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Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease with an unclear etiology. The pathogenesis of IPAH is still unknown. With proteomic techniques, profiling of the human plasma proteome becomes feasible in searching for disease related markers. A comparative proteomic analysis was used to identify differentially expressed proteins in plasma samples of 8 patients with IPAH and compared with 8 healthy subjects after depletion of high abundant proteins. 10 proteins showed significant changes in plasma of IPAH and healthy controls with a fold difference greater than 1.6. The results showed von willebrand factor, complement component 7 (C7), fibrinogen alpha polypeptide, fibrinogen gamma chain isoform, fibrinogen beta chain, haptoglobin related protein and haptoglobin isoform 1 were upregulated while betaglobin, transthyretin and trafficking protein, kinesin binding 2 were downregulated.
A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of normal biologic processes, pathologic processes or pharmacological responses to a therapeutic intervention. Biomarkers are essential tools in translational medicine. Biomarkers can assist in the treatment of patients, by enabling clinicians speculate about the outcome and response to treatment (prognostic biomarkers), who have apparent disease (screening biomarkers), those who are suspected to have disease (diagnostic biomarkers) and categorising disease severity (staging biomarkers). At present, diagnostic and prognostic cardiovascular biomarkers are available, but there are no widely accepted biomarkers for screening. In cardiovascular diseases, diagnostic biomarkers include troponin I and troponin T for myocardial infarction and brain natriuretic peptide for heart failure. These biomarkers also have prognostic value in patients with myocardial infraction and heart failure. Prognostic biomarkers might also have valuable roles as surrogate end points for clinical trials or therapy. An ideal biomarker should be specific and sensitive, predictive and able to bridge preclinical and clinical trials and be easily accessible, using a non invasive method. Not all biomarkers will prove useful in predicting outcomes, but even if they do not, such studies add to the increasing understanding of the pharmacology of the relevant drugs.
Pulmonary arterial hypertension (PAH) is composed of diseases characterized by progressive increase in pulmonary arterial pressure leading to right ventricular failure and eventually death. Idiopathic pulmonary arterial hypertension (IPAH) formerly known as primary pulmonary hypertension, is an idiopathic form of PAH, which has its primary abnormalities localised to the small arterioles (Chin and Rubin, 2008). The mean pulmonary artery pressure for normal adults at sea level is 12-16mmHg and pulmonary artery hypertension is clinically defined by a mean value >25mmHg at rest or 30mmHg during exercise (Strange et al., 2002). The estimated incidence of IPAH in the general population is 1-2 million cases per million people per year and it's twice as common in females as males. The life expectancy for adults with IPAH is less than 3 years from diagnosis (Runo and Loyd, 2003). IPAH can be present at any age with a mean age at diagnosis of 36 years, slightly higher in males than females (Gaine and Rubin, 1998).
The pathological features that are common to IPAH are abnormal pulmonary vasoconstriction, pulmonary vascular remodelling (alterations in pulmonary vascular structure) and thrombosis. These features contribute to pulmonary vascular resistance in IPAH (Humbert et al., 2008). Pulmonary vasoconstriction is reversible but pulmonary vascular remodelling is not. Pulmonary vascular remodelling occurs in all three layers of the blood vessels; the adventitia, the media and the intima. Vascular remodelling is characterised by the thickening of these layers and is due to hypertrophy (cell growth) and hyperplasia (proliferation) of fibroblasts, smooth muscle cells and endothelial cells. Neomuscularisation of non-muscular peripheral cells occurs (Jeffery and Wanstall, 2001). A decrease in the expression of endothelial NO synthase has also been identified in cases of idiopathic pulmonary hypertension, which may result in pulmonary vasoconstriction and endothelium dysfunction (Yu et al., 2007). Endothelium dysfunction has been known to play a major role in the pathogenesis of IPAH. It occurs when there is a difference between the forces that maintains normal vascular tone, vasodilators mainly prostacyclin and nitric oxide and vasoconstrictors produced by the pulmonary endothelium (Yildiz, 2009). Additional features observed in IPAH is the presence of plexiform and neointimal lesions. Plexiform lesions normally appear in small pulmonary artery branches and is made up of a dilated sac which contains a plexus of capillary-like channels separated by proliferating cells whilst neointimal lesions contains smooth muscle cells and extracellular matrix, which are located on the luminal side of the internal elastic lamina. Pulmonary vascular remodelling occurs in response to a wide variety of stimuli, physical (mechanical, stretch, shear stress) and chemical (hypoxia, vasoactive substance and growth factors) (Jeffery and Wanstall, 2001). Thrombosis is a process characterized by the interaction of endothelial cells with both soluble elements such as plasma coagulation proteins and cellular elements of blood such as platelets (Berger et al., 2009).
