human tsp 1 cdna  (Sino Biological)

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 1. (A) Schematic representation of the K14–TSP-1 transgene construct. A 3.6 kb human TSP-1 cDNA fragment was ligated to the BamHI restriction site of the keratin 14 promoter cassette. (B) Overexpression of TSP-1 mRNA in the dorsal and ventral skin of 3-week-old K14–TSP-1 transgenic mice was confirmed by northern blot analysis. TSP-1 mRNA expression was not increased above wild-type (WT) levels in the liver of TSP-1 transgenic mice. Hybridization with a murine β-actin probe served as a control for equal loading. (C) Western blot analysis of skin lysates demonstrates the presence of the intact, 180 kDa TSP-1 protein in TSP-1 transgenic skin. TSP-1, natural human TSP-1. (D) Strong TSP-1 mRNA expression levels in keratinocytes isolated from TSP-1 transgenic mice. TSP-1 expression levels were higher under low Ca2+ conditions than under high Ca2+ conditions that favor keratinocyte differentiation. (E) Western blot analysis confirmed efficient secretion of transgenic TSP-1 protein into culture supernatants and increased protein expression in cell lysates obtained from transgenic keratinocytes, as compared with wild-type (WT) controls.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Construct, Over Expression, Transgenic Assay, Northern Blot, Expressing, Hybridization, Western Blot, Isolation

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 2. (A and B) Twelve-day-old TSP-1 transgenic mice (right) were smaller than their wild-type littermates and showed a delayed first hair cycle. TSP-1 transgenic mice were phenotypically indistinguishable from their wild-type littermates after 4 weeks of age. Little TSP-1 mRNA expression was detected by in situ hybridization in wild-type skin (C and D), mainly restricted to follicular keratinocytes of the outer root sheath and to blood vessels. (E and F) Targeted overexpression of the K14–TSP-1 transgene in the basal keratinocyte layer (arrowheads) and in the outer root sheath keratinocytes of hair follicles (arrows) was confirmed by in situ hybridization. Bar: 250 µm. Giemsa staining of the tail skin of adult 7-week-old TSP-1 transgenic mice (H) showed no differences in skin structure as compared with wild-type mice (G). Bar: 250 µm.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Transgenic Assay, Expressing, In Situ Hybridization, Over Expression, Staining

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 3. Comparable branching pattern and structure of cutaneous blood vessels in adult wild-type mice (A) and TSP-1 transgenic mice (B), visualized in whole-mount preparations of mouse ears after perfusion with fluorescein-labeled L.esculentum lectin. Bar: 1 mm. (C) Microvascular permeability of skin vessels was not altered in TSP-1 transgenic mice (right panels) when compared with wild-type littermates (left panels) in a modified Miles assay. Comparable extravasation of intravenously injected Evans blue was observed after intradermal injection of recombinant VEGF (50 and 100 ng/ml). Injection of PBS served as a control for baseline permeability levels.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Transgenic Assay, Labeling, Permeability, Modification, Injection, Recombinant

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 4. Delayed closure of full-thickness wounds in TSP-1 transgenic mice as compared with wild-type (WT) littermates. (A) At 24 h after injury, wounds were covered by a dry scab, which remained adherent until day 10 in TSP-1 transgenic mice and until day 5–7 in control mice. Bar: 6 mm. (B) A >50% reduction in wound area was observed 3 days after wounding in wild-type mice (n = 30) but only after 9 days in TSP-1 transgenic mice (n = 30) (p <0.001). Complete wound closure occurred after 8 days in wild-type mice and after 14 days in TSP-1 transgenic mice.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Transgenic Assay

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 5. Impaired granulation tissue formation in cutaneous wounds of TSP-1 transgenic mice. In wild-type mice, the wound bed was completely filled with granulation tissue 3 days after wounding (A), and was completely covered by newly formed epidermis after 7 days (C). In contrast, only minor granulation tissue infiltration from the wound margins was observed in TSP-1 transgenic mice after 3 days (B), and by day 7 the wound bed was still only partially filled with granulation tissue (D). Arrows indicate the front line of granulation tissue invading the wound bed. (A–D) Hematoxylin–eosin stain. Magnification: 96×. Bar: 250 µm. (E–H) In situ hybridization confirmed high levels of TSP-1 mRNA expression in transgenic epidermal keratinocytes at day 7 after wounding (F and H), as compared with low TSP-1 mRNA expression in wild-type mice (E and G). Dots indicate the epidermal–dermal junction. Magnification: 192×. Bar: 250 µm. (I and J) Immunofluorescence staining of TSP-1 in 7-day-old wounds reveals strong TSP-1 protein expression in the neoepidermis of TSP-1 transgenic mice (J), as compared with low levels of expression in wild-type wounds (I). Little or no staining of non-resident cells is visible. Vessels are depicted in red (CD31 stain). Magnification: 96×. Bar: 250 µm.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Transgenic Assay, Staining, In Situ Hybridization, Expressing, Immunofluorescence

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 6. Pronounced inhibition of vascular endothelial cell proliferation and neovascularization in granulation tissue of wounds from TSP-1 transgenic mice at 2 days (B) and 4 days (D) after injury, as compared with numerous proliferating endothelial cells in wild-type mice at day 2 (A) and day 4 (C). Immunofluorescence staining demonstrates CD31-stained blood vessels (red), BrdU-labeled proliferating non-endothelial cells (green) and CD31/BrdU double-stained proliferating vascular endothelial cells (yellow, arrows). Staining for CD31 demonstrated reduced neovascularization of 7-day-old granulation tissue in TSP-1 transgenic mice (F), as compared with wild-type mice (E). Bar: 250 µm. (G–I) Computer-assisted morphometric analysis of CD31-stained wound sections revealed a significantly (p <0.01) reduced microvascular density in granulation tissue of TSP-1 transgenic mice (G), as compared with wild-type (WT) controls. (H) Inhibition of vessel size increases through day 14 after injury in granulation tissue of TSP-1 transgenic mice. (I) A strong increase in the total area covered by blood vessels was detected in granulation tissue in wild-type mice, but not in TSP-1 transgenic mice (p <0.001). CD31-stained blood vessels were evaluated in granulation tissue in corresponding 10× fields of five different wounds per time point. Data are expressed as mean ± SEM. **p <0.01; ***p <0.001.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Inhibition, Transgenic Assay, Immunofluorescence, Staining, Labeling

Journal:

Article Title: Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

doi: 10.1093/emboj/19.13.3272

Figure Lengend Snippet: Fig. 7. (A) Dose-dependent inhibition of human dermal microvascular endothelial cell (HDMEC) proliferation by TSP-1 in the presence of 20 ng/ml VEGF. (B) Absence of fibroblast growth modulation by added TSP-1 (20 µg/ml), as compared with untreated controls. (C) TSP-1 dose-dependently inhibited in vitro migration of human dermal fibroblasts (p <0.001) towards a collagen type I matrix. (D) Comparable inhibition of fibroblast migration on collagen type I or fibronectin matrices. Transwell migration chamber assay; TSP-1 concentration: 20 µg/ml. Results are the mean ± SD of two independent experiments. **p <0.01; ***p <0.001.

Article Snippet: 32 P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for TSP-1 were detected with a 3.6 kb human TSP-1 cDNA probe, and a 2.0 kb human β-actin cDNA probe (Clontech, Palo Alto, CA) was used as a control for equal RNA loading.

Techniques: Inhibition, In Vitro, Migration, Boyden Chamber Assay, Concentration Assay