Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: Fibroblasts synthesize abundant thrombospondin 2, whereas ECs do not produce detectable TSP2. (A) TSP2 mRNA (top) is lacking in lung ECs from wild-type (lane 1) and TSP2-null (lane 2) mice and in fibroblasts from TSP2-null mice (lane 4), whereas TSP2 mRNA is highly expressed in lung fibroblasts from wild-type mice (lane 3). Murine S6 mRNA (bottom) is detected in all samples. (B) TSP2 mRNA (top) is present in human fibroblasts isolated from foreskin (lanes 1 and 2), periodontal ligament (lane 3), and gingiva (lane 4), but is only faintly detectable in HMVECs (lane 5). Human S6 mRNA (bottom) is detected in all samples. (C) Western blot analysis, performed under nonreducing (lanes 1–3) and reducing (lanes 4–6) conditions, of TSP2 in conditioned media from lung ECs from wild-type Immortomice (lanes 1 and 4), and fibroblasts isolated from dermis of TSP2-null (lanes 2 and 5) and wild-type (lanes 3 and 6) Immortomice. Each lane contains an equivalent volume of conditioned media obtained from confluent cultures. Arrows indicate unreduced, trimeric, and monomeric murine TSP2. (D) Western blot analysis performed under nonreducing (lanes 1–4) and reducing (lanes 5–8) conditions of TSP2 in conditioned media (6 μg of protein/lane in lanes 4 and 8, and 12 μg of protein/lane in the remaining lanes) from human periodontal ligament (lanes 1 and 5), two separate preparations of human foreskin fibroblasts (lanes 2, 3, 6, and 7), and HMVECs (lanes 4 and 8). Arrows, as described in C.

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Isolation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: Recombinant TSP2 inhibits growth of ECs mediated by multiple mitogens. (A) HMVEC (1 × 10 5 cells/well in six-well plates) were incubated in EBM2 containing 5% FCS supplemented with VEGF (V) or a mixture of bFGF, VEGF, and IGF-1 (FIE), in the presence or absence of recombinant mouse TSP1 or TSP2 (2 μg/ml) for 3 d, and cell number was quantified. TSP2 and TSP1 inhibited proliferation of HMVECs mediated by both growth factors, but did not affect cell number in medium with serum alone. ∗, p < 0.05 by two-tailed t test. Error bars represent SD of the mean (n = 3). The data are representative of three independent experiments. (B) HMVECs (6 × 10 4 cells/well) were incubated in medium with a mixture of bFGF, EGF, and IGF-1, in the presence or absence of recombinant mouse TSP2 (2 μg/ml). Cell number was determined 1, 3, and 5 d thereafter. At this concentration, TSP2 completely arrests proliferation. ∗, p < 0.05 by two-tailed t test for comparison of cells in the presence or absence of TSP2 at the same time point. Error bars represent SD of the mean (n = 3). The data are representative of two independent experiments. (C) HMVECs were incubated in EBM2 containing 5% FCS, bFGF, and IGF-1 as described above and the indicated concentration of TSP2 for 3 d, and cell growth was quantified colorimetrically. Absorbance for cells incubated in EBM2 containing 5% FCS in the absence of growth factors was subtracted. Recombinant mouse TSP2 inhibited proliferation of HMVECs mediated by bFGF and IGF-1 in a dose-dependent manner. ∗, p < 0.05 by two-tailed t test compared with proliferation in the absence of TSP2. (D) HMVECs and HUVECs were plated at equal densities and incubated in EBM2 containing 5% FCS and bFGF as described above in the presence or absence of 5 μg/ml TSP2. After 5 d, cell growth was determined colorimetrically. TSP2 inhibited growth of HMVECs but not HUVECs. ∗, p < 0.01 by two-tailed t test.

