Human metabolism crucially depends on adequate levels of TH for its optimum function. These occur via its central genomic as well as its peripheral, non-genomic actions at the level of the target tissues and cells, including cells of the circulatory and vascular systems. It is well known that THs are involved in modulation, secretion, and degradation of a multitude of plasma proteins. In hyperthyroidism, the latter process is assumed to be predominant owing to the presence of a hyper-metabolic catabolic state characterized by increased resting energy expenditure, lipolysis, gluconeogenesis, and weight loss, as well as reduced cholesterol levels [7
In the present study, we characterized the differences between the plasma proteome of patients during the hyperthyroid state and after becoming euthyroid, following treatment with antithyroid medication, using an untargeted 2D-DIGE proteomics approach in combination with bioinformatic network analysis. We identified significant differences in the quantitative expression level of 20 protein spots, corresponding to 16 unique proteins in between them (Table 2
The proteins identified with a significant increase in abundance between the hyperthyroid compared with euthyroid state were A1BG, HP, HPX, APOL1, C6, CLU, CPN1, FGG, IGHA1, and LRG1, while those with a significantly lower abundance were APOA1, SERPINA1, PLG, FGB, ITIH4, and C1R. These identified proteins are multifunctional in nature and are known to be involved in regulating multiple biochemical and metabolic pathways, individually or in conjunction with the other identified proteins. They are known to function as transport proteins, enzymes, regulators of metabolism, including blood haemeostasis, inflammation, and immunological processes. We broadly grouped these proteins based on their involvement in their major related biochemical pathways as those involved in (i) lipid and lipoprotein metabolism, (ii) the acute phase response (APR), and (iii) the vascular homeostasis and coagulation for ease.
3.1. Differential Regulation of Proteins Involved in Lipid Metabolism
Hyperthyroidism is associated with a decrease in the levels of total cholesterol, triglycerides, HDL, LDL, and apolipoproteins [12
]. We found that six of the differentially abundant proteins identified in our study (APOL1, CLU, and HPX; as well as APOA1, PLG, and SERPINA1) are involved in the regulation of lipid and lipoprotein metabolism. APOA1 is known to participate in regulation of lipid and fatty acid metabolism independently and along with PLG, CLU, and APOL1 in reverse cholesterol transport; uptake; or with PLG, CLU, and SERPINA1 in lipid synthesis and release.
A decrease in the abundances of spots related to APOA1, the primary apolipoprotein of HDL, was noted in the hyperthyroid state, in line with the findings of Muls et al., who also demonstrated that the same [13
] APOA1 is known to facilitate the HDL-mediated diffusion of TH through the cellular and intranuclear membrane for its genomic actions [14
]. It is tempting to speculate that decreased APOA1 levels along with a concurrent decrease in HDL levels, as commonly seen in patients with hyperthyroidism, represent a regulatory mechanism aimed at preventing an excess in flow of both intracellular and intranuclear TH concentrations [14
]. Moreover, APOA1, in association with PLG, acts as a key determinant of the cholesterol efflux capacity, a major process involved in reverse cholesterol transport (RCT) [15
]. Decreased levels of both these proteins indicate that there is a decrease in RCT pathway in the hyperthroid compared with the euthyroid state, which predisposes the former to an increased risk of atherosclerosis and cardiovascular disease. The difference in the abundance of APOA1 between hyperthyroid and euthyroid states was independently confirmed using immunoblot analysis.
In addition to APOA1, protein spots relating to APOL1, a minor HDL3-associated apolipoprotein involved in lipid transport and metabolism, apoptosis, autophagic cell death, and cell lysis by membrane pore formation [16
], were found to have significantly increased abundance in the hyperthyroid compared with the euthyroid state. Increased levels of APOL1 are known to be positively associated with hyperglycemia and plasma triglycerides in patients with coronary artery disease having low HDL, and are a potential factor for premature cardiovascular disease [17
]. The increased levels of circulating plasma APOL1 have not been reported in relation to increased TH levels to date and need to be investigated in future studies.
We also observed an increase in the abundance of spot intensities relating to CLU (Apo J), another apolipoprotein, in the hyperthyroid state compared with the euthyroid. CLU is a secreted, multifunctional chaperone glycoprotein associated with HDL that, along with APOA1, helps in the RCT pathway. Lower CLU levels are known to be associated with insulin resistance, obesity, and dyslipoproteinemia, while higher levels are associated with coronary artery disease [18
]. The concerted action of these proteins in regulation of HDL emphasizes the importance of alteration in HDL metabolism in the hyperthyroid state.
