Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis

Abstract

We report for the first time that saliva of the hard tick and Lyme disease vector, Ixodes scapularis, is a potent inhibitor of angiogenesis. Saliva (< or = 1:500 dilutions) or salivary gland (0.1-0.5 pairs/assay) dose-dependently inhibits microvascular endothelial cell (MVEC) proliferation. Inhibition was also detected with the saliva of the cattle tick Boophilus microplus but not with the salivary gland of Anopheles gambiae, An. stephensi, Lutzomyia longipalpis, Phlebotomus papatasi, Aedes aegypti, Culex quinquefasciatus, and Cimex lectularius. Inhibition of MVEC proliferation by I. Scapularis saliva was accompanied by a change in cell shape (shrinkage of the cytoplasm with loss of cell-cell interactions) and apoptosis which was estimated by expression of phosphatidylserine using the Apopercentage dye, and by a typical pattern of chromatin margination, condensation, and fragmentation as revealed by nuclear staining with Hoechst 33258. The effect of saliva appears to be mediated by endothelial cell alpha5beta1 integrin, because monoclonal antibodies against this but not alphavbeta3, alphavbeta5, alpha9beta1, or alpha2beta1 integrins remarkably block its effect. In addition, SDS/PAGE shows that saliva specifically degrades purified alpha5beta1 but not alphavbeta5 or alphavbeta3 integrins. Incubation of saliva with EDTA and 1,10-phenanthroline, but not phenylmethylsulfonyl fluoride (PMSF), inhibits saliva-dependent degradation of purified alpha5beta1 integrin, suggesting that a metalloprotease is responsible for the activity. Finally, saliva at < or = 1:1,000 dilutions blocks sprouting formation from chick embryo aorta implanted in Matrigel, an in vitro model of angiogenesis. These findings introduce the concept that tick saliva is a negative modulator of angiogenesis-dependent wound healing and tissue repair, therefore allowing ticks to feed for days. Inhibition of angiogenesis was hitherto an unidentified biologic property of the saliva of any blood-sucking arthropod studied so far. Its presence in tick saliva may be regarded as an additional source of angiogenesis inhibitors with potential applications for the study of both vector and vascular biology.

FIG. 1

I. scapularis saliva and salivary gland inhibit microvascular endothelial cell (MVEC) proliferation: comparison with the salivary gland from other blood-sucking arthropods. MVEC were cultured in the presence of EBM-2 Plus for 24 h at 37°C. The relative numbers of cells in each well and the percent inhibition of proliferation were determined using MTS solution. (A) I. scapularis saliva was added as indicated. (B) I. scapularis or other blood-sucking salivary glands were added as indicated. No inhibition was observed with An. gambiae, An. stephensi, Culex quinquefasciatus, Cimex lectularius, Aedes aegypti, Lutzomyia longipalpis, or Phlebotomus papatasi salivary glands (up to 2.5 pairs/assays; n = 8). Partial inhibition was observed with 1 pair/assay of Rhodnius prolixus salivary gland, and total inhibition was detected with ~1 pair/assay of Boophilus microplus salivary gland (not shown; n = 3). Similar results were obtained with human umbilical vein endothelial cells (HUVEC) for all salivary glands tested (not shown; n = 5).

FIG. 2

Tick saliva induces a rapid change in the morphology of MVEC. Tick saliva was added to 80% to 90% confluent MVEC and pictures taken at the indicated time points. (A) Control (buffer, 6 h); (B) saliva (15 min); (C) saliva (30 min); (D) saliva (1 h); (E) saliva (2 h) and (F) saliva (6 h). Magnification: 50× (n = 10).

FIG. 3

Treatment of endothelial cells with anti-integrin antibody prevents change on MVEC caused by tick saliva. Tick saliva was added to 80% to 90% confluent MEVC previously incubated for 1 h with purified mAb (25 μg/ml) when indicated: (A) buffer, no saliva; (B) saliva (5 μl); (C) anti-αvβ3 (LM609); (D) anti-αvβ5 (P1F6); (E) anti-α9β1(Y9A2); and (F) anti-α5β1 (HA6). Similar results were observed with anti-αvβ3 (23C6), anti-αvβ5 (P1F76) mAbs that did not attenuate the effects of saliva, whereas inhibition was observed with ascites anti-α5β1 (JBS4) mAb (pictures not shown). Pictures were taken after 6 hr of incubation with saliva (n = 3). Magnification: 80×. No effects were observed when the following mAb were used: anti-α1, -α2, -α3, -α4, -α5, -α6, -β1, -β2, -αv, -α2β1, or -α3β1 (n = 3). mAbs alone did not affect the MVEC shape.

FIG. 4

Saliva degrades purified integrin α5β1 and α1β1, but not αvβ3 or αvβ5: Effects of inhibitors. In (A) purified integrins αvβ3 or αvβ5 were incubated with saliva overnight at 37°C in EBM-2 and no degradation was observed after SDS/PAGE and staining with 0.2% Coomassie blue (n = 5). In (B) purified integrins α5β1 and α1β1 were incubated with saliva as in (A) and degradation was observed for both α and β chains after SDS/PAGE and staining as above. (C) The metalloprotease inhibitors EDTA (20 mM) or 1,10-phenanthroline (2.5 mM), and the serine protease inhibitor PMSF (2.5 mM) were incubated for 30-min with saliva followed by addition of integrin α5β1 and overnight incubation as in (A) followed by SDS/PAGE and staining as above. The gel shows that degradation of α5β1 did not occur when saliva was treated with EDTA or 1,10-phenanthroline, but was observed in the presence of PMSF (n=4). Results are from a typical experiment.

FIG. 5

Tick saliva induces apoptosis in endothelial cells. (A) MVEC were incubated in the absence or (B) presence of saliva for 4 hours and stained with Apopercentage dye (see Methods). In (A) cells are not stained confirming its viability and non-apoptotic nature, whereas in (B) arrows indicate the red-purple stained cells indicate apoptosis. Magnification: 50x (n=3). (C) MVEC were incubated in the absence or (D) presence of saliva for 6 hours and stained with Hoechst 33258 (see Methods). In (C), nuclear contour is regular and smooth, whereas in (D), arrows indicate cells with irregular nuclear contour, with nuclear hyperchromaticity, chromatin condensation, and fragmentation typical of apoptosis. Magnification: 200× (n = 3).

FIG. 6

Tick saliva inhibits sprouting formation from 12-day-old chick embryo aorta. Saliva was added to chick embryo aorta as described. After 3 days of incubation at 37°C, 5% CO₂, pictures were taken. (A) No saliva. (B) Saliva (0.1 μl). (C) Saliva (0.3 μl). (D) Saliva (1 μl). (E) Dose-response curve for the inhibition of sprouting formation by saliva (n = 8). Magnification: 50×.

FIG. 7

Orderly phases of wound healing and the effects of salivary components. Wound healing is divided in three phases: inflammatory (inflammation), proliferative (granulation tissue), and remodeling (wound contraction) phases. Salivary molecules Ixolaris (7), Penthalaris (18), SALP14 (19), ISAC (20), a salivary inhibitor of neutrophil function (6, 21), and a bradykinin-degrading kininase (22)—work in concert to effectively block the acute phase of inflammation. Later stages of inflammation, during which endothelial cell-dependent granulation tissue formation takes place, appear to be counteracted by tick salivary components displaying anti-angiogenesis activities. Modified from R. A. Clark, 1991 (50).