Mutations in the gene encoding the bone morphogenetic protein receptor type II gene (BMPRII), a member of the transforming growth factor β (TGF-β) family which plays a key role in the regulation of apoptosis and cell growth, have been identified as the main cause of familial and sporadic forms of idiopathic pulmonary hypertension (Machado et al., 2009).
The aetiology of IPAH remains unclear. There are different stimuli that may trigger IPAH and this includes appetite suppressants, cocaine, monocrotaline extracts, infections such as HIV and inflammatory disorders. In the IPAH, there is an imbalance between mediators for vasoconstriction and vasodilatation. It is likely that this imbalance plays a key role in the development of IPAH and thereby contributes to an increase in vascular resistance and vascular remodelling (Strange et al., 2002). A number of vasoconstriction mediators such as increased plasma serotonin, dysfunction in voltage gated potassium channel, increased blood concentrations of endothelin 1, abnormal expression of angiotensin-converting enzyme and impaired fibrinolysis may contribute to the pathogenesis of IPAH (Runo and Loyd, 2003). Increased vascular endothelial growth factor (VEGF) expression has been found to play a role in vascular remodelling. VEGF is an endothelial-cell-specific mitogen produced by macrophages and vascular smooth muscle, is known to increase the production of nitric oxide and prostacyclin, thereby leading to vasodilation (Lahm et al., 2007). Expression of platelet-derived growth factor (PDGF) may play a role in the progression of PAH by promoting proliferation and migration of pulmonary artery smooth muscle cells. PDGF is produced by many different cell types such as endothelial cells, smooth muscle cells and macrophages (Perros et al., 2008).
Patients with IPAH show dyspnoea, exertional chest pain, fatigue, syncope and peripheral oedema and significant exercise intolerance despite current treatments (Mainguy et al., 2009). The diagnosis of IPAH requires evaluation which includes pulmonary function testing and echocardiography (Chin and Rubin, 2008). Right heart catheterization is also used in the diagnosis of IPAH. The severity of IPAH has been assessed by 6 minute walk test (6MWT) and cardiopulmonary exercise testing (CPET). The 6MWT is simpler and cheaper and the most commonly used test to assess exercise capacity. The 6MWT is predictive of survival in IPAH and also correlates with WHO functional class severity, the baseline cardiac output and total pulmonary resistance. CPET measures gas exchange and has been shown to correlate with outcome of IPAH (Barst et al., 2004).
Standard therapies for the treatment of IPAH consists of the use of anticaogulants, prostacyclin analagoues and calcium channel blockers, while the newer thearpies for includes the use of endothelin-1 receptor antagonists, nitric oxide, phosphodiesterase-5-inhibitors and gene therapy (Runo and Loyd, 2003). The existing pharmacological treatment of IPAH is focussed at preventing or reversing vasoconstriction, thrombosis and vascular remodelling (Strange et al., 2002).
