Supplementary MaterialsSupplemental data Supp_Data. oxide GPX1 synthase (eNOS) to promote production of nitric oxide (?NO). The importance of ?NO for angiogenesis is well established by studies showing decreased angiogenesis in response to hind-limb ischemia and decreased Matrigel plug angiogenesis (19, 15) in mice lacking eNOS (26, 29, 31). In addition, decreased ?NO bioavailability due to disease may contribute to increased vascular permeability (22); decreased ?NO-induced vessel relaxation (4, 21); and decreased angiogenesis (18). However, the mechanisms of ?NO action in ECs and angiogenesis are not well understood. Innovation Endothelial cell (EC) migration is required for both physiological and pathological angiogenesis. Nitric oxide (?NO)\dependent signaling is required for vascular endothelial growth factor (VEGF)\induced EC migration, but the protein targets that may be redox regulated are poorly understood. Here, we present novel evidence that control. DETA NONOate, diethylenetriamine NONOate; HAEC, human aortic endothelial cell; LNAME, N (G)-nitro-L-arginine methyl ester; ?NO, nitric oxide; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; VEGF, vascular endothelial growth factor. Knockdown of the SERCA2 isoform inhibits Ca2+ uptake and prevents VEGF-induced increase in EC migration Because ECs contain both SERCA3 and the SERCA2 splice isoform, SERCA2b (5, 13, 20), the specific contribution of SERCA2b to VEGF-induced signaling, was assessed by knockdown of SERCA2 in HAECs using selective siRNA. Knockdown was confirmed by both quantitative real time-polymerase chain reaction (qRT-PCR) for SERCA2 mRNA and western blot for SERCA2b with an isoform-specific antibody (Fig. 2A, B). Cell viability was confirmed by trypan blue exclusion (data not shown). In addition, the basal SERCA activity, assessed by 45Ca2+ uptake, was markedly inhibited by knockdown of SERCA2, indicating that it is the major isoform in these cells (Fig. 2C). Wound-healing migration assays over 6?h showed no change in unstimulated migration between ECs treated with scrambled siRNA controls BILN 2061 cost and siRNA against SERCA2, indicating that basal migration is not dependent on SERCA2 levels (Fig. 2D). However, knockdown of SERCA2 entirely prevented the VEGF-induced increase in HAEC migration, demonstrating an essential role for SERCA2 in VEGF-stimulated EC migratory signaling. Although SERCA3 was also present in these cells, as shown by Western blot and low levels of mRNA as measured by qRT-PCR, ECs treated with SERCA3 siRNA displayed 45Ca2+ uptake similar to that of those treated with scrambled siRNA BILN 2061 cost and migrated similarly to controls in response to VEGF (Supplementary Fig. S2). Open in a separate window FIG. 2. siRNA-mediated knockdown of SERCA2 prevents VEGF-induced endothelial cell migration. HAECs were treated with specific siRNA to SERCA2. Knockdown was confirmed by qRT-PCR for SERCA2 (A) and immunoblot for SERCA2 [(B), Control. Stimulation of EC migration by VEGF is mediated by S-glutathiolation of SERCA2b and can be prevented by overexpression of BILN 2061 cost Glrx-1 To assess control). HAECs overexpressing LacZ or Glrx-1 were seeded on Matrigel in the presence or absence of VEGF (D) and assessed for tube formation over 24?h (LacZ control, ***control). Migration over 6?h in response to DETA NONOate [(C), vehicle control, **(9) was given at the time of the scratch in a serum-free medium. Images were taken at 0 and 6?h at three fixed locations along the scratch (Supplementary Fig. S1). Migration distances were averaged from the three measurements per condition using ImageJ software, and this was considered as capillary tube-like formation on Growth Factor Reduced Matrigel (BD Biosciences) was performed as previously described (24). Briefly, 96-well plates were coated with Matrigel according to the manufacturer’s instructions. Appropriately treated HAECs were seeded at a density of 1104 cells/cm2 with or without 50?ng/ml VEGF in low-serum endothelial growth media (Lonza) and incubated at 37C overnight. Images were taken at 24?h with the scale indicated in the images, and tube formation was quantified by scoring for the tube number by observers blinded to sample.