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A proteomic profile of synoviocyte lesions microdissected from formalin-fixed paraffin-embedded synovial tissues of rheumatoid arthritis.

Hayashi J, Kihara M, Kato H, Nishimura T - Clin Proteomics (2015)

Bottom Line: Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear.With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues.Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1.

View Article: PubMed Central - PubMed

Affiliation: Niizashiki Central General Hospital, Saitama, Japan.

ABSTRACT

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control.

Results: A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc.

Conclusions: Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein-protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10(-45)), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10(-5)). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.

No MeSH data available.


Related in: MedlinePlus

Examples of the laser microdissections (LMDs) of targeted lesions from a OA and b RA synovial tissues (1, before; 2, after) on the DIRECTOR® slides. The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest is carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube.
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Fig3: Examples of the laser microdissections (LMDs) of targeted lesions from a OA and b RA synovial tissues (1, before; 2, after) on the DIRECTOR® slides. The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest is carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube.

Mentions: Targeted synoviocyte lesions were identified on serial sections of synovial tissues stained with hematoxylin and eosin (HE). For proteomic analysis, a 10-μm thick section prepared from the same tissue block was attached onto DIRECTOR®slides (OncoPlexDx, Rockville, MD, USA), de-paraffinized twice with xylene for 5 min, rehydrated with graded ethanol solutions and distilled water, and stained by hematoxylin. Those slides were air-dried and subjected to laser microdissection with a Leica LMD6000 (Leica Micro-systems GmbH, Ernst-Leitz-Strasse, Wetzlar, Germany). The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest are carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube. At least 30,000 cells (ca. 8.0 mm2) were collected directly into a 1.5-mL low-binding plastic tube. Proteins were extracted and digested with trypsin using Liquid Tissue® MS Protein Prep kits (OncoPlexDx, Rockville, MD, USA) according to the manufacturer’s protocol. Targeted lesions were laser-microdissected from FFPE synovial tissues as exemplified in Fig. 3.Fig. 3


A proteomic profile of synoviocyte lesions microdissected from formalin-fixed paraffin-embedded synovial tissues of rheumatoid arthritis.

Hayashi J, Kihara M, Kato H, Nishimura T - Clin Proteomics (2015)

Examples of the laser microdissections (LMDs) of targeted lesions from a OA and b RA synovial tissues (1, before; 2, after) on the DIRECTOR® slides. The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest is carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4527102&req=5

Fig3: Examples of the laser microdissections (LMDs) of targeted lesions from a OA and b RA synovial tissues (1, before; 2, after) on the DIRECTOR® slides. The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest is carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube.
Mentions: Targeted synoviocyte lesions were identified on serial sections of synovial tissues stained with hematoxylin and eosin (HE). For proteomic analysis, a 10-μm thick section prepared from the same tissue block was attached onto DIRECTOR®slides (OncoPlexDx, Rockville, MD, USA), de-paraffinized twice with xylene for 5 min, rehydrated with graded ethanol solutions and distilled water, and stained by hematoxylin. Those slides were air-dried and subjected to laser microdissection with a Leica LMD6000 (Leica Micro-systems GmbH, Ernst-Leitz-Strasse, Wetzlar, Germany). The DIRECTOR® slide is similar to a standard glass (uncharged) microscope slide, but has an energy transfer coating on one side of the slide. Tissue sections are mounted on top of the energy transfer coating, and when the slide is turned over, the tissue faces down under the microdissection system. Targeting cells or tissue areas of interest are carried out on computer display. The laser energy is converted to kinetic energy upon striking the coating, vaporizing it and instantly propelling selected tissue features into the collection tube. At least 30,000 cells (ca. 8.0 mm2) were collected directly into a 1.5-mL low-binding plastic tube. Proteins were extracted and digested with trypsin using Liquid Tissue® MS Protein Prep kits (OncoPlexDx, Rockville, MD, USA) according to the manufacturer’s protocol. Targeted lesions were laser-microdissected from FFPE synovial tissues as exemplified in Fig. 3.Fig. 3

Bottom Line: Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear.With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues.Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1.

View Article: PubMed Central - PubMed

Affiliation: Niizashiki Central General Hospital, Saitama, Japan.

ABSTRACT

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control.

Results: A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc.

Conclusions: Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein-protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10(-45)), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10(-5)). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.

No MeSH data available.


Related in: MedlinePlus