Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: FFPE lung tissue sections from control and COPD donors were stained using immunohistochemistry for ECM and ECM-associated proteins with specific signals being detected with Nova red (red) and counterstained with hematoxylin (blue). Sections were scanned at 40x magnification using a digital slide scanner. Scale bar = 400um. Images are representative of protein detection patterns seen in control donors (n=18), SEO-COPD (n=12), and moderate COPD (n=14). ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Control, Staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Parenchyma was isolated and analyzed for percentage positive tissue area and mean intensity of positive pixels of expression for each protein. All 44 donors were available for analysis of COL1A1, COL6A2, FBLN2, LTBP4, LUM, DCN, VCAN, AND ELN, while for COL6A1, COL14A1, and FBLN5 43 donors were available. For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins present in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Airway walls were isolated and analyzed for percentage area and mean intensity of expression for each protein. The number of airway walls available for the analysis for each protein were COL1A1 (n= 155), COL6A1 (n=150), COL6A2 (n=149), COL14A1 (n=152), FBLN2 (n=158), FBLN5 (n=173), LTBP4 (n=163), LUM (n=158), DCN (n=156), VCAN (n=165), and ELN (n=158). For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM differences noted in the different analyzes throughout this study have been summarized here. In the staining and ECM signatures, red or blue arrows indicate higher or lower proportional levels or component scores in COPD respectively, while they denote positive or negative associations with FEV1 respectively. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; COL1A1: type I collagen α chain 1; COL6A1: type VI collagen α chain 1; COL6A2: type VI collagen α chain 2; COL14A1: type XIV collagen α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin; FEV1: forced expiratory volume in 1 second.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Staining, Binding Assay

Journal: EMBO Reports

Article Title: Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

doi: 10.15252/embr.202051913

Figure Lengend Snippet: A Box plot showing the quantitative protein expression results of nanoLC‐MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4 −/− compared to the Ndrg4 +/+ ENS cell secretome ( n = 4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann–Whitney U ‐test. B Data derived from the Linnarsson mouse database (http://mousebrain.org/) display the expression of Nid1 and Fbln2 within cells of the central, peripheral, and enteric nervous system. Unit color scale: Expression (log(UMI)); UMI, unique molecular identifier. Abbreviations: ENS, enteric nervous system (i.e., enteric neurons and glial cells); OL, oligodendrocytes; Exc N, excitatory neurons in CNS; Hippo, hippocampus; MB, midbrain; HB, hindbrain; Str, striatum; Hyp, hypothalamus; BG, basal ganglia; OB, olfactory bulb; CB, cerebellum; SC, spinal cord; PNS, peripheral nervous system; Glia, glial cells; and Vasc, vasculature. C–E Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS cells ((C), n = 3, black arrowheads, scale bars, 50 µm; (D, E), n = 3, scale bars, 10 µm).

Article Snippet: To assess the influence of the two identified extracellular matrix (ECM) proteins, Nidogen‐1 (NID1) and Fibulin‐2 (FBLN2), on the proliferation and migration capacities of the human CRC cell lines HCT116 and Caco‐2, a mixture of NID1 (2.0 μg/ml in PBS, 2570‐ND‐50, R&D systems) and FBLN2 (1.0 μg/ml in PBS, 9559‐FB‐050, R&D Systems) was added to the cell culture medium.

Techniques: Expressing, Tandem Mass Spectroscopy, MANN-WHITNEY, Derivative Assay, Immunohistochemistry, Immunofluorescence, Labeling

Journal: EMBO Reports

Article Title: Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

doi: 10.15252/embr.202051913

Figure Lengend Snippet: Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium ( n = 3). Scale bar, 50 µm. Addition of NID1&FBLN2 to the Matrigel® dome significantly enhanced the relative growth rate of HIOs after 5 days of culture as compared to the PBS control condition. Compared to the PBS control condition (white), stimulation with NID1&FBLN2 (black) enhanced the proliferation rate of HCT116 cells after 72 h, but did not affect Caco‐2 cell proliferation. The migration rate of HCT116 cells was already significantly increased 24 h after addition of NID1&FBLN2 (black), whereas the migration of Caco‐2 cells only significantly increased after 48 h of culture with NID1&FBLN2 (black). Box plot reflecting the quantitative nanoLC‐MS/MS analysis results shows the up‐regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome ( n = 17) compared to the normal colon tissue secretome ( n = 17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10‐transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range. Representative images of human colonic cancer epithelium ( n = 3) show a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm. Data information: Data in (B) are derived from three independent experiments and represented as relative mean percentage ± SEM versus day 0, with the P ‐value determined using a two‐tailed, unpaired t ‐test. Data in (C and D) are derived from three independent experiments and are represented as mean ± SEM, with P ‐values determined using a two‐tailed, unpaired t ‐test. To compare the paired normal colon and CRC secretome data in (E) ( n = 17), a paired Beta‐Binominal test, taking into account the sample origin (i.e., comparing protein signatures between tissues derived from the same patient), was performed (Pham & Jimenez, ; de Wit et al, ). Source data are available online for this figure.

Article Snippet: To assess the influence of the two identified extracellular matrix (ECM) proteins, Nidogen‐1 (NID1) and Fibulin‐2 (FBLN2), on the proliferation and migration capacities of the human CRC cell lines HCT116 and Caco‐2, a mixture of NID1 (2.0 μg/ml in PBS, 2570‐ND‐50, R&D systems) and FBLN2 (1.0 μg/ml in PBS, 9559‐FB‐050, R&D Systems) was added to the cell culture medium.

Techniques: Control, Migration, Tandem Mass Spectroscopy, Transformation Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: LTBP-2 competes with tropoelastin for binding to fibulin-5 and heparin, and is a negative modulator of elastinogenesis.

doi: 10.1016/j.matbio.2013.10.007

Figure Lengend Snippet: Fig. 5. Confocal co-localisation of LTBP-2 with fibulin-5, fibrillin-1, fibronectin and heparan sulfate proteoglycans in fibroblast matrix. Human skin fibroblast cultures were cultured for 21 days and then stained for confocal microscopy with antibodies to LTBP-2 in combination with antibodies to fibulin-5, fibrillin-1, fibronectin, perlecan or syndecan-4. Primary antibody was detected using an appropriate secondary antibody conjugated to fluorophore Alexa488 (red) or Cy3 (green). (see Experimental section). A,D,G,J, and M, rabbit anti-[LTBP-2 peptide] antibody 3504 [16 μg/ml]; B, mouse anti-fibrillin-1 antibody MAB1919 [5 μg/ml]; C = A and B merged; E, mouse anti-fibulin-5 antibody MAB3095 [25 μg/ml]; F = D and E merged; H, mouse anti-[cellular fibronectin] antibody ab6328 [10 μg/ml]; I = G and H merged; K, mouse anti-perlecan antibody [2.5 mg/ml]; L = J and K merged; N, mouse syndecan-4 antibody [4 μg/ml]; O = M and N merged; P, rabbit IgG [15.6 μg/ml] control for A,D,G,J and M; Q, mouse IgG (25 μg/ml) control for B,E,H,K and N; R = P and Q merged. Bar = 200 μM.

Article Snippet: Anti-fibulin5 (DANCE) antibody MAB3095 was purchased from R and D systems Inc., Minneapolis, MN, USA.

Techniques: Cell Culture, Staining, Confocal Microscopy, Control