Regulation of vascular smooth muscle cell contraction by TSP1. Agonist-stimulated VSMC contraction involves Ca-dependent activation of myosin light chain kinase (MLCK) and inactivation of myosin light chain phosphatase via its regulatory subunit MYPT1 and phosphorylation of its inhibitor CPI-17. MLCK phosphorylates the regulatory subunit of myosin (MLC2) on Ser¹⁹ to activate myosin/actin-mediated VSMC contraction. NO relaxes VSMC via increasing cGMP, which simultaneously activates cGMP-gated potassium channels and cGMP-dependent protein kinase-I (cGK-1). The former reduces intracellular calcium levels, and the latter phosphorylates MYPT1 on Ser⁶⁹⁵ and leads to dephosphorylation and inactivation of CPI-17 via protein phosphatase 2A (PP2A) [139]. TSP1 signaling through CD36 and CD47 prevents cGMP accumulation by inhibiting sGC and simultaneously inhibits cGK-1 to prevent downstream signaling [22,86]. Thus, TSP1 increases MLC2 phosphorylation (inset) and increases VSMC contractility by blocking the relaxing activities of NO.

Thrombospondin-1 inhibits angiogenic responses by inhibiting the fatty acid translocase activity of CD36. Several ligands of CD36 including TSP1, a D-Ile derivative of a sequence from its type 1 repeats, the mimetic ABT-510, and the CD36 antibody SMΦ are known to inhibit endothelial cell motility and survival in vitro and angiogenesis in vivo. These ligands inhibit uptake of fatty acids including myristate via CD36 [27]. Myristate plays an important role in membrane anchoring of the ~160 proteins in the human genome that contain N-terminal Met-Gly motifs that are substrates for methionine aminopeptidases (MetAP2) and the N-myrisotyltransferases (NMT) that transfer myristate from myristoyl-CoA onto the exposed subterminal Gly residue [89]. Limiting myristate uptake by binding to CD36 prevents membrane anchoring of Fyn and presumably some of the other known or predicted myristoylated proteins. CD36 ligands thereby inhibit myristate- or VEGF-stimulated of activation of Fyn assessed by phosphorylation on Tyr⁴¹⁶. TSP1 and other CD36 ligands also prevent myristate-stimulated activation of eNOS, presumably by limiting membrane localization and activation of an unidentified myristoylated target [27].

Concentration-dependent control of NO/cGMP signaling by TSP1. Physiological levels of TSP1 in tissues and blood (100–200 pM) are sufficient to tonically limit NO/cGMP signaling. Hemostasis, wounding, and aging are known acutely or chronically increase TSP1 to levels sufficient to prevent all activation of sGC. Conversely, malignant progression is frequently associated with loss of TSP1 expression in tumor cells, which may relieve negative regulation of NO/cGMP signaling in tumor cells. However, stroma in some tumors also exhibit elevated TSP1 expression, which may limit NO signaling in the tumor vasculature and infiltrating immune cells as well as suppress angiogenesis in distant metastases [49].

Pro- and anti-angiogenic functions of TSP1 and its endothelial cell receptors. The N-terminal domain of TSP1 stimulates angiogenic responses via α3β1 [9], α4β1 [10], α6β1 [12], and α9β1 [13]. The type 1 repeats contain two distinct anti-angiogenic sequences that interact with sulfated glycoconjugates [134], fibronectin [135], TGFβ[136], and the receptor CD36 [16]. The type 3 repeats contain an RGD sequence that interacts with α5β1 and αvβ3 integrins [8,137] This sequence can stimulate endothelial cell adhesion, but its role in angiogenesis is unclear. The C-terminal G domain interacts with CD47. Some CD47-binding peptides derived from this domain inhibit angiogenesis [138] and CD47 is necessary for anti-angiogenic activities of TSP1 in the context of nitric oxide signaling [86].

Regulation of NO/cGMP signaling by TSP1 in endothelial cells. VEGF signaling through its receptor activates the kinase Akt, which phosphorylates eNOS on Ser¹¹⁷⁹ [140]. This increases NO synthesis by decreasing the Ca-dependence of eNOS. NO activates sGC to increase cGMP synthesis. cGMP acts on several downstream target in endothelial cells to stimulate pro-angiogenic responses including survival, growth, motility, and adhesion. At physiological concentrations, TSP1 acts primarily through its receptor CD47 to limit sGC activation. At nM concentrations, TSP1 also signals through CD36 to inhibit the same responses, but CD47 is also necessary for these signals to inhibit cGMP signaling. Therefore, engaging either receptor is sufficient to inhibit NO/cGMP signaling, but CD47 is the necessary receptor of TSP1 action.

Acute effects of endogenous TSP1 on hind limb perfusion following ischemic injury. Wild type and TSP1-null mice underwent ligation of the femoral artery at the level of the inguinal ligament, and limb perfusion was measured during the first post-operative hour. An increased persistence of limb perfusion is demonstrated in the TSP1-null limb.