Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies

Abstract

In addition to long-term regulation of angiogenesis, angiogenic growth factor signalling through nitric oxide (NO) acutely controls blood flow and haemostasis. Inhibition of this pathway may account for the hypertensive and pro-thrombotic side effects of the vascular endothelial growth factor antagonists that are currently used for cancer treatment. The first identified endogenous angiogenesis inhibitor, thrombospondin 1, also controls tissue perfusion, haemostasis and radiosensitivity by antagonizing NO signalling. We examine the role of these and other emerging activities of thrombospondin 1 in cancer. Clarifying how endogenous and therapeutic angiogenesis inhibitors regulate vascular NO signalling could facilitate development of more selective inhibitors.

Figure 1. The central role of nitric oxide (NO) signaling in angiogenesis, vascular tone, and hemostasis

a | Vascular endothelial growth factor (VEGF) binding to its receptor on endothelial cells activates nitric oxide synthase (eNOS) to produce the diffusible signaling molecule NO. NO acts in an autocrine manner to stimulate endothelial cell growth and motility leading to angiogenesis. VEGF signaling via NO also contributes to increasing vascular permeability. NO diffuses into vessel walls, causing arterial vessels to relax and increase blood flow. NO also acts in a paracrine manner to prevent thrombosis by inhibiting platelet adhesion and aggregation. b | Different vascular activities of NO occur on different time scales. c | In endothelial cells, VEGF signaling through VEGFR2 activates the phosphatidyinositol 3-kinase (PI3K) pathway; Akt then phosphorylates human eNOS at Ser¹¹⁷⁷ , , activating eNOS and decreasing its calcium dependence. The kinase Src, which is activated by VEGF, also activates eNOS through two mechanisms: phosphorylation of Tyr, and phosphorylation of heat shock protein 90 (Hsp90), which then binds to eNOS and activates NO synthesis . Simultaneously, VEGFR2 signaling through phospholipase-Cγ (PLCγ) mobilizes intracellular Ca²⁺, which further activates eNOS in a calmodulin (CaM)-dependent manner, and increases AMP kinase (AMPK)-mediated eNOS phosphorylation at Ser^1177161. NO produced by eNOS binds to the prosthetic heme on soluble guanylate cyclase (sGC) to stimulate cGMP synthesis, activating cGMP-dependent protein kinase (cGK-I) and cGMP-gated channels to regulate downstream targets that increase endothelial cell proliferation, migration, survival, and permeability. Additional parallel signaling through Src, Akt, and the protein kinase C-mitogen-activated protein kinase pathway (PKC-Raf1-MEK-ERK) synergizes with NO/cGMP signaling to support each of these endothelial cell responses. In VSMC and platelets, the same downstream pathways are activated by exogenous NO diffusing from endothelium.

Figure 1. The central role of nitric oxide (NO) signaling in angiogenesis, vascular tone, and hemostasis

a | Vascular endothelial growth factor (VEGF) binding to its receptor on endothelial cells activates nitric oxide synthase (eNOS) to produce the diffusible signaling molecule NO. NO acts in an autocrine manner to stimulate endothelial cell growth and motility leading to angiogenesis. VEGF signaling via NO also contributes to increasing vascular permeability. NO diffuses into vessel walls, causing arterial vessels to relax and increase blood flow. NO also acts in a paracrine manner to prevent thrombosis by inhibiting platelet adhesion and aggregation. b | Different vascular activities of NO occur on different time scales. c | In endothelial cells, VEGF signaling through VEGFR2 activates the phosphatidyinositol 3-kinase (PI3K) pathway; Akt then phosphorylates human eNOS at Ser¹¹⁷⁷ , , activating eNOS and decreasing its calcium dependence. The kinase Src, which is activated by VEGF, also activates eNOS through two mechanisms: phosphorylation of Tyr, and phosphorylation of heat shock protein 90 (Hsp90), which then binds to eNOS and activates NO synthesis . Simultaneously, VEGFR2 signaling through phospholipase-Cγ (PLCγ) mobilizes intracellular Ca²⁺, which further activates eNOS in a calmodulin (CaM)-dependent manner, and increases AMP kinase (AMPK)-mediated eNOS phosphorylation at Ser^1177161. NO produced by eNOS binds to the prosthetic heme on soluble guanylate cyclase (sGC) to stimulate cGMP synthesis, activating cGMP-dependent protein kinase (cGK-I) and cGMP-gated channels to regulate downstream targets that increase endothelial cell proliferation, migration, survival, and permeability. Additional parallel signaling through Src, Akt, and the protein kinase C-mitogen-activated protein kinase pathway (PKC-Raf1-MEK-ERK) synergizes with NO/cGMP signaling to support each of these endothelial cell responses. In VSMC and platelets, the same downstream pathways are activated by exogenous NO diffusing from endothelium.

Figure 1. The central role of nitric oxide (NO) signaling in angiogenesis, vascular tone, and hemostasis

