Idiopathic pulmonary arterial hypertension (IPAH) is characterised by sustained elevation of pulmonary artery pressure which leads to right heart failure and death in untreated patients (Runo and Loyd, 2003). It is defined as a mean pulmonary artery pressure > 25 mm Hg, pulmonary vascular resistance > 3 Wood units and pulmonary capilliary wedge pressure <15 mmHg at rest, leading to increase in pulmonary vascular resistance (Archer et al., 2010). The aetiology is unknown but the pathology includes thrombosis, smooth muscle hypertrophy, medial hypertrophy and endothelial dysfunction (Gaine and Rubin, 1998).
As the symptoms are non-specific and overlap with other cardiorespiratory diseases, diagnosis of IPAH is commonly delayed, sometimes for more than 2 years; estimated mean survival in untreated disease is 2.8 years. Diagnosis is usually confirmed by right heart catheterization (Chin and Rubin, 2008). The 6 minute walk test (6MWT), a measure of exercise capacity and the New York Heart Association (NYHA)/World Health Organization (WHO) based functional classification, are used to follow the clinical course of the disease and these both correlate with disease severity and prognosis (Warwick et al., 2008).
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Recently, a range of biomarkers have been proposed as diagnostic and prognostic indicators in IPAH. These include brain natriuretic peptide, troponin T, von Willebrand factor, serum uric acid, endothelin-1 and metabolites of nitric oxide (Heresi and Dweik, 2010).
A system-based screening method such as proteomics, is suitable in capturing the changes that occur within a complex disease such as IPAH (Yildiz, 2009). A previous proteomics study on the analysis of plasma proteome for biomarkers of idiopathic pulmonary arterial hypertension using the liquid chromatography tandem mass spectrometry (LC-MS/MS) method identified von Willebrand factor, fibrinogen, haptoglobin and complement component 7 as upregulated proteins. Complement component 7 was considered as a biomarker for further investigation and in this study, attempts were made to confirm the initial observation by western blot analysis (Umukoro, 2010).
The human complement system is a part of the innate immune system that plays a key role in the elimination of pathogens (Agah et al., 2000). It is made up of plasma proteins, cell surface regulatory molecules and cellular receptors which play a key role in host defence (Seifert and Kazatchkine, 1988).The complement system is a major mediator of inflammation and host protection (Barrington et al., 2001).
Activation of the complement system takes place via any of the three pathways; classical, the mannan binding lectin or the alternative pathway. Following complement activation, proinflammatory peptides like the anaphylatoxins C3a, C4a and C5a are generated and the membrane attack complex, C5b-9, is formed (Kirschfink, 1997). The formation of C5b-9 is initiated when C5 is cleaved into C5a and C5b. C5a is a potent anaphylatoxin that mediates different inflammatory events, while C5b binds to C6 to form a stable complex C5b-6. C7 binds to the C5b-6 complex to form C5b-7, leading to a rapid loss of haemolytic activity of both proteins due to complex formation. The reaction is followed by a conformational transition, thereby allowing its insertion into lipid membranes. The C5b-7 complex enables C8 and C9 to insert themselves into the target membrane which results in the formation of a transmembrane pore, C5b-9 (figure 1) (Podack and Tschopp, 1984).
As a result, formation of C5b-9 can induce changes in lipid bilayer permeability and ultimately result in cellular activation or death. Sublytic amounts of C5b-9 leads to the activation of endothelial, neutrophils and epithelial cells, thereby leading to a proinflammatory state (Morgan, 1989). Also, C5b-9 has been found to induce endothelial expression of P- selectin and IL-8, enhances TNF-induced ICAM-1 and E-selectin expression (Kilgore et al., 1996). C5b-9 has been shown to be associated with lesions thereby suggesting that complement activation may contribute to tissue injury and perpetuation of inflammatory response (Peerschke et al., 2004). Jointly, this information shows an important role of C5b-9 in inflammation.