3.2. Differential Regulation of Proteins Involved in the Acute Phase Immune Response
In the present study, we also found a significant differential abundance in twelve of the identified proteins that function as acute phase proteins (APPs) and are involved in the regulation of acute phase response. Seven of them (HP, HPX, CLU, APOL1, FGG, A1BG, and LRG1) showed a significant increase in abundance, while another six showed a significant decrease in abundance (APOA1, PLG, SERPINA1, ITIH4, FGB, and C1R) in patients with hyperthyroidism before and after treatment.
Hyperthyroidism is characterized by increased levels of THs, of which T3 is known to increase the hepatic APPs [20
]. APPs comprise a large, heterogeneous group of proteins and polypeptides whose concentrations increase during inflammatory states, largely in response to inflammation-associated cytokines. This relationship between these proteins and the pro-inflammatory cytokine was highlighted in the network pathway, which is deregulated in the hyperthyroid state. Classically, APPs are classified either as positive or negative APP based on their increased or decreased concentrations during inflammation. Recent studies have shown that the concentrations of specific APPs do not necessarily follow the same rule, and their varying concentrations are characteristic, reflecting the different disease conditions [11
]. Increased TH levels have been shown to increase the concentration of APPs, including HP, A1BG, and fibrinogen and its end products, indicating an inflammatory state [21
We observed an increase in spot intensities related to HP in the plasma of patients between hyperthyroid compared with the euthyroid state. HP is a positive APP that is known to scavenge free hemoglobin, prevent development of oxidative stress, and selectively bind APOA1. An increase in HP abundance, in the hyperthyroid state, may serve as an anitoxidant and to protect the diminishing APOA1 from further oxidative damage and loss of function [22
]. In addition to these, HP functions as a regulator for vascular homeostasis and angiogenesis, and in regulating inflammation as previously described [11
We also noted a decrease in abundance for the protein spot intensities relating to ITIH4 in the hyperthyroid state compared with the euthyroid. ITIH4 belongs to a group of IL-6-regulated serine protease inhibitors and is a known positive APP. It has been suggested to play a role in stabilization of extracellular matrix by binding to hyaluronic acid, may be involved in modulating inflammation and inhibit apoptosis. Previous studies have identified ITIH4 as a potential diagnostic and prognostic marker for a number of diseases that include acute ischemic stroke, ovarian cancer, interstitial cystitis, liver fibrosis [23
] and its increased levels were seen in cases of thyroid cancer [24
]. The decrease in the abundance of ITIH4 in our study was confirmed by immunoblotting.
Another serine protease inhibitor and a positive APP, SERPINA1, was identified with a decreased abundance in the plasma of hyperthyroid patients before and after treatment. SERPINA1 is known to possess the ability to not only inhibit serine proteases, such as collagenase, elastase, and proteinase-3, but also acts as an antioxidant and exerts anti inflammatory tissue-protective effects independent of protease inhibition. Decreased levels of SERPINA1 in hyperthyroid state may indicate a decrease in the inhibitory activity of the enzyme on the proteases and an increased inflammatory state [25
]. Alternatively, elevated levels of SERPINA1 have been observed in inflammatory diseases and different types of malignancies, including thyroid cancer [26
In addition to these proteins, circulating extracellular CLU, identified in our study with increased abundance, also acts as an anti-inflammatory agent. Its overexpression is known to attenuate the expression of proinflammatory chemokines, cell adhesion molecules, and matrix-degrading endopeptidases stimulated by the NF-κB signaling pathway [28
], and its levels are elevated with oxidative stress and metabolic disease associated with systemic inflammation [29
] similar to that seen in the hyperthyroid state.
Aside from the known APPs, we also identified C1R, a complement protein known to regulate the inflammatory process. C1R is a modular serine protease, a subunit of the complement initiating complex C1 and regulator of C1 activity and the complement cascade. The complement system not only acts as a rapid and efficient immune surveillance system, but is also involved in coagulation and the inflammatory process [31
]. A decreased level of C1R suggests that increased TH levels predispose the patients with hyperthyroidism to a chronic inflammatory state [33
3.3. Differential Regulation of Proteins Involved in Vascular Homeostasis and Coagulation
In the present study, we also found a significant differential abundance of proteins that influence vascular homeostasis, angiogenesis (SERPINA1, HP, and HPX), and the coagulation pathways (APOA1, PLG, SERPINA1, FGG, FGB, and IGHA1). It is known that increased circulating levels of TH, as seen in hyperthyroidism, impact proteins involved in the coagulation process, increasing the tendency for thrombosis accompanied by a parallel decrease in fibrinolysis [34
]. Earlier studies have demonstrated that subclinical and iatrogenic hyperthyroidism are clinically associated with myocardial infarction, recurrent pulmonary embolism, and atrial fibrillation [35
Recent studies indicate that endothelial cell dysfunction is an important pathogenic feature of hyperthyroidism that causes it to be associated with increased arterial stiffness (arteriosclerosis) [36
]. The decrease in abundance of SERPINA1 (alpha 1 antitrypsin) identified in our study may also contribute to this pathology. The decrease levels of the enzyme lead to its decreased capacity to regulate neutrophil elastases, causing increased elastin degradation and increased collagen deposition in the endothelial cells, inducing arterial stiffness. This in turn creates a higher risk for developing arteriosclerosis [37
], development of a more prothrombotic, procoagulant state leading to an increased risk of cardiovascular complications.
