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Altered angiogenesis and survival in human tumor-derived endothelial cells¹

Dipartimento di Medicina Interna and Dipartimento di Scienze Cliniche e Biologiche, Università di Torino and Centro Ricerca Medicina Sperimentale (CeRMS), Ospedale S. Giovanni Battista, Italy

²Correspondence: Dipartimento di Medicina Interna, Ospedale S. Giovanni Battista, Corso Dogliotti 14, 10126, Torino, Italy. E-mail: giovanni.camussi{at}unito.it

SPECIFIC AIMS

The concept that newly formed and remodeled vessels are different from the quiescent vessels present in adult tissues is at the basis of the use of specific angiogenesis inhibitors in the treatment of cancer. However, the phenotypic and functional characteristics of tumor-derived endothelial cells are poorly known. The aim of the present study was to establish and characterize at a functional and molecular level endothelial cell lines derived from highly vascularized human renal carcinomas. We studied in vitro and in vivo the angiogenic characteristics, sensitivity to apoptosis, and the proadhesive properties of two different tumor endothelial cell (TEC) lines compared with normal endothelial cells. To investigate the differences at a molecular level, we studied by gene array technology the expression of genes related to the oncogenic transformation, cell survival, adhesion, and angiogenesis and the role of the PI3K/Akt survival-dependent pathway. Using soluble chimeric receptor inhibitors, we evaluated whether autocrine angiopoietin-1 (Ang-1) and vascular endothelial growth factor-D (VEGF-D) stimulation was involved in the functional properties of TEC.

PRINCIPAL FINDINGS

1. TEC did not undergo senescence and showed constant expression of markers of endothelial activation and angiogenesis
TEC were purified from nine renal cell carcinomas using magnetic beads coupled to endoglin (CD105). Six endothelial cell lines were established whereas three primary endothelial cultures lasted for only few passages. The established TEC lines did not undergo the process of senescence in vitro normally observed for endothelial cells and were able to proliferate for >50 culture passages. All TEC were characterized by cytofluorometric analysis on the basis of positive expression of a panel of endothelial markers, such as vWF, CD105, Pal-E, Muc-18, E-selectin, ICAM-1, and the fucosylated receptors for plant lectins and by negative staining with mAbs against renal carcinoma (Uro-3) and cytokeratin. Similar expression of endothelial markers was detected on normal human microvascular endothelial cells (HMEC). Expression of cytokeratin, but not of vWF or CD105, was detected in the carcinoma cells derived from the corresponding tumor. TEC showed constitutive expression of growth factor receptors known to be up-regulated in tumor vessels, as they expressed VEGF receptor (VEGFR) -1, VEGFR-2, VEGFR-3, and the angiopoietin receptor tie-2. TEC expressed, besides HLA class I, the HLA-G antigen, a nonclassical class I antigen expressed mainly at the maternofetal interface where it is thought to protect the fetus from the maternal cellular immunity.

2. TEC showed enhanced survival, proadhesive, and angiogenic properties
Two representative cell lines,TEC-25 and TEC-28, were used for further functional and molecular studies. One of the most peculiar characteristics of endothelial cells is to undergo apoptosis in the absence of serum. At a variance of normal HMEC and endothelial cells derived from the umbilical vein (HUVEC), TEC were resistant to serum starvation and vincristine-induced apoptosis, as detected by TUNEL assay and cell cycle analysis. TEC were able to proliferate at low serum concentration (0–5%). In dynamic conditions, labeled renal tumor epithelial cells incubated with an endothelial monolayer exhibited enhanced adhesion to TEC compared with normal HMEC or HUVEC. Like normal HMEC, TEC-25 and TEC-28 showed the ability to form tubes when plated onto Matrigel with serum. In contrast to normal HMEC, which rapidly undergo apoptosis when plated onto Matrigel in the absence of serum, the structures formed by TEC persisted 7 days in 0% serum. In vivo, TEC-25 and TEC-28, when s.c. injected in diluted Matrigel in SCID mice, grew and spontaneously organized within 1 wk in functional human microvessels connected to the mouse vasculature (Fig. 1 ). In contrast, normal endothelial cells in the same conditions did not form vascular structures.

