Activation of endothelial ras signaling bypasses senescence and causes abnormal vascular morphogenesis

Abstract

Angiogenesis is crucial for embryogenesis, reproduction, and wound healing and is a critical determinant of tumor growth and metastasis. The multifunctional signal transducer Ras is a proto-oncogene and frequently becomes mutated in a variety of human cancers, including angiosarcomas. Regulation of Ras is important for endothelial cell function and angiogenesis. Hyperactivation of Ras is linked with oncogene-induced senescence in many cell types. Given links between vascular malformations and angiosarcoma with activated Ras signaling, we sought to determine the consequence of sustained Ras activation on endothelial cell function. We find that sustained Ras activation in primary endothelial cells leads to prolonged activation of progrowth signaling, accompanied by a senescence bypass, enhanced proliferation, autonomous growth, and increased survival. Moreover, Ras severely compromises the ability of these cells to organize into vascular structures, instead promoting formation of planar endothelial sheets. This abnormal phenotype is regulated by phosphoinositide 3-kinase signaling, highlighting the therapeutic potential of agents targeting this axis in dealing with vascular morphogenic disorders and vascular normalization of tumors.

Figure 1. Inducible expression of activated Ras in primary endothelial cells

(A) pSLIK-Ras^V12-Venus lentiviral vectors were used to obtain HUVECs stably expressing activated Ras (B) infection efficiencies are shown by the histograms for Venus expression. (C) Cells were induced with doxycycline and Ras expression was measured. (D) Serum starved HUVECs were induced with 50 ng/ml doxycycline and whole cell lysates were probed as indicated. Data are representative of three independent infections on independent lots of endothelial cells.

Figure 2. Activated Ras induces growth arrest in primary melanocytes but not in endothelial cells

(A) Control (uninduced) and Ras expressing melanocytes and endothelial cells were grown in complete growth media and population doublings monitored. This is representative data from one experiment. Similar results were obtained in at least three additional experiments. (B) In parallel, lysates were probed for p16, Pan-Ras, and ERK2 after 7 days. (C) Control and Ras expressing endothelial cells were serum starved for 24 h after which BrdU incorporation was measured. The data is from one representative experiment and the error bars represent standard error (**P< 0.01) of triplicate determinations. Similar results were obtained in at least three additional experiments. (D) Cell lysates were made simultaneously to the experiments in (C) and probed for Cyclin D1, Pan-Ras, and Akt.

Figure 3. Activated Ras confers a survival advantage to primary endothelial cells

(A) Control (uninduced) and Ras expressing endothelial cells were cultured in minimal media and photographed at 0 and 72 h. (B) Cells at indicated times of serum-free culture were stained with crystal violet and extracted dye was quantified by measuring absorbance at 590 nm. Error bars represent standard error (**P< 0.01) of triplicate determinations from a single experiment. Similar results were obtained in three additional experiments. (C) Apoptosis was induced in endothelial cells by treating with serum-free M199 after which lysates were probed for cleaved caspase-3, HA-tag, and total caspase-3. (D) Normalized cleaved caspase-3 values obtained from at least three different experiments. The error bars represent standard error (**P< 0.01).

Figure 4. Effect of activation of Raf and Akt on endothelial cell proliferation and survival

(A) Cells were serum deprived in the absence (U) or presence (I) of doxycycline to induce protein expression. Cell lysates were probed as indicated. (B) Cells treated as above were stimulated with complete growth medium (GM) as indicated and pulsed with BrdU. Data represents the % BrdU positive cells compared to total cell number. The error bars represent standard error of triplicate determinations in one experiment. Similar results were obtained in two additional experiments. (C) Apoptosis was induced in endothelial cells by treating with serum free M199, and probing lysates for cleaved and total caspase 3.

Figure 5. Activated Ras prevents normal vascular morphogenesis

Uninduced and Ras expressing endothelial cells co-cultured with fibroblasts for 14 days and visualized by staining live endothelial cells with a FITC-tagged UEA-1 lectin. Ras expression was turned on in the uninduced cells by adding doxycycline and shut down in the induced cells by withdrawing doxycycline and the cells were again stained after an additional 14 days. The pictures (at 200X) are representative of data obtained from at least three independent experiments.

Figure 6. PI-3′-Kinase and Akt activation are principle contributors to the failed morphogenesis following Ras activation

(A) Uninduced and Ras expressing endothelial cells were co-cultured for 14 days after which the cells were treated with 5 μM U0126 or 5 μM LY294002 and cultured for an additional 7 more days. Cells were fixed and stained with CD31. The pictures (at 200X) are representative of data obtained from at least three independent experiments. (B) Endothelial cells expressing Ras effector mutants or GFP alone (control) were co-cultured with fibroblasts for 14 days prior to staining with CD31. The pictures (at 200X) are representative of data obtained from at least two independent experiments. (C) HUVECS infected with inducible lentiviruses coding for either active Raf1 or active Akt were plated with fibroblasts in the absence (uninduced) or presence (induced) of doxycycline. Visualization of ΔN-Raf1 vascular structures was done at 100X by staining with FITC- UEA-1 lectin. The myr-Akt infected cultures were visualized at 200X with FITC-UEA-1. (D) Lower power (50X) visualization of myrAkt expressing co-cultures compared to uninduced.