Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium - PubMed (original) (raw)
Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium
Natalya V Narizhneva et al. FASEB J. 2005 Jul.
Abstract
Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor alpha (TNF-alpha). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies.
Figures
Figure 1
A) SDS-PAGE analysis of TSP-1 purified from platelets and recombinant TSP-1 expressed in the baculovirus system: Aa) Coomassie staining (reducing conditions, 50 mM DTT); Ab) Western blot (nonreducing conditions). Expression of CAM on HUVEC. B) Time-dependence of ICAM-1 (■) and VCAM-1 (○) surface expression on TSP-1-treated cells. TSP-1 concentration was 3 μg/ml. C) Western blot shows the level of ICAM-1 in control-untreated HUVEC (lane 1) or in HUVEC after 17 h of treatment with rTSP-1 (lane 2) or TNF-α (lane 3). The cell lysate was subjected to Western blot using rabbit polyclonal antibodies against ICAM-1. β-actin was used as a loading control. Effect of various concentrations of TSP-1 on ICAM-1 (D), VCAM-1 (E), and E-selectin (F) expression on HUVEC. Cells were incubated in the presence of recombinant TSP-1 (D, open symbols; E and F, shaded bars) or TSP-1 purified from platelets (D, closed symbols; E and F, solid bars) for 4 or 17 h to assess the expression of E-selectin or ICAM-1 and VCAM-1, respectively. The surface expression of CAM was analyzed by flow cytometry, as described in Materials and Methods. The increases in MFI over control (PBS buffer) are shown (_P_≤0.008). The data are means ±
sd
of three to seven separate experiments.
Figure 2
Expression of CAM on HAEC and HMVEC. Cells were incubated for 17 h in the presence of 3 μg/ml TSP-1 or rTSP-1, 10 ng/ml TNF-α, or PBS buffer as a negative control. A) Immunostaining of formaldehyde-fixed HAEC using anti-ICAM-1 mAb as described in Materials and Methods. B, C) Expression of ICAM-1 on HAEC and VCAM-1 on HMVEC, respectively. The data were analyzed by flow cytometry. The increases in MFI over control (PBS) are shown (_P_≤0.01).
Figure 3
Expression of CAM and monocyte adhesion to HMVEC. A, B) Expression of ICAM-1 (A) and VCAM-1 (B) on HMVEC. Cells were incubated for 17 h in the presence of 10 ng/ml TNF-α, 3 μg/ml TSP-1, 3 μg/ml N-terminal fragment of TSP-1 (NoC-1; amino acids 1–356 of TSP-1), or 50 μM TSP-1-derived peptides: CD-36 binding peptide CSVTCG, IAP binding peptide VV (RFYVVMWK), and control peptide GG (RFYGGMWK). The expression of CAM was analyzed by flow cytometry. The increases in MFI over control (PBS) are shown (_P_≤0.02). The data are means ± SD of three to five separate experiments. C, D) Adhesion of monocytes to endothelium. HMVEC grown in 24-well plates were treated with TSP-1 for 17 h or remained untreated. THP-1 cells were added to HMVEC at 3.75 × 105 cells per well. After 45 min incubation at 37°C, the unbound monocytes were removed by repeated washing, and bound cells were quantified by measuring radioactivity remaining in wells. Data are normalized on background level. C) Effect of various concentrations of TSP-1 on monocyte adhesion. The data are means ± SD of three separate experiments. D) Effect of TSP-1 and TSP-1-derived peptides on adhesion of THP-1 cells. The concentration of TSP-1 as well as NoC-1 fragment of TSP-1 (amino acids 1–356) was 3 μg/ml; IAP binding peptide VV (RFY
VV
MWK), control peptide GG (RFY
GG
MWK), and CD-36 binding peptide (CSVTCG), 75 μM; TNF-α, 10 ng/ml. The data are means ± SD of three separate experiments.
Figure 4
The infection of EC by lentivirus-expressing siRNA. A) Micrographs of uninfected HMVEC and HMVEC infected with lentiviruses expressing the GFP gene along with siIAP or siLuc under visible and ultraviolet light. B) IAP expression in HMVEC. Cell lysates were subjected to Western blot using B6H12 mAb against IAP: (lane 1) cells infected with siIAP; (lane 2) noninfected cells; (lane 3) cells infected with siLuc. β-actin was used as a loading control.
Figure 5
Expression of CAM on EC infected with siIAP or siLuc. A, B) The expression of ICAM-1 on HMVEC (A) or HUVEC (B) in the presence of 3 μg/ml TSP-1 (17 h of treatment). C, D) The expression of VCAM-1 on HUVEC (C) and ICAM-1 on HMVEC (D) in the presence of 10 ng/ml TNF-α (17 h of treatment). E) Effect of Ab-3 TSP-1-blocking antibodies or normal IgG on the expression of ICAM-1 on HUVEC in the presence of 2, 5, and 10 ng/ml TNF-α. Antibody concentration was 4 μg/ml. Data were analyzed by flow cytometry: The increases in MFI over control (PBS) are shown (_P_≤0.01). The data are means ±
sd
of five separate experiments.
Figure 5
Expression of CAM on EC infected with siIAP or siLuc. A, B) The expression of ICAM-1 on HMVEC (A) or HUVEC (B) in the presence of 3 μg/ml TSP-1 (17 h of treatment). C, D) The expression of VCAM-1 on HUVEC (C) and ICAM-1 on HMVEC (D) in the presence of 10 ng/ml TNF-α (17 h of treatment). E) Effect of Ab-3 TSP-1-blocking antibodies or normal IgG on the expression of ICAM-1 on HUVEC in the presence of 2, 5, and 10 ng/ml TNF-α. Antibody concentration was 4 μg/ml. Data were analyzed by flow cytometry: The increases in MFI over control (PBS) are shown (_P_≤0.01). The data are means ±
sd
of five separate experiments.
Figure 6
A) The effect of rTSP-1 and TNF-α on NF-κB activity in HUVEC (solid bars), HUVEC infected with adenovirus-encoding inhibitor of NF-κB (I-κB; shaded bars), and HUVEC infected with adenovirus-encoding GFP (striped bar). rTSP-1 concentration was 5 μ g/ml; TNF-α concentration was 10 ng/ml. NF-κB activity was analyzed using a luciferase reporter assay. Effect of TNF-α on the expression of IAP and TSP-1 on the EC. B) Western blot shows the level of TSP-1 synthesis in HUVEC after 17 h of treatment with TNF-α. The cell lysate was subjected to Western blot using TSPB-7 mAb against TSP-1. β-actin was used as a loading control. C) Effect of various concentrations of TNF-α on ICAM-1 (solid box) and TSP-1 (shaded circle) expression on HUVEC. The increases in MFI over control (PBS buffer) are shown. The data are means ±
sd
of three experiments. D) Representative data of FACS analysis. B6H12 mAb were used to assess the surface expression of IAP: shaded spectra, nontreated cells; open spectra, cells treated with 10 ng/ml TNF-α for 17 h.
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