Complement component 7 is an important component of the terminal complement components and its main role is to contribute to the formation of the membrane attack complex and the cytolytically inactive C5b-9 (SC5b-9) is involved in host defence against pathogens and promotion of inflammation (Podack and Tschopp, 1984). Bossi et al suggested that C7 expressed on the cell membrane may have an additional function by acting as a trap for the late complement components on endothelial cells and as a regulator of the excessive proinflammatory stimulation induced by SC5b-9 (Bossi et al., 2009). C7 is widely distributed on endothelial cells from various tissues, including brain, kidney, skin and endometrium, thereby suggesting a physiologic role for cell bound C7 in vivo. Human umbilical vein endothelial cells (HUVEC) cells have also been shown to actively synthesise C7, extending previous studies which showed that endothelial synthesis of a functional terminal pathway but not of C7 (Langeggen et al., 2000). This is of important significance because of the position of endothelial cells along the surface of vessel walls, allowing such cells to supply both the circulating blood and the extracellular fluids with C7 (Wurzner, 2000).
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Complement activation is recognised as a major contributor to vascular inflammation. Complement deposition has been observed in atherosclerotic lesions and a growing number of evidence suggests that complement plays a significant role in ischemia/reperfusion injury (Peerschke et al., 2010). In physiological conditions in situ, complement activation contributes to the removal of apoptotic cells from vascular lesions to prevent necrosis and vascular damage associated with thrombosis. Under pathologic conditions, dysregulated complement activation may enhance vascular inflammation and thrombosis, thereby influencing the adaptive immune response in the arterial wall (Peerschke et al., 2004). Complement activation have also been found to be associated with vascular injury (Peerschke et al., 2008).
Other complement mediators such as C3, C4a and C1q have been shown to be associated with the pathogenesis of IPAH. C3 is produced by endothelial cells, fibroblasts and macrophages. C3 induces circulating monocytes to express tissue factor, which is a potent procoagulant. C3 complement levels in the serum of IPAH patients may be helpful in the diagnosis of IPAH (Zhang et al., 2009). Complement 4a (C4a) des Arg was shown as a possible diagnostic biomarker of IPAH using the relatively high throughput SELDI-TOF MS technique. C4a des Arg was detected in plasma and was an indirect measure of C4a and the increase in its levels is consistent with the pathogenesis of IPAH, where increases in a number of other inflammatory mediators have been found. C4a des Arg is produced when the weak anaphylatoxin, C4a is inactivated in the circulation by the activity of carboxypeptidase N (Abdul-Salam et al., 2006). C1q is synthesized by monocytes, macrophages and myeloid cells and circulates in the blood. C1q has been found in pulmonary arterial walls and known to contribute to inflammation and thrombosis (Nakagawa et al., 2003).
Complement component 7 levels are elevated in IPAH patients compared to healthy controls.
Materials and Method
NuPAGE MES running buffer and NuPAGE lithium dodecyl sulphate (LDS) sample buffer were purchased from Invitrogen Ltd (Paisley, UK). Transblotting buffer contained 0.025 M Tris and 0.192 M glycine at pH8.3 with 20% (v/v) methanol and incubation buffer contained 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Blocking solution contained 3% BSA in PBS while washing solution contained 0.05% BSA and 0.05% Tween-20 in PBS. All reagents were purchased from Sigma-Aldrich Company Ltd (Dorset, UK).
The plasma samples for this study were obtained using the informed consent of patients and healthy volunteers and the approval of the Brompton Harefield & NHLI and Hammersmith Hospitals Research Ethics Committees. Eight IPAH patients and eight healthy volunteers serving as a control were included in this study. The mean age of the patients and controls was 48.0 Â± 15.7 years (range: 25-76 years) and 37.7 Â± 10.5 (range: 24 - 57 years) respectively, (P value = 0.092, Student's t-test).
Purified human C7 protein was purchased from Quidel (San Diego, CA).
Monoclonal and polyclonal antiserum to human C7 protein were used as primary antibodies (Quidel San Diego,USA). Secondary antibodies used were anti-mouse IgG-peroxidase to whole antibody produced from sheep (GE healthcare, London, UK) and anti-goat IgG-peroxidase antibody produced in rabbit (Sigma-Aldrich Company Ltd., Dorset, UK). All primary antibodies were used at a 1:4000 dilution while the secondary antibodies were used at 1:200 dilution for the anti-goat IgG-peroxidase antibody and 1:3000 dilution for the anti-mouse IgG-peroxidase antibody
Test protein for validation
Purified human C7 protein was prepared by serial dilutions (0.01 Âµg/ml, 0.1 Âµg/ml and 1Âµg/ml) by adding 9 Âµl of phosphate buffered saline (PBS), 15 Âµl of lithium dodecyl sulphate (LDS) (Invitrogen Ltd., Paisley, UK)and 6 Âµl of 0.5 M dithiothreitol (DTT) (Sigma-Aldrich Company Ltd., Dorset, UK) to 30 Âµl of protein and heated at 100Â°C for 2 min. Monoclonal and polyclonal antibodies against human C7 were screened for specificity by western blot. To assess the minimum difference detected and for western blot validation studies, C7 polyclonal antibody was used to observe the binding effect of IPAH and control plasma samples using different volumes of plasma (0.1Âµl and 0.5Âµl).