IPAH is incurable. Hence, there is need for available and robust biomarkers for IPAH that can be used for early diagnosis and assessing prognosis of the disease with novel surrogate markers for monitoring the efficacy and toxicity of drugs. Presently, there are no available diagnostic biomarkers for IPAH, though some advances has been made in the development of prognostic biomarkers, they include c reactive protein (Quarck et al., 2009), troponin T (Torbicki et al., 2003), big endothelin-1 (Rubens et al., 2001), uric acid (Nagaya et al., 1999) and brain natriuretic peptide (Nagaya et al., 2000).
Proteomics approaches used to characterize plasma proteins includes both traditional (two-dimensional polyacryamide gel electrophoresis (2D-PAGE) and new high throughput (mass spectrometry (MS), liquid chromatography, mass spectrometry (LC-MS/MS), surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF MS), matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) etc) techniques (Yildiz, 2009). With such proteomics techniques, profiling of the human plasma proteome has become more feasible in searching for disease related biomarkers (Anderson and Anderson, 2002). Recently, a specific serum protein expression pattern discriminates IPAH patients had been identified and some of the proteins were identified with LC-MS/MS. Some of the proteins identified such as alpha-1 antitrypsin and vitronectin, could be potential candidates for a role in the pathogenesis of IPAH but further analysis and functional examinations are needed in order to differentiate other candidates (Yu et al., 2007). It was also reported that the plasma levels of complement 4a des Arg were significantly higher in IPAH patients when compared with normal controls by SELDI-TOF MS (Abdul-Salam et al., 2006). Zhang et al showed that there was a significant difference in protein expression in the serum of patients with IPAH and normal subjects by MALDI-TOF-MS. They deduced that the proteins identified such as leucine-rich α-2-glycoprotein , RAF1 and complement component 3 may be helpful for the diagnosis of IPAH and leucine-rich α-2-glycoprotein could be a specific prognostic biomarker of IPAH (Zhang et al., 2009).
Proteomics is the complementary technology to genomics and can be used to analyze body fluids such as plasma, urine and cerebrospinal fluid. It encompasses a broad range of technologies aimed at determining the identity and quantity of expressed proteins in cells and their three dimensional structure. Proteomic studies have the potential to transform how we diagnose disease, assess risk, determine prognosis, and target therapeutic strategies among individuals with this life-threatening disease (Cho, 2007).
Plasma proteins provide a foundation for identification of candidate protein markers for disease diagnosis, prognosis and development of new therapeutic products (Shen et al., 2005). Human blood plasma is easy to obtain, standardized, a large range of dynamic state in property of plasma proteins (Li et al., 2009). However, the proteomic analysis of plasma samples represents a challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulin which tend to mask those of lower abundance, therefore preventing their detection and identification in proteomic studies. Thus, depletion of major proteins is one possible approach for enhancing detection sensitivity in plasma (Yu et al., 2007).
This study describes the proteomic analysis and changes in the expression of plasma proteins in patients with IPAH using a label free LC tandem MS method after depletion of the high abundant proteins in plasma.
Plasma samples and Patients
8 IPAH patients and 8 healthy subjects were included in the study (Table 2). The mean age of the patients and controls was 48.00 ± 15.71 years (range: 25-76 years) and 37.75 ± 10.55 (range: 24 - 57 years) respectively. The healthy subjects had their blood pressure measured and had no indication of any cardiopulmonary risk factors. The IgY-12 kit (Beckman Coulter) was used to remove 12 highly abundant proteins; albumin, IgG, transferrin, fibrinogen, IgA, α1, antitrypsin, haptoglobin, α1-acid glycoprotein, IgM, apolipoprotein A-I and apolipoprotein A-II.
SDS/PAGE and LC-MS/MS
To identify IPAH-specific proteins, plasma samples from 8 patients with IPAH and 8 healthy subjects were used. SDS/PAGE and LC-MS/MS were performed. Gels for each plasma samples were stained and each sample containing lane cut into a series of regions (R1-R10) based on the position of molecular weight markers and the distribution of proteins observed in the gel. Each gel was digested with trypsin, peptides extracted and dried. Extracted peptides were then separated by LC-MS/MS.