Another protein participating in the coagulation cascade and identified with decreased abundance in our study is PLG, an inactive precursor of plasmin found normally stored in platelets. Physiological concentrations of TH are known to act on platelets through their non-genomic mechanisms by binding to the peripheral receptors, resulting in degranulation, aggregation, and clot formation [38
]. Activated plasmin is involved in clot lysis, while decreases in PLG levels are associated with thrombotic complications. The decrease in abundance of PLG detected in our proteomic study indicates the presence of a hypofibrinolytic state with an increased fibrin deposition and increased coagulation. Substantial clinical evidence indicates that TH modulates the complex process of blood coagulation and increases the risk of pathologic intravascular coagulation at different sites, including the brain (cerebral venous thrombosis) [39
In addition to PLG, we also detected a significant differential abundance in two chains of fibrinogen glycoprotein, FGB and FGG, that is involved in coagulation cascade and tissue repair. Previous studies have shown a strong association between subclinical as well as overt hyperthyroidism with increased levels of fibrinogen, which shifts the haemostatic balance towards a hypercoagulable and hypofibrinolytic state. We found a decrease in the abundance of protein spot intensities relating to FGB, while an increase in abundance was noted for FGG. Marongiu et al. reported significantly increased plasma levels of fibrinogen, and specifically the Bβ chains in hyperthyroid patients that correlated with an increased risk of thromboembolism [40
]. Lancellotti et al. on the other hand reported that the FGG chain reduces thrombin-induced platelet aggregation and is antithrombotic [41
]. The differential regulation of these chains, decreased FGB, and increased FGG in our group of patients might point to compensatory regulation of the fibrinogen chains to reduce thrombotic events in our patients. Additional mechanistic studies need to be carried out to explore the molecular mechanisms and implications detail.
3.4. Network Pathway Analysis of the Significant Differentially Abundant Proteins
The bioinformatics and network pathway analysis was carried out using IPA to demonstrate the biological significance of the proteins and relate them to known pathways. The proteins identified in our study converged on two central nodes with highest connectivity; the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Both signaling pathways are known to be crucial for normal thyroid growth and function and for normal immune and inflammatory responses and are adversely affected in the case of thyroid autoimmune disease, thyroid cancer, and inflammation. NF-κB is a ubiquitous transcription factor involved in inflammatory and immune responses [42
], as well as in regulating the expression of genes related to cell survival, proliferation, and differentiation Previous studies have identified the involvement of NF-κB in thyroid-specific gene regulation, thyroid autoimmunity, thyroid cancer, and thyroid orbitopathy, and vice versa. Increased NF-κB activity has been previously demonstrated in patients with hyperthyroidism and in livers from rats treated with exogenous T3 [20
]. p38 MAPKs, on the other hand, are members of the MAPK family that are activated by a variety of environmental stresses and inflammatory cytokines. Involvement of the p38 MAPK signaling pathway suggests an increase in the non-genomic, rapidly acting thyroid signaling pathway that regulates cellular physiology, induces angiogenesis through hemodynamic effects, and promotes cell growth [44
In summary, our proteomics analysis of the plasma of hyperthyroidism patients revealed changes in the abundance of several proteins involved in maintaining both homeostatic and hemostatic processes. Increased levels of circulating TH alter the transport and composition of lipids, lipoproteins, acute phase response proteins, and proteins involved in coagulation. Changes in the levels of these proteins can have serious clinical implications owing to endothelial dysfunction, the development of cardiovascular disease, and the increased risk of venous thrombosis or pulmonary embolism. Therefore, biochemical measurement of the proteins identified in our study could serve as clinical markers for the early detection of side effects related to the development of serious clinical complications in patients with hyperthyroidism. Future mechanistic studies are required to fully understand the clinical implications associated with changes in the levels of these differentially abundant proteins.