Journal: PLoS ONE

Article Title: Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

doi: 10.1371/journal.pone.0106732

Figure Lengend Snippet: A: Immunofluorescent staining for collagen IV, XV and XVIII (green) and neutrophils (red) in WT and double mutant mice at day 1 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of neutrophils around these capillaries. Less neutrophil influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signals (in green). Nuclei are shown in blue. Scale bars 20 µm. B: Immunofluorescent staining for neutrophils (red) and nuclei (blue) in WT and double mutant sham operated mice at day 1 after I/R showed the absence of neurophils in renal tissues. Scale bars 20 µm. C: Number of neutrophils per HPF (High Power Field) at different timepoints after I/R. At day 1 after reperfusion double mutant mice showed a decreased number of neutrophils compared to WT mice (p<0.05). At day 5 significantly less neutrophils were observed in kidneys of Col15a1 −/− and Col18a1 −/− mice compared to WT (p<0.05) as well as in those of double mutant mice compared to WT (p<0.01). Data is presented as mean ± SEM. *: p<0.05, **: p<0.01.

Article Snippet: The entire N-terminal non-collagenous domain of short collagen XVIII comprising the amino acid residues 1–325 and including Tsp-1 sequence was amplified by PCR from murine Col18a1 cDNA(26) using the primers 5′-ctagata∧agcttctggggagatggcgcccaggtggcacctc-3′ (HindIII cleavage site) and 5′-tcattgt∧ctaga cta a tggtgatggtgatgatg ctttatcaagcc-3′ (XbaI cleavage site, Stop codon underlined, histidine tag in italics) and cloned into mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

doi: 10.1371/journal.pone.0106732

Figure Lengend Snippet: A: Immunofluorescent staining for collagen IV, XV and XVIII (green) and macrophages (red) in WT and double mutant mice at day 5 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of macrophage/monocytes around these capillaries. Less macrophage influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signal (in green). The nuclei are stained in blue. Scale bars 20 µm. B: Immunofluorescent staining for macrophages (red) and nuclei (blue) in WT and double mutant sham operated mice at day 5 after I/R showed no macrophage/monocyte in renal tissues. Scale bars 20 µm. C: Number of macrophage/monocytes per HPF (High Power Field) at different timepoints after I/R. At day 5 significantly less macrophage/monocytes were observed in kidneys of Col15a1 −/− ×Col18a1 −/− mice compared to WT (p<0.05). Data is presented as mean ± SEM. *: p<0.05.

Article Snippet: The entire N-terminal non-collagenous domain of short collagen XVIII comprising the amino acid residues 1–325 and including Tsp-1 sequence was amplified by PCR from murine Col18a1 cDNA(26) using the primers 5′-ctagata∧agcttctggggagatggcgcccaggtggcacctc-3′ (HindIII cleavage site) and 5′-tcattgt∧ctaga cta a tggtgatggtgatgatg ctttatcaagcc-3′ (XbaI cleavage site, Stop codon underlined, histidine tag in italics) and cloned into mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Staining, Mutagenesis

Journal: PLoS ONE

Article Title: Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

doi: 10.1371/journal.pone.0106732

Figure Lengend Snippet: A: Solid phase binding assay showed a dose dependent binding of L-selectin to full-length short collagen XVIII (Short-XVIII), and to its N terminal non-collagenous portion (Tsp1-C18) bearing HS side chains. The interaction of L-selectin with HS side chains on collagen XVIII is stronger when the HS side chains are longer. B: The interaction of MCP-1 with Short-XVIII and Tsp1-C18 bearing HS side chains in solid phase binding assay. MCP-1 interacts with collagen XVIII in a dose-dependent manner and the interaction is dependent on the length of HS side chains. Both assays were performed at least three times and the graphs show a representative experiment. PBS coated wells were used as negative controls (not shown).

Article Snippet: The entire N-terminal non-collagenous domain of short collagen XVIII comprising the amino acid residues 1–325 and including Tsp-1 sequence was amplified by PCR from murine Col18a1 cDNA(26) using the primers 5′-ctagata∧agcttctggggagatggcgcccaggtggcacctc-3′ (HindIII cleavage site) and 5′-tcattgt∧ctaga cta a tggtgatggtgatgatg ctttatcaagcc-3′ (XbaI cleavage site, Stop codon underlined, histidine tag in italics) and cloned into mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Basement Membrane Zone Collagens XV and XVIII/Proteoglycans Mediate Leukocyte Influx in Renal Ischemia/Reperfusion

doi: 10.1371/journal.pone.0106732

Figure Lengend Snippet: A: MCP-1 dose dependently increased the migration of monocytes over a porous membrane. Immobilization of heparin-albumin, mimicking an artificial BM HSPG, promotes monocyte transmigration. Spontaneous migration over albumin-coated membrane in the absence of MCP-1 was set as 1 and the other values were calculated accordingly. The error bars represent SEM. B: Transmigration of monocytes towards MCP-1 (10 ng/ml) was increased in the presence of heparin-albumin and collagen XVIII with long GAG chains. Heparin-albumin increased the monocyte migration significantly compared to albumin coated membrane (p<0.01). N-terminal fragment of short collagen XVIII with long GAG chains promoted transmigration significantly compared to albumin and N-terminal fragment without GAG chain (both p<0.05). Relative to albumin, also the full-length short collagen XVIII promoted MCP-1-induced monocyte transmigration to some extent (not significant). Data is calculated relative to migration over albumin-coated membrane towards 10 ng/ml MCP-1. The error bars represent SEM. *: p<0.05, **: p<0.01.

Article Snippet: The entire N-terminal non-collagenous domain of short collagen XVIII comprising the amino acid residues 1–325 and including Tsp-1 sequence was amplified by PCR from murine Col18a1 cDNA(26) using the primers 5′-ctagata∧agcttctggggagatggcgcccaggtggcacctc-3′ (HindIII cleavage site) and 5′-tcattgt∧ctaga cta a tggtgatggtgatgatg ctttatcaagcc-3′ (XbaI cleavage site, Stop codon underlined, histidine tag in italics) and cloned into mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).

Techniques: Migration, Transmigration Assay