Raw data files were collected from R1-R10 and analysed using progenesis software. Progenesis software (Nonlinear Dynamics, Newcastle upon Tyne, UK) was used to measure the relative abundance (intensity) of peptides between the control and IPAH groups. Data were archsinh transformed and analysed by ANOVA. The identity of peptides was determined using SEQUEST (BioWorks Browser 3.2; Thermo Electron Corporation, Waltham, USA) and interrogating it against the NCBI human Refseq database. Protein identification results were filtered with the Xcorr >1 peptide, +2 charge, ANOVA (P<0.05) and fold difference > 1.6.
To investigate the differences in protein expression in plasma samples from patients with IPAH (n=8) and healthy controls (n=8) were subjected to SDS-PAGE and LC-MS/MS. Comparative proteomic analysis was performed on both IPAH and healthy control plasma samples. A total of 329 different plasma proteins were found on the basis of Xcorr (cross correlation score) >1 peptide, +2 charge, these included each protein and its fragments. Each protein was matched with the corresponding molecular weight on the regions R1-R10. A total of 140 different plasma proteins that were suitable for comparative analysis of the levels of expression between the IPAH and control groups were identified from these regions. Proteins of interest were selected based on the following boundaries; statistical significance (ANOVA) of P < 0.05 and a fold difference greater 1.6 (standard deviation of fold difference divided by 2). On this basis the levels of 10 proteins were found to be affected, of which 7 were higher and 3 were lower in the IPAH group (Table 1). To confirm these findings, a volcano plot was performed. -log 10 P-value was plotted against log 2 fold difference to identify the upregulated and downregulated proteins (Figure 1).
In this study, a label-free proteomics approach was used to define the protein profile in plasma samples from patients with IPAH and identify proteins that were differentially expressed when compared with control samples. Overall, from 140 proteins analysed, 10 proteins were found that varied in IPAH. Of these, 7 were upregulated and 3 were 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. The upregulated proteins could be associated with blood coagulation and immunological and acute inflammatory response. The downregulated proteins include betaglobin, transthyretin and trafficking protein, kinesin binding 2. The downregulated proteins are involved in iron homeostasis and intracellular transport.
Haptoglobin is one of the acute phase proteins and is synthesised in the liver. Plasma levels of haptoglobin increase significantly in pathologic state, for example, carcinoma, inflammation, infection and so on. Haptoglobin has been considered to have a role in iron homeostasis due to its anti-oxidant and anti-inflammatory properties, due to its ability to bind haemoglobin and heme respectively. Haptoglobin is also known to protect against reactive oxygen species and aids in wound repair by stimulating angiogenesis (Gabay and Kushner, 1999).
Fibrinogen is a large soluble glycoprotein synthesised in the liver. It is made up of three pairs of non-identical polypeptide chains linked together by disulphide bonds; alpha, beta and gamma. Fibrinogen affects plasma viscosity and induces reversible cell aggregation, thereby limiting blood fluidity. Fibrinogen plays a crucial role in a variety of physiopathological processes in the body such as inflammation, thrombogenesis and atherogenesis (Kamath and Lip, 2003).
Fibrinogen, an acute phase protein, is a maker for systemic inflammation. It can also influence wound healing and can cause endothelial-cell adhesion, spreading and proliferation (Gabay and Kushner, 1999). Fibrinogen, a substrate for thrombin, is the final product in the coagulation cascade and maintains homeostasis by promoting platelet aggregation. Fibrinogen is also in the final pathway of platelet aggregation. During platelet aggregation, fibrinogen binds to receptors on the platelet membrane; glycoprotein IIb-IIIa complex (Ko et al., 1997).