One-dimensional gel electrophoresis (SDS/PAGE)
Diluted samples were loaded onto a 10% bis-tris NuPAGE gel (Invitrogen Ltd., Paisley, UK) with MES buffer for 35 min at constant voltage (200V). Gels were stained with InstantBlueÂ® (Novexin Ltd, Cambridgeshire, UK) for 15 min to establish the relative molecular weight of human C7.
Western blot analysis
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After gel migration, proteins were electrotransferred onto a nitrocelluose membrane and the membranes were blocked with 50 ml 3% BSA in PBS overnight. After this, the blocking solution was poured off and rinsed by the addition of 50 ml PBS. The membrane was incubated with monoclonal antibody to human C7 (1:4000 dilution) and polyclonal antibody to C7 (1:2000 and 1:4000 dilutions) (Quidel, San Diego, USA) as primary antibodies in 0.1% BSA in PBS for 1 hour at room temperature. The membranes were then washed five times with 30 ml washing solution each time over a period of about 15 min. The membranes were then incubated with anti-mouse IgG-peroxidase antibody (1:3000 dilution) and anti-goat IgG-peroxidase antibody (1:200 dilution) as secondary antibodies in 0.1% BSA in PBS for 1 hour at room temperature. The membranes are washed five times as described above and developed with the ECL system (GE healthcare, London, UK).
Plasma samples preparation
Plasma samples were denatured by the addition of 160 Âµl of 9 M urea/2% [(3-cholamidopropl) dimethylammonio]-1-propanesulfonic acid (CHAPS) to 40 Âµl of neat plasma to make 200 Âµl of stock. Samples were then reduced by the addition of 10 Âµl of 0.5 M DTT (Sigma-Aldrich Company Ltd., Dorset, UK), 25 Âµl of LDS (Invitrogen Ltd., Paisley, UK) sample buffer, 55 Âµl of water and 10 Âµl of the stock previously made prior to heating at 100Â°C for 2 min.
One-dimensional gel electrophoresis (SDS/PAGE) and western blot analysis
Denatured plasma samples were loaded on a 10% bis-tris NuPAGE gel (Invitrogen Ltd., Paisley, UK). After gel migration (35 min at 200V), proteins were electrotransferred onto a nitrocelluose membrane. The membranes were incubated with 1:4000 polyclonal anti-serum to human C7 protein as primary antibody and 1:200 diluted anti-goat IgG-peroxidase antibody as secondary antibody. After washing, the membrane was developed with the ECL system (GE healthcare, UK).
Statistical analysis was performed by using Graphpad prism software (version 4.0; Graphpad Software Inc., San Diego, USA). Two sided student's t-test was carried out to investigate the statistical differences in C7 expression level obtained via western blot analysis in the healthy controls and IPAH patients. The level of significance was set at P<0.05.
Using the different dilutions of C7 protein, bands were observed at the appropriate region. The monoclonal antibody to C7 recognised a single band, thereby indicating its low affinity to bind C7. The polyclonal antibody recognised multiple bands for both 1:2000 and 1:4000 dilutions. 1:4000 dilution was used for this validation study because it showed much clearer bands when compared to 1:2000 dilution and it is more cost efficient. 0.5Âµl of plasma was selected for validation because it shows a clearer blot (figure 2).
Western blot validation
Western blot analysis was performed on 8 IPAH and 8 control plasma samples to confirm the proteomic observations from the previous report and to validate its potential as a candidate biomarker for IPAH. Western blot analysis demonstrated that C7 was increased in IPAH, when compared with control tissues. When analysed quantitatively, the protein expression determined by western blot analysis was not statistically significant (P value - 0.28) (figure 3). When compared with the proteomics result (the protein abundance as calculated from LC-MS/MS data), there was no correlation observed (figure 4). This explains why there was no difference observed between IPAH patients and controls.
