Kirsten O. Hannesson

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (Salmo salar L.) individuals displaying spinal fusions

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.

Type of carbohydrate in feed affects the expression of small leucine-rich proteoglycans (SLRPs), glycosaminoglycans (GAGs) and interleukins in skeletal muscle of Atlantic cod (Gadus morhua L.)

Aquaculture requires feed that ensures rapid growth and healthy fish. Higher inclusion of plant ingredients is desirable, as marine resources are limited. In this study we investigated the effects of higher starch inclusion in feed on muscular extracellular matrix and interleukin expression in farmed cod. Starch was replaced by complex fibers in the low-starch diet to keep total carbohydrate inclusion similar. Blood glucose and fructosamine levels were elevated in the high-starch group. The group fed a high-starch diet showed up-regulation on mRNA level of proteoglycans biglycan and decorin. ELISA confirmed the real-time PCR results on protein level for biglycan and also showed increase of lumican. For decorin the protein levels were decreased in the high-starch group, in contrast to real-time PCR results. Disaccharide analyses using HPLC showed reduction of glycosaminoglycans. Further, there was up-regulation of interleukin-1β and -10 on mRNA level in muscle. This study shows that the muscular extracellular matrix composition is affected by diet, and that a high-starch diet results in increased expression of pro-inflammatory genes similar to diabetes in humans.

Small leucine-rich proteoglycans in the vertebrae of Atlantic salmon Salmo salar

We analysed the distribution and expression of the small leucine-rich proteoglycans (SLRPs) decorin, biglycan and lumican in vertebral columns of Atlantic salmon Salmo salar L. with and without radiographically detectable deformities. Vertebral deformities are a reoccurring problem in salmon and other intensively farmed species, and an understanding of the components involved in the pathologic development of the vertebrae is important in order to find adequate solutions to this problem. Using immunohistology and light microscopy, we found that in non-deformed vertebrae biglycan, lumican and decorin were all expressed in osteoblasts at the vertebral growth zones and at the ossification front of the chondrocytic arches. Hence, the SLRPs are expressed in regions where intramembranous and endochondral ossification take place. In addition, mRNA expression of biglycan, decorin and lumican was demonstrated in a primary osteoblast culture established from Atlantic salmon, supporting the in vivo findings. Transcription of the SLRPs increased during differentiation of the osteoblasts in vitro and where lumican mRNA expression increased later in the differentiation compared with decorin and biglycan. Intriguingly, in vertebral fusions, biglycan, decorin and lumican protein expression was extended to trans-differentiating cells at the border between arch centra and osteoblast growth zones. In addition, mRNA expression of biglycan, decorin and lumican differed between non-deformed and fused vertebrae, as shown by quantitative PCR (qPCR). Western blotting revealed an additional band of biglycan in fused vertebrae which had a higher molecular weight than in non-deformed vertebrae. Fourier-transform infrared (FTIR) spectroscopy revealed more spectral focality in the endplates of vertebral fusions and significantly more non-reducible collagen crosslinks compared with non-deformed vertebrae, thus identifying differences in bone structure.