Fibrinogen is associated with atherosclerotic lesions, where it is converted to fibrin and fibrinogen degradation products. Fibrin binds to lipoprotein and sequesters more fibrinogen in the vascular intima and may also improve the accumulation of extracellular lipids in fibrous plaques. Both fibrinogen and its degradation products have been shown to cause endothelial damage and also stimulate smooth muscle cell migration and proliferation (Onohara et al., 2000). Increasing evidence from studies suggests that increased plasma levels of fibrinogen are associated with cardiovascular disorders such as coronary heart disease. This elevated plasma fibrinogen levels may promote hypercoagulant or prothrombotic state. In situ thrombosis is thought to have a role in the pathogenesis of IPAH.
Von Willebrand factor (vWf) is synthesized by endothelial cells and megakaryocytes. vWF is found in the endothelial basement membrane, where it is involved with the attachment of endothelial cells and platelets to the matrix. It is also found in plasma where it serves as a carrier for coagulation factor VIII, thereby protecting it from proteolysis. vWF and factor VIII circulates in the plasma as a non-covalent complex. In its activated state, factor VIII plays an important role as a cofactor in the activation of factor X. In physiological conditions, vWF is released from endothelial cells into the plasma and to the albuminal cell surface, where it binds to the subendothelium. After the realease of vWF into the plasma, endothelial cell derived vWF is converted to a series of multimers by cleaving activities present in granulocytes. In pathological conditions, stimulation of endothelial cell by different agents such as thrombin, fibrin, endotoxin, cytokines and components of the complement system, leads to a rapid release of vWF from storage granules into the circulation (Lopes and Maeda, 1998). vWF plays a role in platelet aggregation with binding sites for glycoproteins Ib and IIb/IIIa. vWF also binds to monomeric and fibrillar collagen type III and to heparin in the subendothelial matrix (Blann and McCollum, 1994). Abnormalities or lack of vWF results in the inherited bleeding disorders von willebrand disease and haemophilia A while an increase in the plasma concentrations of vWF has been associated with a moderately increased risk of thrombosis (arterial) (Martinelli, 2005).
vWF is the most useful marker for the assessment of endothelial cell injury. Endothelial cell injury and dysfunction have been found to play a major role in the pathology of pulmonary arterial hypertension. Kawat et al reported that increased levels of vWF at baseline and follow up are associated with worse survival in PAH patients. Levels of circulating vWF are increased following endothelial cell damage and may also increase during acute phase responses. As an acute phase reactant, elevated levels of PAH may simply reflect worse systemic inflammation. Higher levels may cause in situ thrombosis in PAH (Kawut et al., 2005). In pulmonary hypertension, quantitative and structural abnormalities in the multimeric structure of circulating vWF have been identified. Lopes and colleagues showed that patients with pulmonary hypertension who have abnormalities in circulating vWF have reduced 1 year survival. They also reported that biochemical biomarkers of endothelial dysfunction may have prognostic value in pulmonary hypertension (Lopes et al., 1998). Lopes and colleagues also showed that IPAH patients have higher vWF levels than patients with other aetiologies of PAH. However, these studies did not show statistically significant associations between vWF levels and survival in IPAH patients (Lopes et al., 2000) (Lopes and Maeda, 1998). Collados et al reported VWF as a prognostic marker in patients with IPAH (Collados et al., 1999). In this study, the level of vWF is downregulated in IPAH patients compared to controls, suggesting that it might be associated with endothelial dysfunction in IPAH.
Complement component 7 (C7) was upregulated in this study. The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. Complement activation via the classical, the lectin or the alternative pathway leads to the formation of the terminal complement components (TCC), both in the extracellular fluid and as the membrane attack complex (MAC) on the surface of target membranes. After cleavage of C5 by either the classical or alternative C5 convertase, the terminal complement components C6, C7, C8 and C9 are sequentially activated (figure 2) (Wurzner, 2000). C7 plays a vital role in the hydrophilic-amphiphlic transition of MAC due to the transient ability of C5b-7 to bind directly to the target cell membrane (Papanastasiou and Zarkadis, 2005).
