Endothelial TMEM16F lipid scramblase regulates angiogenesis

Abstract

Dynamic loss of lipid asymmetry through the activation of TMEM16 Ca²⁺-activated lipid scramblases (CaPLSases) has been increasingly recognized as an essential membrane event in a wide range of physiological and pathological processes, including blood coagulation, microparticle release, bone development, pain sensation, cell-cell fusion, and viral infection. Despite the recent implications of TMEM16F CaPLSase in vascular development and endothelial cell-mediated coagulation, its signaling role in endothelial biology remains to be established. Here, we show that endothelial TMEM16F regulates in vitro and in vivo angiogenesis through intracellular signaling. Developmental retinal angiogenesis is significantly impaired in TMEM16F deficient mice, as evidenced by fewer vascular loops and larger loop areas. Consistent with our in vivo observation, TMEM16F siRNA knockdown in human umbilical vein endothelial cells compromises angiogenesis in vitro. We further discovered that TMEM16F knockdown enhances VE-cadherin phosphorylation and reduces its expression. Moreover, TMEM16F knockdown also promotes Src kinase phosphorylation at tyrosine 416, which may be responsible for downregulating VE-cadherin expression. Our study thus uncovers a new biological function of TMEM16F in angiogenesis and provides a potential mechanism for how the CaPLSase regulates angiogenesis through intracellular signaling.

Keywords: CaPLSase; TMEM16F; angiogenesis; endothelial cells; scramblase.

Figure 1.. TMEM16F is functionally expressed in endothelial cells.

(A) Representative western blots of TMEM16F in HUVECs with and without siRNA knockdown. (B) Densitometry quantifications of TMEM16F and loading control β-actin (n=4). (C) Schematic representation of fluorescence-based scrambling assay. PS: phosphatidylserine, AnV: Annexin V. (D) Representative images of Ca²⁺ and AnV in control (left) and TMEM16F knockdown (right) HUVECs stimulated with 2.5 μM ionomycin. (E, F) Quantifications of the time course (E) or the maximum fluorescence intensity of AnV at 10 minutes post ionomycin stimulation (F) for control siRNA (n=37) and TMEM16F (n=46). Each dot represents AnV signals from one cell. Data are presented as mean±S.E.M. ****P<0.0001, two-tailed t-test. (G) Representative currents recorded in control siRNA- and TMEM16F siRNA-treated HUVECs. The currents were elicited by a voltage step protocol from −100 mV to +160 mV with a 20 mV increment. Holding potential was set at −60 mV. (H) Current-voltage (I-V) relationship of currents recorded in (G). Data are presented as mean±S.E.M. Currents at +160 mV were compared with two-tailed t-test ****P<0.0001 (n=7 for each group).

Figure 2.. TMEM16F deficiency suppresses angiogenesis in vivo.

(A-B) IB4 (green) staining of retinal whole mount from P5 WT (A) and TMEM16F KO (B) littermate. White boxes indicate enlarged area shown in the middle panels. The bottom panels illustrate processed enlarged area used for the calculations of number of loops and hole areas. White areas are defined as holes and vasculature (black lines) encircling the holes as loops. (C-E) Quantification of retinal vasculature coverage (C), average hole areas (D), and number of loops (E) calculated from four random fields of each retina. Data are presented as mean±S.E.M. Each dot represents data from one animal (n=7 pairs of WT and TMEM16F KO littermates). *P<0.05, **P<0.01, ***P<0.001, two-tailed t-test.

Figure 3.. TMEM16F deficiency in HUVEC impairs in vitro angiogenesis.

(A) Representative images of tube-forming control and TMEM16F knockdown HUVECs after seeded on Matrigel. Yellow arrowheads indicate the thin and stretched tubes. (B) Quantification of loop numbers of control (n=20) and TMEM16F (n=20) knockdown HUVECs at 24 hours after seeding. Each dot represents data from one well of tube formation μ-Slide. Data are presented as mean±S.E.M. ****P<0.0001, two-tailed t-test.

Figure 4.. TMEM16F knockdown reduces VE-cadherin expression and promotes the phosphorylation of VE-cadherin and Src.

(A,B) Representative western blot of anti-VE-cadherin (A) and densitometry quantifications (n=9) (B). (C,D) Representative western blot of anti-Tyr685 phosphorylated and anti-total VE-cadherin (C) and densitometry quantifications (n=5) (D). (E,F) Representative western blot anti-Tyr416 phosphorylated and anti-total Src (E) and densitometry quantifications (n=4) (F). (G) Proposed role of TMEM16F scramblase in angiogenesis. TMEM16F deficiency increases Src phosphorylation and promotes its activation, which downregulates VE-cadherin and leads to defective angiogenesis. Data are presented as mean±S.E.M. **P<0.01, ***P<0.001, ****P<0.0001, two-tailed t-test.