Currently, the severity of GOV caused by PH is critical for the prognosis of patients with decompensated liver cirrhosis. Most of the need lies in the detection of liver cirrhosis patients at risk of occurrence and the progression of PH, and early diagnosis and treatment may greatly contribute to enhancing the survival of such patients. Decompensated liver cirrhosis presents not only as GOV but also PHG. The severity of swelling and reddening of gastric mucosa(8), interstitial vascular ectasia, spindle cell proliferation and fibrohyalinosis(9) reflect the degree of PHG and are associated with severity of liver disease. Monitoring changes in the gastric mucosa proteome may have an advantage in the diagnosis of decompensated liver cirrhosis. However, targeted proteomics, which identifies promising biomarkers of disease activity and organ involvement, is rarely involved in non-invasive method for detection and diagnosis of liver cirrhosis induced PH. Our group devoted to the research of proteomics in liver disease(10, 11). To the best of our knowledge, no proteomic analysis of gastric mucosa has been reported for the discovery of biomarkers and to describe the involvement of related biological pathways in liver cirrhosis induced PH. In this study, we performed a comparative analysis of the gastric mucosa proteome to obtain insights into the quantitative assessment and prognosis in liver cirrhosis patients via LC-MS/MS and PRM. As a result, we found that 423 proteins were differentially expressed in gastric from PHG patients before and after endoscopic cyanoacrylate injection compared to that from normal controls. Due to the importance of pathogenesis and early diagnosis of PHG, we studied the characterization and function of some differentially expressed proteins in depth.
According to KEGG function classification analysis, focal adhesion and vascular smooth muscle contraction pathway are enriched in PHG. In the process of liver fibrogenesis, focal adhesion connects hepatic stellate cells (HSCs) and ECM, and provides a direct sensor to the integrity of interaction with the extracellular environment, causing cell adhesion and migration(12, 13). The patients with liver cirrhosis exhibit a progression of vascular smooth muscle contraction dysfunction, which presents as an increased intrahepatic vasoconstriction and reduced systemic vascular resistance(14). The progress of PHG is related to hemodynamic alterations in the gastric mucosa, presenting as hemangiectasis(15), abnormal formation of arteriovenous shunts and blood flow augmentation(16).
Among these DEPs, PDLIM4 is the only protein which alters among the three groups, indicating cytoskeleton organization and stress fiber formation(17) may relate to development of PHG. Moreover, 17 DEPs about liver diseases and gastrointestinal system were further chosen and validated by PRM. There DEPs are up-regulated in PHG patients and even more after endoscopic cyanoacrylate injection, indicating their expression level is consistent with severity of PHG. Among these DEPs, MYLK, TNC, ITGA1, FLNA, COL6A1, COL6A2 and COL6A3 are enriched in focal adhesion pathway. MYLK participate in smooth muscle contraction pathway at the same time. Activation of MYLK triggers gastrointestinal smooth muscle contraction with an increase of intracellular Ca2+ [Ca2+]I. Compared with antrum muscles, fundus muscles have a higher level of MYLK expression(18), which may contribute to the mechanism of gastric motility. In addition, MYLK also mediates cell-cell and cell-matric adhesion(19). TNC can promote arterial smooth muscle cells proliferation and ECM elements deposition after stimulation of inflammatory cytokines and growth factors in cerebral arteries(20) and pulmonary arteries(21) by enhancing focal adhesion kinase phosphorylation(22). These mechanisms may contribute to TNC mediated vascular remodeling(23, 24) and participate in the pathogenesis of subarachnoid hemorrhage and pulmonary arterial hypertension. FLNA couples cell cytoskeleton to ECM and regulates integrin alpha-1 (ITGA1), thus affects the function of collagen and ECM remodeling(25–28). With the development of liver fibrosis, FLNA can be activated in HSCs and may serve as a biomarker(29). Integrin modulates angiogenesis, regulates inflammation, and translates biliary injury into stimulation of matrix-producing mesenchymal cells in the process of fibrosis development(30). Thereinto, ITGA1 is a receptor for laminin and collagen, it is involved in cell adhesion and correlates with inflammatory activity and fibrosis(31). Collagen IV contributes to ECM structure and expresses extracellularly. It is structured by a heterotrimer of the alpha1(VI), alpha2(VI), and alpha3(VI) chains which are encoded by COL6A1, COL6A2, COL6A3, respectively(32).
Other validated proteins, though not enriched in the two pathways mentioned above, also play a role in the process of liver and gastrointestinal diseases. CPT1A and ACSL3, which are responsible for fatty acid oxidation, have been proved to stimulate gastric cancer and hepatocellular carcinoma cell growth and migration(33–35). MCAM (CD146) involves in actin cytoskeleton rearrangement, blood vessel endothelial cells remodeling and intercellular junction(36). CSRP2 expresses specifically in HSCs in liver and mediates the transdifferentiation of HSCs into myofibroblasts with the activation of TGF-β(37). Focal adhesion protein TGFB1I1 (also known as Hic5) is induced by TGF-βand regulates myofibroblast differentiation in liver(38). TNS1 is also localized to focal adhesions and acts as a bridge linking ECM and the actin cytoskeleton(39). EFEMP1 and FBLN5 are ECM proteins and highly express in portal fibroblasts, they have been proved to play a role in progressive liver fibrosis(40, 41). SORBS1 involves in cytoskeleton organization and insulin signaling pathway in human hepatoma cell line(42). PDLIM7 is composed of PDZ and LIM7 domain, the LIM7 domain is associated with progression of liver fibrosis in hepatitis C virus infected patients(43). Although the association with PHG is not quite clear, this study identified these DEPs as potential biomarkers on gastric mucosa for liver cirrhosis induced PH. However, more studies are required to make sure their availability as predictive biomarkers and diagnostic targets.