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Recombinant, Incubation, Two Tailed Test, Concentration Assay, Comparison

Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: TSP2 impairs G 1 /S phase progression in HMVECs. Sparsely plated HMVECs were incubated in basal medium (EBM2/5% FCS) alone or supplemented with a combination of bFGF, EGF and IGF-1, VEGF alone, or all growth factors combined, in the absence (top) or presence of 2.5 μg/ml recombinant mouse TSP2 (bottom) for 24 h. Cells were stained with propidium iodide and analyzed for DNA content ( x -axis) by FACS as described in MATERIALS AND METHODS. Note that in media with all combinations of growth factors, the proportion of cells in S phase and G 2 /M phase is reduced in cells incubated in the presence of TSP2. Data are representative of three independent experiments.

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Incubation, Recombinant, Staining

Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: TSP2 and TSP1 inhibit cell cycle progression equally in HMVECs. HMVECs were incubated in basal medium alone or supplemented with bFGF, EGF, IGF-1, and VEGF (GF), in the absence or presence of TSP1 or TSP2. Cells were analyzed for cell cycle distribution as described above. TSP1 and TSP2 have a similar ability to arrest HMVECs in the G 0 /G 1 phase of the cell cycle. ∗, p < 0.0005, compared with cell-cycle phase in the presence of growth factors alone, by a two-tailed t test (n = 3). Error bars represent SD of the mean. Data are representative of two independent experiments.

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Incubation, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: VEGF blocks TSP2-mediated impairment of HMVEC viability. (A) Sparsely plated HMVECs were incubated for 72 h in medium containing 5% serum alone, or supplemented with a mixture of bFGF, IGF-1, and EGF (FIE), or with VEGF alone (V), in the presence or absence of 2 μg/ml TSP2 or TSP1. Adherent and detached cells were collected and stained with trypan blue. The proportion of nonviable, trypan blue-positive cells was determined. Error bars represent SD of the mean (n = 3). ∗, p < 0.005 by two-tailed t test. TSP2 and TSP1 impair viability of HMVECs in the presence of bFGF, IGF-1, and EGF, but not in the presence of VEGF. (B) HMVECs plated and incubated with growth factors and TSP2 as described above for 7 h were analyzed for caspase activity. TSP2 induced caspase activity in basal media, but both VEGF and the combination of bFGF, IGF-1, and EGF blocked TSP2-mediated caspase activation. ∗, p < 0.05 by two-tailed t test (n = 3). (C) HMVECs plated and incubated in the presence or absence of TSP1, TSP2, or VEGF were analyzed for caspase activity. TSP1 and TSP2 induce activation of caspases to similar degrees, but VEGF blocks TSP1- and TSP2-mediated caspase activation. ∗, p < 0.05 by two-tailed t test (n = 3).

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Incubation, Staining, Two Tailed Test, Activity Assay, Activation Assay

Journal: Molecular Biology of the Cell

Article Title: Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

doi: 10.1091/mbc.E01-09-0066

Figure Lengend Snippet: Inhibition of caspases does not affect TSP2-mediated inhibition of DNA synthesis by HMVECs. Top, HMVECs were incubated in basal media alone or with growth factors (bFGF, IGF-1, EGF, and VEGF), in the presence or absence of TSP2 (2 μg/ml), and zVAD-fmk (100 μM) or an equivalent volume of DMSO, for 22 h, and pulsed in the same media containing [ 3 H]thymidine for 2 h. Incorporation of [ 3 H]thymidine into DNA was determined. ∗, p < 0.05; ∗∗, p < 0.005 by two-tailed t test. Bottom, HMVECs treated in an identical manner were assayed for total caspase activity 4 h after addition of indicated factors. ∗, p < 0.05 by two-tailed t test. Error bars represent the SD of the mean (n = 3).

Article Snippet: Human foreskin fibroblasts (a gift from David Morris, University of Washington, Seattle, WA) and human gingival and periodontal ligament fibroblasts (gifts from Dr. A.S. Narayanan, University of Washington) were propagated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HMVECs (BioWhittaker) and human umbilical vein endothelial cells (HUVECs) (provided by the cell culture core group of the Biology of the Artery Wall Program Project, University of Washington) were propagated in EGM2-MV in a 37°C incubator under 5% CO 2 , and used between passages 4 and 10.

Techniques: Inhibition, DNA Synthesis, Incubation, Two Tailed Test, Activity Assay