The primary function of MAC is the destruction of invading organisms and also leads to the promotion of inflammation. MAC has the ability to insert itself into the phospholipids bilayer of the target cells as a sublytic complex. Another example of MAC among all other cytolytic and phagocytic systems of immune defence is the finding that genetic deficiencies of terminal complement proteins are often associated by recurrent and fatal Nesserial infections (Podack and Tschopp, 1984). The precise mechanism of complement-mediated cytotoxicity remains unresolved. There are two hypotheses; one hypothesis states that the polar domains of inserted complement proteins may cause distortion of the phospholipid bilayer thereby leading to leaky patches. The other alternative pore hypothesis proposes that the polar surfaces of the individual complement components come together to form a hydrophilic channel through the membrane (Barroso et al., 2006). The properties of TCC in a biological system include the stimulation of host cell protein biosynthesis of proinflammatory mediators and also the potential to induce procoagulant activity (Wurzner, 2000).
Reinartz and colleagues showed that C7 is a plasminogen- binding protein. Plasminogen is found in plasma and interstitial fluid and binds specifically and saturably to the C7 leading to plasminogen activation by tPA. However, when bound to plasmin, C7 was protected against inhibition by its natural inhibitor α2- anti-plasmin. They also looked at whether plasminogen interacts with C7 within the C5b-9 complex. Their results showed that even after the incorporation of C7 into the C5b-9 complex, C7 still retains plasminogen binding capability. In view of procoagulant sublytic MAC-induced activities, this interaction offers a link to the plasminogen activator system. The functional importance of these interactions under in vivo conditions remains unclear (Reinartz et al., 1995).
Bossi et al showed the expression of C7 on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function (Bossi et al., 2009). Langeggen et al showed that the functional active C7 is sythesised by the endothelium, extending previous studies which showed that the synthesis of endothelium of a functional terminal pathway but not of C7 in particular (Langeggen et al., 2000).
In this study, the levels of C7 were increased in the plasma samples from IPAH patients compared to healthy controls subjects. C7 concentration increases in response to inflammation. These results suggest that in vivo settings were in conditions with increased inflammation. Vascular pathology which leads to thrombosis is influenced by a number of known risk factors such as arterial injury, inflammation and activation of the immune response. Local pulmonary thrombosis, possibly initiated by microscopic endothelial surface coagulation, is often seen in patients with IPAH.
Transthyretin (TTR) also known as prealbumin is a homotetrameric protein of 55kDa. TTR belongs to a group of proteins such as albumin and thyroxine-binding globulin which binds and transports thyroid hormones in the blood. It is found mostly in the plasma and cerebrospinal fluid. It major sites of synthesis are the liver, choroid plexus, retinal pigment epithelium and pancreas. TTR in the plasma is synthesized and secreted by the liver. It transports about 15% of the total thyroxine (T4) and retinol through the formation of a complex with retinol binding protein (RBP) (Buxbaum and Reixach, 2009).
TTR has been established to have two clinical significance; one, its plasma concentration has been utilized as a marker of nutritional and inflammatory status in different conditions; two, it is associated with the amyloidoses, a group of disorders defined pathologically by the formation and aggregation of misfolded proteins which result in extracellular deposits that impair organ function (Buxbaum and Reixach, 2009). As a marker of nutritional status, its plasma levels have been proposed as sensitive biochemical parameters of subclinical protein malnutrition, because the levels of protein, adequacy and energy intakes are reflected in plasma levels. Diseases associated with acute-phase responses are affected by plasma levels of TTR. For example, liver activity is converted to acute-phase response proteins, resulting in a dramatic drop in visceral proteins, despite nutritional support. TTR also plays an important role in vitamin A homeostasis (Schweigert et al., 2004).