Analysis of the Peptide list for Complement component 7
Further studies were carried out by examining the peptide lists of C7 protein. Three peptides unique to complement component 7 protein were found (table 1) and the peptide abundance for each peptide were plotted against the average densitometry (arbitrary units: AU) of western blot for each patient and control, no correlation was observed. The identity of the peptides unique to complement component 7 were determined by the generation of SEQUEST results files using BioWorks Browser (version3.2; Thermo Electron Corporation, Waltham, USA). To analyse this further, the peptide abundance for individual peptides of patients and controls were plotted against each other to observe any existing correlation. There was also no correlation observed (figure 4).
The search for biomarkers in IPAH has been carried out in recent years using several technologies. At the moment, there are a few biomarkers that have been shown to be associated with IPAH. Proteomics is the complementary technology to genomics and can be used to analyze body fluids such as plasma, urine and cerebrospinal fluid. It includes a broad range of technologies aimed at determining the identity and quantity of expressed proteins in cells and their three dimensional structure (Cho, 2007). Proteomic researches of lung or plasma samples from IPAH patients have led to the identification of prognostic and diagnostic biomarkers, including serum uric acid, von Willebrand factor, brain natriuretic peptide, troponin T and endothelin-1, which could provide a basis of developing new methods for early diagnosis and detection of IPAH (Heresi and Dweik, 2010).
In a previous study (Umukoro, 2010), comparative proteomics analysis was used to compare plasma samples from IPAH patients and healthy control using the liquid chromatography MS tandem (LC/MS-MS) method. This method of approach enables reliable identification of plasma proteins with a high dynamic range of more than three orders of magnitude (Levin et al., 2007). A total of 10 differently expressed proteins were identified of which seven were upregulated and three downregulated. The upregulated proteins includes von Willebrand factor, complement component 7, fibrinogen alpha polypeptide, fibrinogen gamma chain, fibrinogen beta chain, haptoglobin related protein and haptoglobin and are associated with blood coagulation and immunological and acute inflammatory response. The downregulated proteins include betaglobin, transthyretin and trafficking protein kinesin binding 2 and are associated with iron homeostasis and intracellular transport. Complement component 7 was chosen for validation because fibrinogen and haptoglobin, were depleted during the plasma sample preparation using the IgY-12 kit (Beckman Coulter, ?) and von Willebrand factor has been known already with supporting evidence to have a possible role in the prognosis and follow up of patients with pulmonary arterial hypertension (Heresi and Dweik, 2010).
In this study, the observation for complement component 7 was investigated further using an independent technique to measure the plasma levels. Hence, western blot analysis was used to assess the levels of complement component 7 in the plasma of IPAH patients and healthy controls. The results showed that the mean plasma levels of complement component 7 in IPAH patients was not significantly higher (P value = 0.28, Student's t-test) than healthy controls and did not correlate with the proteomics (LC-MS/MS) results. When analysed further, the mean plasma levels of three peptides unique to complement component 7, did not also correlate with the proteomics (LC-MS/MS) results. Possible explanations for this result have been considered.
The proteomics (LC-MS/MS) technique showed some inconsistencies with complement component 7. When individual peptides were plotted to investigate for correlations, no correlation was observed. To investigate this further, when the individual peptides for both IPAH patients and controls of von Willebrand factor were plotted, correlation was observed.
The western blot analysis method is less quantitative and other more quantitative methods such as ELISA could have been used but there are no available ELISA kits for C7 and preparing one is time consuming. The arbitrary units measured from the western blot analysis could have not been calculated properly. The antibodies specific to complement component 7 might be binding to complement component 7 and also to other protein. This may also lead to variations.
The plasma samples used for the proteomics technique were depleted while the plasma samples used for western blot analysis did not undergo immunodepletion. This was exemplified by C7 where a difference was seen with the depleted sample in the proteomics method but not with the neat samples. This may have introduced some differences in the observed results. The plasma sample size was too small. Increasing the samples size could be an option in getting a better result and thereby reducing any variations.
The increase in plasma levels of complement component 7 found using a proteomic approach in IPAH patients could not be confirmed by western blot analysis. This study demonstrated that proteomics (LC-MS/MS) is able to detect low-level plasma proteins despite the complexity of the sample.