a | Vascular endothelial growth factor (VEGF) binding to its receptor on endothelial cells activates nitric oxide synthase (eNOS) to produce the diffusible signaling molecule NO. NO acts in an autocrine manner to stimulate endothelial cell growth and motility leading to angiogenesis. VEGF signaling via NO also contributes to increasing vascular permeability. NO diffuses into vessel walls, causing arterial vessels to relax and increase blood flow. NO also acts in a paracrine manner to prevent thrombosis by inhibiting platelet adhesion and aggregation. b | Different vascular activities of NO occur on different time scales. c | In endothelial cells, VEGF signaling through VEGFR2 activates the phosphatidyinositol 3-kinase (PI3K) pathway; Akt then phosphorylates human eNOS at Ser¹¹⁷⁷ , , activating eNOS and decreasing its calcium dependence. The kinase Src, which is activated by VEGF, also activates eNOS through two mechanisms: phosphorylation of Tyr, and phosphorylation of heat shock protein 90 (Hsp90), which then binds to eNOS and activates NO synthesis . Simultaneously, VEGFR2 signaling through phospholipase-Cγ (PLCγ) mobilizes intracellular Ca²⁺, which further activates eNOS in a calmodulin (CaM)-dependent manner, and increases AMP kinase (AMPK)-mediated eNOS phosphorylation at Ser^1177161. NO produced by eNOS binds to the prosthetic heme on soluble guanylate cyclase (sGC) to stimulate cGMP synthesis, activating cGMP-dependent protein kinase (cGK-I) and cGMP-gated channels to regulate downstream targets that increase endothelial cell proliferation, migration, survival, and permeability. Additional parallel signaling through Src, Akt, and the protein kinase C-mitogen-activated protein kinase pathway (PKC-Raf1-MEK-ERK) synergizes with NO/cGMP signaling to support each of these endothelial cell responses. In VSMC and platelets, the same downstream pathways are activated by exogenous NO diffusing from endothelium.

Figure 2. CD47 and CD36 mediate inhibition of NO/cGMP signaling by thrombospondin-1

Thrombospondin-1 (TSP1) redundantly inhibits cGMP signaling driven by exogenous or endogenous NO in vascular cells via 2 receptors. The C-terminal G-module of TSP1 contains two peptide motifs that have been shown to bind CD47. The yellow highlighted residues in these peptides are conserved across all 5 TSPs, but studies using recombinant domains of TSP2 and TSP4 indicate that high affinity binding to CD47 is specific to TSP1. Binding of TSP1 or the peptides signals via CD47 to inhibit both NO-mediated activation of soluble guanylate cyclase (sGC) in endothelial cells, VSMC, and platelets and cGMP-mediated activation of cGMP-dependent protein kinase (cGK-I) in platelets. At elevated levels, TSP1 also interacts with CD36 via its central type 1 repeats and thereby inhibits its fatty acid translocase activity, limiting myristate uptake into the cytoplasm and thereby inhibiting membrane translocation of Src family kinases in endothelial cells. CD36-dependent myristate uptake activates two known eNOS kinases: Src and AMP kinase (AMPK), , but the relative importance of these two pathways in controlling eNOS activation through CD36 has not been established. The TSP1 mimetic drug ABT-510 also binds to CD36 and inhibits angiogenesis via both translocase-dependent and caspase-dependent mechanisms, .

Figure 3. Vascular responses outside the tumor primarily determine effects of TSP1 on tumor perfusion

a | Tumor vasculature is much less responsive than normal vasculature to physiological regulation by vasodilators such as NO and vasoconstrictors such as epinephrine. Thus, vasodilation indirectly decreases blood flow through the tumor, and vasoconstriction increases flow through the tumor. b | These responses to vasoactive agents are not sensitive to endogenous TSP1 in the tumor vasculature, but over-expression of TSP1 in the tumor either locally or by release into the circulation tempers responses to vasoactive agents in the host vasculature and decreases the “steal” phenomenon in mice bearing TSP1-expressing tumors. The relative contribution of local versus systemic effects of TSP1 on tumor vascular resistance remains to be defined.

Figure 3. Vascular responses outside the tumor primarily determine effects of TSP1 on tumor perfusion

a | Tumor vasculature is much less responsive than normal vasculature to physiological regulation by vasodilators such as NO and vasoconstrictors such as epinephrine. Thus, vasodilation indirectly decreases blood flow through the tumor, and vasoconstriction increases flow through the tumor. b | These responses to vasoactive agents are not sensitive to endogenous TSP1 in the tumor vasculature, but over-expression of TSP1 in the tumor either locally or by release into the circulation tempers responses to vasoactive agents in the host vasculature and decreases the “steal” phenomenon in mice bearing TSP1-expressing tumors. The relative contribution of local versus systemic effects of TSP1 on tumor vascular resistance remains to be defined.

Figure 4. Convergence of angiogenesis inhibitor signaling on the nitric oxide (NO)/cGMP cascade

The current US Food and Drug Administration (FDA)-approved angiogenesis inhibitors for cancer therapy block vascular endothelial growth factor (VEGF) binding to the VEGF receptor (VEGFR2) (the inhibitor bevacizumab) or inhibit the tyrosine kinase activity of VEGFR2 (the inhibitors sorafenib and sunitinib). VEGFR2 activates several parallel signaling pathways that control angiogenesis, but inhibiting NO/cGMP signaling blocks most of these responses. Therapeutic responses to VEGF inhibitors may be limited by parallel activation of the downstream pathways by additional angiogenic factors produced by tumors that can activate NO/cGMP signaling independent of the VEGF receptor such as adrenomedullin, angiopoietin-1, sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA), angiopoietin-related growth factor (AGF), estrogens, insulin, and fibroblast growth factor-2 (FGF2), –. Therefore, inhibitors that act on essential downstream pathways may be more effective than the existing VEGF/VEGFR antagonists for controlling tumor angiogenesis. Endostatin is one such agent that acts via the phosphatase PP2A to inhibit nitric oxide synthase (eNOS) activation and downstream to inhibit soluble guanylate cyclase (sGC) expression. Thrombospondin 1 (TSP1) is a redundant inhibitor of the NO/cGMP cascade, and the experimental drug ABT-510, a TSP1 mimetic, inhibits angiogenesis via the TSP1 receptor CD36. However, native TSP1 also acts through the more potent inhibitory pathway mediated by CD47. Proangiogenic factors and signaling pathways are shown in green, and anti-angiogenic factors and pathways are in red.