TTR has been established as a cryptic protease that cleaves apolipoprotein A-I (Apo A-I) in vitro. TTR plays an important role in the nervous system. It has been reported that TTR is involved in preventing A-Beta fibrillization by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity (Costa et al., 2008). Costa et al further confirmed that TTR could act as a protective molecule in AD and prompted A-Beta proteolysis, by TTR as a protective mechanism in Alzheimer's disease (AD) (Costa et al., 2008). Recently, it was reported that TTR has a protective role in transgenic murine models of Alzheimer's disease (Buxbaum et al., 2008) and that the absence of TTR leads to a distinct behavioural phenotype, even in the absence of a pathologic amyloid beta gene, which has not been fully characterized (Sousa et al., 2004). The relationship of these observations to human Alzheimer's disease has not yet been established.
TTR has been reported to influence spatial reference memory in young adult wild type mice (Sousa et al., 2007). Meistermann et al reported that transthyretin could act as a biomarker for gentamicin-induced nephrotoxicity in rat (Meistermann et al., 2006). Farias-Eisner et al reported that TTR could act a biomarker for the detection of endometrial cancer (Farias-Eisner et al., 2010). Some studies showed that TTR was over expressed in blood serum in lung squamous cell carcinoma when compared with normal lung. Li et al reported that the content of TTR in plasma greatly declined in the formation of lung cancer (Li et al., 2009). In this study, the levels of TTR were decreased in the plasma samples from IPAH patients compared to healthy controls subjects. A decrease in plasma may be pro-inflammatory. TTR inhibits the production of interleukin-1 by monocytes and endothelial cells (Gabay and Kushner, 1999).
Betaglobin, the beta subunit of hemoglobin was downregulated. Hemoglobin is the main oxygen carrying protein in the blood and the most abundant heme-protein in humans. Heme molecule consists of an iron atom that has a high affinity for oxygen (Belcher et al., 2009). The potential dysregulation of iron homeostasis is of interest as increasing data indicates that iron levels can have an important effect on human pulmonary vascular tone and response to hypoxia (Smith et al., 2008). PAH is associated with hemoglobinopathies such as sickle cell anemia. Hemolysis accompanying sickle cell anemia may lead to the destruction of bioactive nitric oxide by free haemoglobin or reactive oxygen species. As a result of the lack of available nitric oxide, an inflammatory and proliferative cascade may ensue with culmination in PAH. Accordingly, decreased nitric oxide bioavailability with development of pulmonary hypertension has been reported after haemolysis in a murine model of sickle cell disease (Chan and Loscalzo, 2008).
Trafficking protein, kinesin binding 2 (TRAK 2) also known as GRIF-1 (GABAA receptor interacting factor-1) was downregulated in this study. GRIF-1 was first identified from a yeast two-hybrid screen searching for GABAA receptor clustering and trafficking proteins in a rat's brain. GRIF1 belongs to the new identified family of coiled-coil proteins. GRIF-1 is protein that is involved in motor-dependent trafficking of proteins (Pozo and Stephenson, 2006). It may regulate endosome to lysosome trafficking of membrane cargo, including EGFR. However, the role of GRIF-1 in the development of IPAH remains unclear and need further investigation.
There are several limitations to the study. First, the plasma levels of haptoglobin, fibrinogen were removed but were still detected, suggesting that they were either charge isoforms or fragments of the same protein. Haptoglobin for example, may be different in IPAH patients, that is maybe cleaved at different regions or post modification. This also suggests that the high abundant proteins in plasma were not completely removed, thereby masking the presence of low abundant proteins. To remove multiple high abundant proteins in the prefractionation step, more efficient methods may be needed. This may also depend on the depletion efficiency. The depletion efficiency might be different for the proteins. Secondly, some low abundant proteins released into plasma from diseased tissues may not be detected.
In conclusion, this study showed that there are significant differences in the expression of proteins in the plasma of patients with IPAH and normal subjects. 10 proteins were identified by LC-MS/MS. Complement component 7 may be considered as a candidate for further investigation of pathophysiological mechanism, and as potentially therapeutic target for IPAH.