A peptide profile of amniotic fluid in a fetal lamb model of gastroschisis

To investigate roles of short peptides in gastroschisis (GS), we comprehensively analyzed peptides in amniotic fluid (AF), creating a fetal lamb model of GS. We created GS in 4 fetal lambs at 60 days of gestation. Three GS and 4 normal fetuses were delivered at term (145 days) by cesarean section, when AF samples were collected. Short peptides in the AF samples were detected and identified by mass spectrometry. One of the identified peptides was synthesized and it’s functions were investigated. In total, 77 peptide peaks were detected in the AF samples. Of these, 12 peptides showed significantly different intensity between the GS and control groups. Three of the 12 peptides were identified. One of the identified peptides with high intensity in the GS group was amino acids (AA) 135-185 of lamb annexin 7 (ANX7). A synthesized peptide for AA168-211 of human ANX7, which corresponded to AA135-185 of lamb ANX7, decreased anti-inflammatory cytokine secretion from mesothelial cells by an cytokine array study. We report a unique AF peptide profile in a GS model. One of the peptides increased in GS was suggested to possess pro-inflammatory potential. These peptides would be related to the pathophysiology of GS. Introduction Gastroschisis (GS) is a congenital disease characterized by an abdominal wall defect through which bowel protrudes. The defect is almost always located to the right of umbilicus [1]. The antenatal diagnosis of GS is usually made by ultrasonographic observation of bowel loops floating in amniotic fluid (AF). The frequency of GS has increased over the last few decades, increasing from 0.6/10,000 live births in 1980-1984 to 2.33/10,000 live births in 2000-2002 [2]. GS occurs sporadically, appearing to have no genetic links. The pathogenesis of GS remains unclear, although it is possibly associated with a rupture of the physiological umbilical hernia and the regression of the umbilical vein [3]. Recently, the prognosis of GS has been improved significantly by the use of a Silo during closure [4] and the ready availability of total parenteral nutrition [5]. However, GS associated with bowel edema, intrauterine growth retardation, oligohydramnios, and/or bowel atresia still has a poor prognosis [6]. In many infants with GS the protruding bowel is covered by a fibrinous “peel”. There may be ischemic changes and dilatation macroscopically. Histologically, there is thickening and edema of the muscularis mucosae and serosal layers [7]. There are several theories put forward to explain the bowel inflammation and the development of “peel” in GS, with exposure to AF being most commonly implicated [8]. The level of ferritin, a marker of chronic inflammation and the levels of interleukin (IL)-6 and IL8, proinflammatory cytokines, have been reported to be increased significantly in AF of patients with GS [7]. Many inflammatory cells such as macrophages are also detected in AF of patients with GS [7]. In this context, it would be of great interest to determine whether there are bioactive molecules in AF that may affect the protruding bowel. Possibilities include proteins like IL-6 and IL-8 as mentioned above. Even though protein components in AF have been comprehensively analyzed in humans in vivo [9], AF proteins from patients with GS has not been analyzed, to our knowledge. Another important type of molecule would be short peptides. Short peptides are thought to be generated by physiological cleavage of precursor proteins and by pathological degradation of various proteins. The former includes essential bioactive peptides like substance P [10] and defensin [11]. As examples of the pathological degradation of proteins, the level of C3f-des-arginine, a degraded product of C3 was reported to be increased in systemic sclerosis and C3f-des-arginine also enhances proliferation of vascular endothelial cells [12]. The level of AC13, a degraded peptide of apolipoprotein A, was reported to be increased in microscopic polyangiitis. Also, AC13 enhances production of IL-6 and IL-8 from endothelial cells [13]. However, such degradationmediated peptides have been poorly understood until now. To our Correspondence to: Tomohiro Kato, MD, PhD, Professor/Director, Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki, Kanagawa, 2168511, Japan; Tel: +81-44-977-8111 (ext. 3522); FAX: +81-44-976-7553; E-mail: [email protected]