Nitric oxide regulates angiogenesis through a functional switch involving thrombospondin-1

Abstract

Nitric oxide (NO) donors have been shown to stimulate and inhibit the proliferation, migration, and differentiation of endothelial cells in vitro and angiogenesis in vivo. Recently, we have shown distinct thresholds for NO to regulate p53-Ser-15P, phosphorylated extracellular signal-regulated kinase (pERK), and hypoxia inducible factor 1alpha in tumor cells. Because these signaling pathways also promote the growth and survival of endothelial cells, we examined their roles in angiogenic responses of venous endothelial cells and vascular outgrowth of muscle explants elicited by NO. An additional protein involved in the regulation of angiogenesis is thrombospondin-1 (TSP1), a matricellular glycoprotein known to influence adhesion, migration, and proliferation of endothelial cells. Here we demonstrate a triphasic regulation of TSP1 mediated by a slow and prolonged release of NO that depends on ERK phosphorylation. Under conditions of 5% serum, a 24-h exposure of NO donor (0.1-1,000 microM) mediated a triphasic response in the expression of TSP1 protein: decreasing at 0.1 microM, rebounding at 100 microM, and decreasing again at 1,000 microM. Under the same conditions, we observed a dose-dependent increase in P53 phosphorylation and inverse biphasic responses of pERK and mitogen-activated protein kinase phosphatase-1. Both the growth-stimulating activity of low-dose NO for endothelial cells and suppression of TSP1 expression were ERK-dependent. Conversely, exogenous TSP1 suppressed NO-mediated pERK. These results suggest that dose-dependent positive- and negative-feedback loops exist between NO and TSP1. Limiting TSP1 expression by positive feedback through the ERK mitogen-activated protein kinase pathway may facilitate switching to a proangiogenic state at low doses of NO.

Fig. 1.

NO-mediated proangiogenic response as characterized by vascular cell outgrowth and endothelial cell proliferation. C57B16 WT mouse muscle fragments were embedded in 3D collagen matrices and exposed to control, 1 mM l-Arg, 500 μM l-NAME, or 0.1–1,000 μM DETA/NO and migration distance evaluated. [Bar, 500 μm(A–C).] (D) Vascular cell invasion of collagen matrices was quantified in each of four quadrants as the distance of farthest cell invasion from the muscle border in response to l-Arg (LA), l-NAME (LN), or DETA/NO. Results are presented as mean ± SD, n ≥ 3. (E) Proliferative effects of chronic exposure to NO were determined in HUVECs exposed to 0.1–1,000 μM DETA/NO in the presence and absence of the MEK[1/2] inhibitor U0126. Proliferation was quantified by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and results are presented as mean ± SD, n ≥ 3.

Fig. 2.

NO-mediated down-regulation of the antiangiogenic factor TSP1. HUVECs were plated as described in Materials and Methods and grown to 70% confluence (≈2 × 10⁶ cells). (A and C) Cells were exposed to 0.1–1,000 μM DETA/NO for 24 h and the sample media concentrated on heparin columns as described in Materials and Methods and then immunoblotted for TSP1 levels vs. μM DETA/NO. (B) Quantification of the protein bands in A. (D) Real-time PCR was performed on cDNA prepared from cells exposed to 1 μM DETA/NO for 2 h.

Fig. 3.

NO modulation of molecular target proteins in HUVECs. (A) Representative immunoblot of protein from cells exposed to 1–1,000 μM DETA/NO for 24 h (n = 3). (B and C) Quantification of the NO-induced changes in target protein levels shown in A. (D) Modulation of the levels of pERK by endogenous NO after a 24-h exposure of HUVECs to 1 mM l-Arg or 5 mM l-NAME. (E) Steady-state nanomolar levels of NO generated by 10–1,000 μM DETA/NO in treatments media.

Fig. 4.

Inhibition of ERK phosphorylation reverses NO-mediated down-regulation of TSP1. (A) Cells were plated as described in Fig. 1A and exposed to 1 μM DETA/NO for 10 h in the presence and absence of the MEK[1/2] inhibitor U0126. The sample media were concentrated and immunoblotted for TSP1 levels as described in the Fig. 2 legend. Data are representative of two experiments. (B) Quantification of the immunoblot shown in A.

Fig. 5.

Exogenous TSP1 suppresses NO-mediated ERK phosphorylation. (A) Cells were exposed to 10 μM DETA/NO for 4 and 10 h in the presence and absence of 300 ng/ml TSP1 and then immunoblotted for pERK. (B) Quantification of immunoreactive protein levels demonstrated TSP1 suppression of pERK.