buffer containing protease inhibitors (EDTA-free) and incubated for 30 minutes at room temperature.
• EDTA-free protease inhibitors
• Microvesicles containing protein markers were separated, via ultracentrifugation on a continuous sucrose gradient from large aggregates which sedimented in the pellet as shown in Figure 11. After centrifugation the different layers were taken out from the centrifugation tube and (after density measurement) used for Western Blot analysis.
• a panel of antibodies directed against CD14 were used for Western Blot analysis.
• CD9 protein is used as the vesicle protein marker since it is one of the most abundant protein families found in the membrane of
• CD14 signal was detected in the densities fractions comprised between 1.176 g ml “1 and 1.216 g ml “1 .
• Floating vesicles containing Serpin F2 and Serpin Gl were also found in densities fractions ranging from 1.176 g ml “1 to 1.245 g ml “1 and 1.196 g ml “1 to 1.216 g ml “1 , respectively.
• Cystatin C differed on their molecular weight.
• the 50 kD Cystatin C were found in the collected densities fractions of 1.196 g ml "1 and 1.176 g ml "1 whereas Cystatin C of 180-200 kD was indicated in densities fractions from 1.196 g ml "1 to 1.245 g ml "1 .
• the results confirmed successful isolation of micrdvesicles containing CD14 Cystatin C, Serpin F2, and Serpin Gl via flotation experiment and verified by Western Blot analysis. The occurrence of each marker in more than one density fraction is likely due to different
• Microvesicles were isolated by overnight incubation of plasma samples obtained from 25 patients suffered a secondary coronary event during follow-up and 25 controls that did not have an event during follow-up with ExoQuick Precipitation Solution, resulting in a pellet at the bottom of the tube. This microvesicle pellet was lysed with Roche lysis -M buffer and used for CD14, Cystatin C, Serpin F2, and Serpin Gl Luminex detection and quantification.
• biomarkers showed a significant difference (p ⁇ 0.05) between patients with a secondary cardiovascular event and controls.
• the significant difference between events and controls was lost in supernatant 1 while for Cystatin C the significant difference between events and controls was no longer detected in supernatant 2.
• Serpin F2 maintained its significant difference between events and controls in supernatant 1, Exo-pellet 2, supernatant 2, and Exo-pellet 3 but not in supernatant 3 as indicated in Figure 13.

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