Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin

Abstract

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ∼22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.

FIGURE 1.

Sequences of overlapping FRET peptides and proteolysis-sensitive hinge region of the NC-1 domain of human and mouse collagen XVIII. A, design of FRET-peptides. Ten overlapping 10-mer peptides (Endo-01 to Endo-10), with a four-residue stagger and spanning the proteolysis-sensitive hinge region of the NC-1 domain of collagen XVIII, were synthesized as FRET peptides and are flanked by an N-terminal fluorescence donor (Abz), and a C-terminal quenching acceptor (3-Tyr-NO2). B, hinge region of both murine and human NC-1 domains. Positions of cleavage sites identified by in vitro assays are compared with in vivo circulating endostatin (identified by N-terminal sequencing) found in human plasma (residues 1311–1354: human collagen XVIII numbering: Swissprot access no. 39060). Upper characters for cathepsins L, S, and B relate to cleavage sites matching with previously described circulating forms of human endostatin (9); their corresponding N-terminal residues are Pro-1325, Ser-1330, and Val-1346.

FIGURE 2.

Consequences of gene silencing of cathepsins L and S. A, consequences of transient inhibition of cathepsins L, S, and B on level of endogenous 22-kDa endostatin. At 48 h post-transfection, HUVECs were lysed, and the protein level of endogenous endostatin was analyzed by Western blot in the presence of siRNAs. siCTL, control siRNA; siCatB, siRNA directed against cathepsin B; siCatL, siRNA directed against cathepsin L; siCatS, siRNA directed against cathepsin S. B, proliferation assays: At 48 h post-transfection, proliferative properties of HUVECs were analyzed in the presence of VEGF (20 ng/ml). Assays were conducted in the presence of exogenous human endostatin as described under “Experimental Procedures.” E-64 (20 μm) was added in the medium for a control (CTL) experiment (n = 6, results are expressed as median). C, migration assays. At 48-h post-transfection with siRNAs directed against cathepsins, anti-migrative properties of exogenous endostatin were analyzed in a Boyden chamber. VEGF (20 ng/ml) was used as chemoattractant. A control experiment was made in the presence of E-64 (20 μm) (n = 6, results are expressed as median).

FIGURE 3.

Hydrolysis of human endostatin by cathepsins L and S: location of cleavage sites. Following incubation with cathepsins L and S, hydrolysis products were separated by reverse-phase HPLC and identified by mass spectrometry, and cleavage sites were deduced from mass analysis (human collagen XVIII numbering: Swissprot access no. 39060). Positions of both 10-mer G10T (¹⁴⁵⁵GSDPNGRRLT¹⁴⁶⁴) and 15-mer K15T (¹⁴⁵⁰KSVWHGSDPNGRRLT¹⁴⁶⁴) peptides are highlighted in gray and light gray, respectively. Arrowheads correspond to identified cleavage sites.

FIGURE 4.

Anti-angiogenic properties of hydrolysis products of human endostatin. A, proliferation assays. Following incubation for 4 h at 37 °C in the presence of cysteine cathepsins (enzyme/substrate molar ratio, 1:5), hydrolysis products of endostatin (0.1 mm) were assayed on HUVECs in the presence of VEGF (20 ng/ml). Native recombinant endostatin (0.1 mm) was used as control. After a 2-h incubation in the presence of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,absorbance was measured (A = 490 nm). Results are expressed as the percentage of the difference in absorbance observed in the presence or not of VEGF (n = 12, data reported as median ± quartiles). B, caspase-3 activity: Experiments were done in the presence of VEGF (20 ng/ml). Ability of native endostatin (0.1 mm) or its hydrolysis products (0.1 mm) to induce apoptosis in HUVECs was determined by measurement of the proteolytic activity of caspase toward Z-DEVD-AMC (50 mm). Results were normalized with respect to the hydrolysis of Z-DEVD-AMC in the presence of VEGF alone. The specificity of caspase-dependent AMC release was checked by addition of 50 mm Z-DEVD-FMK (not shown here) (n = 6, data expressed as median ± quartiles). C, migration assays. Briefly, HUVEC cells were treated for 15 min at 37 °C with endostatin (0.1 mm) or its hydrolysis fragments (0.1 mm) and then placed in fibronectin-coated filter inserts for 5 h (24-well plates) in the presence of VEGF (20 ng/ml). Cells that have migrated in the lower section of the Boyden chamber were counted as described under “Experimental Procedures.” Results are expressed as the ratio of cells that have migrated in the absence of chemoattractant (n = 6, data expressed as median ± quartiles). D, immunoblotting analysis. Residual uncleaved endostatin was revealed by an anti-endostatin antibody following its incubation with cathepsins. *, p < 0.1; **, p < 0.05.

FIGURE 5.

Angiostatic activities of endostatin-derived G10T and K15T. A, proliferation assays of HUVECs. Dose-response effects of G10T and K15T peptides were determined in the presence of VEGF (20 ng/ml). EC₅₀ were calculated by using GraphPad Prism software (La Jolla, CA). Data are reported as median ± quartiles (n = 10). B, caspase-3 activity. Dose-response proapoptotic effects of peptides G10T and K15T were determined in the presence of VEGF (20 ng/ml) (n = 9, data expressed as median ± quartiles). C, migration assays. HUVECs were treated for 15 min at 37 °C with peptides G10T and K15T then placed in fibronectin-coated filter inserts for 5 h (24-well plates) in the presence of VEGF (20 ng/ml). Cells that have migrated in the Boyden chamber were counted, and results were reported as previously described (n = 9, data correspond to median ± quartiles). White bar, G10T; gray bar, K15T. *, p < 0.1; **, p < 0.05. Dashed lines correspond to control values (100%).

FIGURE 6.

Inhibition of APN/CD13 and impairment of tube formation by the G10T peptide. A, recombinant human APN (20 ng/assay) was incubated for 15 min at 37 °C in the presence of bestatin, G10T, and Sc-G10T (final concentration, 0–200 mm) before adding H-Ala-AMC (100 mm). Residual enzymatic activity of APN was deduced from the release of AMC (excitation wavelength, 380 nm; emission wavelength, 460 nm) (experiments done in triplicate; n = 3). Results were reported as means ± S.D. B, endothelial tube formation assay. 1 × 10⁵ endothelial cells per well were applied to Matrigel-coated 48-well plates. Cells were further incubated with G10T peptide, its scrambled form (Sc-G10T), or a vehicle-only control. Total tubule branching was counted as described under “Experimental Procedures,” and results were expressed as the average tube length per field of view (×20 magnification) (16). **, p < 0.05. C, inhibition of tube formation by G10T and bestatin. The G10T peptide and bestatin were applied alone or in combination to evaluate additive effects of the two inhibitors. Results were normalized using as reference vehicle-only control. White bar, 100 nm; gray bar, 500 nm. **, p < 0.05.

FIGURE 7.

Release of angiostatic peptides by cysteine cathepsins S and L. Both G10T (¹⁴⁵⁵GSDPNGRRLT¹⁴⁶⁴) and K15T (¹⁴⁵⁰KSVWHGSDPNGRRLT¹⁴⁶⁴) peptides enclose an NGR tumor-homing motif (highlighted in light gray) with enhanced anti-proliferative and proapoptotic potencies compared with the parent endostatin toward endothelial cells (human collagen XVIII numbering). Experimental data also support that biological effects of G10T could be mediated by molecular interactions with APN/CD13, which is already known to be the pharmacological target of bestatin.