Three Distinct d-Amino Acid Substitutions Confer Potent Antiangiogenic Activity on an Inactive Peptide Derived from a Thrombospondin-1 Type 1 Repeat

Abstract

Mal II, a 19-residue peptide derived from the second type 1 properdin-like repeat of the antiangiogenic protein thrombospondin-1 (TSP-1), was inactive in angiogenesis assays. Yet the substitution of any one of three L-amino acids by their D-enantiomers conferred on this peptide a potent antiangiogenic activity approaching that of the intact 450-kDa TSP-1. Substituted peptides inhibited the migration of capillary endothelial cells with an ED50 of 8.5 nM for the D-Ile-15 substitution, 10 nM for the D-Ser-4 substitution, and 0.75 nM for the D-Ser-5 substitution. A peptide with D-Ile at position 15 could be shortened to its last seven amino acids with little loss in activity. Like whole TSP-1, the Mal II D-Ile derivative inhibited a broad range of angiogenic inducers, was selective for endothelial cells, and required CD36 receptor binding for activity. A variety of end modifications further improved peptide potency. An ethylamide-capped heptapeptide was also active systemically in that when injected i.p. it rendered mice unable to mount a corneal angiogenic response, suggesting the potential usefulness of such peptides as antiangiogenic therapeutics.

CD36: Implications in cardiovascular disease

“…In addition to facilitating fatty acid transport into adipocytes and cardiac and skeletal muscle (and perhaps other cells), CD36 was recently shown to be a sensor for fatty acids in taste buds, eliciting a secretory response in the gut (Laugerette, et al, 2005). CD36 interacts with ligands which contain a thrombospondin 1 (TSP-1) type 1 repeat element (TSR) to inhibit a pro-angiogenic signal, resulting in endothelial cell apoptosis (Dawson, Pearce, Zhong, Silverstein, Frazier, & Bouck, 1997,Dawson, et al, 1999,Jimenez, Volpert, Crawford, Febbraio, Silverstein, & Bouck, 2000,Miao, Seng, Duquette, Lawler, Laus, & Lawler, 2001,Simantov, Febbraio & Silverstein, 2005.…”

“…Thus, in a hyperlipidemic setting, expression of CD36 may play a role in ischemia, and may contribute to ischemia in the aftermath of myocardial infarction and stroke. Inhibition of angiogenesis by exploiting this pathway has led to the development of TSP-1 peptides and mimetics that are currently in clinical trials for application in cancer and diabetic retinopathy (Dawson, et al, 1999,Rusk, et al, 2006,Gietema, et al, 2006. The idea of up regulation of CD36 to inhibit angiogenesis by TSP-1 has been explored to increase the efficacy of the antiangiogenic effect in a tumor model (Huang, et al, 2004).…”

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment

“…8 One of them, DI-TSP is active at nanomolar concentrations that interacts with CD36 and causes endothelial cell apoptosis via the same signaling cascade 9 (Zaichuk and Volpert, unpublished observations) while lacking undesirable effects due to binding receptors other than CD36. 8,9 This is a C-ter capped nonapeptide based on the GVITRIR sequence from the second properdin repeat, containing an L-to D-isoleucyl substitution. 9 TSP1, or DI-TSP binding to CD36 triggers signaling cascade that begins with recruitment of Src-related kinase p59 fyn and ultimately causes endothelial cells apoptosis.…”

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

“…We also quantified the activity of the predicted peptide fragments in the proliferation experiments relative to known short peptides derived from the type I thrombospondin repeats of the thrombospondin 1 protein Mal II (SPWSSCSVTCGDGVITRIR) and Mal III (SPWDICSVTCGGGVQKRSK) (Dawson et al, 1999;Tolsma et al, 1993). Those short peptides exhibited maximum activity of 25% and 35%, respectively, at 10 μg/ml (data not shown).…”

“…Both can inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane assay as well as fibroblast growth factor (FGF-2)-induced neo-vascularization in the corneal micro-pocket assay Vazquez et al, 1999). Also two peptides, Mal II and Mal III, derived from the type I thrombospondin repeats of the thrombospondin 1 protein have been shown to exert antiangiogenic activities both in vitro and in vivo (Dawson et al, 1999;Tolsma et al, 1993). There is also an increasing interest in using the thrombospondin-1 protein as a prototype for designing anti-angiogenic peptides with optimized activities (Haviv et al, 2005).…”