Laminar shear inhibits tubule formation and migration of endothelial cells by an angiopoietin-2 dependent mechanism

Abstract

Objective: Fluid shear stress plays a role in angiogenesis. Laminar shear stress (LS) promotes endothelial cell (EC) quiescence, whereas oscillatory shear stress (OS) promotes EC turnover and dysfunction, which could lead to pathological angiogenesis. We hypothesized that LS inhibits EC migration and tubule formation, 2 functions important in angiogenesis, by inhibiting the secretion of proangiogenic factors.

Methods and results: Human umbilical vein ECs (HUVECs), human microvascular ECs (HMECs), or bovine aortic ECs (BAECs) were subjected to either LS (15 dyn/cm2) or OS (+/-5 dyn/cm2) for 24 hours and used in Matrigel tubule formation or scratch migration assays. Exposure of HUVECs, HMECs, but not BAECs, to LS inhibited tubule formation compared with OS. LS also inhibited migration of HUVECs and BAECs compared with OS. Angiopoietin-2 (Ang2), a known angiogenic protein, was found to be downregulated by LS both in cultured ECs and mouse aortas. Using Ang2 siRNA, Ang2 knockdown blocked OS-mediated migration and tubule formation and the LS-inhibited tubule formation was partially rescued by recombinant Ang2.

Conclusions: Our data suggests that Ang2 produced by OS in ECs plays a critical role in migration and tubule formation, and may play an important role in diseases with disturbed flow and angiogenesis.

Figure 1

LS inhibits tubule formation compared with OS in HUVECs and HMECs but not BAECs. A, HUVECs were sheared at 5 and 15 dyn/cm² unidirectional LS or ±5 and ±15 dyn/cm² OS for 24 hours with static condition (ST) as a control and then used in a Matrigel tubule formation assay. B, Conditioned media (CM) collected from HUVECs that were sheared at 15 (LS), ±5 (OS) dyn/cm², or ST for 24 hours were added to static HUVECs in a Matrigel tubule formation assay. HMEC-1 (C) or BAECs (D) were sheared at 15 (LS) or ±5 dyn/cm² (OS) for 24 hours and then used in the Matrigel tubule formation assay. E, CM collected from sheared BAECs as in D were added to static HUVECs in a Matrigel tubule formation assay. Shown are representative images (10× magnification) and tubule length was quantified over 4 high powered fields at 5× magnification and normalized to percent static. (mean±SEM, n=3 to 9; *P<0.05 LS vs OS; #P<0.05 compared with ST).

Figure 2

LS inhibits migration compared with OS in HUVECs and BAECs. HUVECs (A) or BAECs (B) were sheared at 15 (LS) and ±5 (OS) dyn/cm² for 24 hours. The monolayers were scratched, and photographed immediately (0 hour) (HUVECs: a, b; BAECs: e, f) and again after 6 hours incubation in fresh reduced serum medium (HUVECs: c, d; BAECs: g, h). For C and D, confluent static cultured HUVECs were scratched and the media were replaced with CM obtained from sheared HUVECs or BAECs as in A and B. Cells were photographed at 0 and 6 hours incubation. The center lines indicate the original scratch margins and the arrows indicate flow directions. Graphs represent number of cells migrated into the scratched area as a percent of static. (mean±SEM, n=3; *P<0.05 LS vs OS; #P<0.05 compared with ST).

Figure 3

Ang2 is upregulated by OS compared with LS in vitro and at sites of disturbed flow in vivo. Confluent HUVECs were exposed to LS, OS, and ST as described in Figure 2. Cell lysates and CM were used for Western blots with antibodies to Ang2 (A) and total RNA was used for real-time PCR analyses of Ang2 mRNA (B). C57/BL6J mice were pressure fixed with 10% formalin, and the aortic arch and thoracic aorta were isolated. The tissue was incubated with primary antibodies against Ang2 (C & D), Ang1 (F & G) or secondary antibody only (rhodamine-red conjugated secondary; E & H). The greater curvature (GC) and lesser curvature (LC) were isolated and splayed open on a microscope slide, endothelium facing up. Blue indicates nuclei stained with DAPI. Shown are representative confocal images at 63× magnification (n=5).

Figure 4

Ang2 knockdown by siRNA inhibits endothelial cell migration. A, HUVECs were treated with 50 nmol/L of nonsilencing siRNA (ns) or Ang2 siRNA (Si, siAng2) for 2 days. Cells were then sheared (LS, OS, and ST) for 24 hours as in Figure 2. Cell lysates and CM were Western blotted with an Ang2 antibody, whereas Ang1 and β-actin antibodies were used as controls (A). Ang2 bands were quantified by densitometry (B, C). D, HUVECs transfected with nonsilencing siRNA or siAng2 were sheared for 24 hours and sheared cells were used in a scratch migration assay (mean±SEM, n=3 to 10; *P<0.05; #P<0.05 compared with ST).

Figure 5

Ang2 knockdown inhibits OS-mediated tubule formation and LS-inhibited tubule formation can be rescued by exogenous addition of Ang2. A, HUVECs treated with nonsilencing siRNA (NS) or siAng2 (si) as in Figure 4 were sheared (LS, OS, and ST for 1 day), and CM were collected. CM were then added to static HUVECs in a tubule formation assay. B, CM collected from sheared HUVECs (LS, OS, and ST for 1 day) were added to static HUVECs for tubule formation assay and Ang2 was added at the concentrations indicated. Shown are mean±SEM, n=3 to 6; *P<0.05; +P<0.05 compared with ST; ‡P<0.05 compared with LS-0; #P<0.05 compared with OS-0).