View larger version (70K): [in this window] [in a new window]   Figure 1. In vivo angiogenesis induced by TEC. TEC implanted s.c. in SCID mice within diluted Matrigel organized in vascular structures connected with the mouse circulation within 7 days. A, B) Light microscopy presenting TEC organized in vascular structures containing red blood cells and leukocytes (x200). Inset: A sequential section shows immunofluorescence positivity for smooth muscle actin of the two vessels of panel B indicating the presence of smooth muscle cells. C, D) Double immunofluorescence staining for vWF (C) and human HLA class I (D) indicating the human origin of endothelial cells forming vessels within Matrigel (x400). E) Expression of human Pal-E by endothelial cells forming vessels in Matrigel (x200). F, G) Presence of anastomosis among human TEC (stained in red with the vital PKH26 cell dye before injection) and mouse endothelial cells (stained with an anti-mouse FITC labeled-CD31 mAb) at the periphery of the implant. Arrows shown chimeric vessels formed by human and mouse endothelial cells at lower (F, x160) and higher (G, x250) magnification.

3. TEC showed a differential expression of genes related to cell survival, adhesion, and angiogenesis in respect to normal endothelial cells
With twofold expression as the cutoff threshold, gene array analysis showed a differential expression between TEC and normal HMEC of several genes related to apoptosis, adhesion, and angiogenesis. We found in TEC-25 and TEC-28, but not in HMEC, the expression of genes known to be involved in endothelial survival, such as the IAP and the bcl-2 families. Several genes related to cell adhesion such as integrins Muc-18, ICAM-1, CD44, and the neural cell adhesion molecule NCAM were up-regulated. Regarding angiogenesis, gene array confirmed the increase in VEGFR-2 in TEC compared with HMEC. TEC up-regulated the genes for Ang-1, for angiogenin and for the lymphatic growth factor VEGF-D and down-regulated the VEGF-A gene. A similar up-regulation of Ang-1, angiogenin, and VEGF-D was observed in the endothelial-like tumor cells obtained by a Kaposi's sarcoma patient. The increased protein expression of VEGF-D and Ang-1 in TEC was confirmed by Western blot analysis and immunofluorescence staining on TEC in culture and on vascular structures in vivo.

4. TEC showed enhanced Akt activation and decreased expression of PTEN
We compared, after serum starvation, Akt phosphorylation and Akt kinase activity of TEC and HMEC. TEC exhibited enhanced levels of Akt phosphorylation and kinase activity (Fig. 2 ). That Wortmannin, an inhibitor of PI3 kinase, abrogated Akt phosphorylation and Akt kinase activity in TEC as well as the resistance of apoptosis induced by serum starvation suggests an involvement of the PI3 kinase/Akt pathway in the survival signal of TEC. Expression of the tumor suppressor PTEN, which functions as a direct antagonist of PI3 kinase-dependent signaling, was reduced relative to normal HMEC (Fig. 2) . Blockade of VEGF-R-(2–3)-VEGF-D interaction by soluble chimeric Flt-4 (sFlt-4), but not of Tie-2/Ang-1 interaction by soluble Tie-2 (sTie-2), reduced Akt activation and TEC resistance to apoptosis (Fig. 2) . Expression of PTEN by TEC was enhanced after incubation with sFlt-4 and, to a lesser extent, with sTie-2 (Fig. 2) . Both sFlt-4 and sTie-2 inhibited the ability of TEC to form and maintain for up to 7 days tubular structures on Matrigel in the absence of serum.

View larger version (33K): [in this window] [in a new window]   Figure 2. Effect of sFlt-4 and sTie-2 on Akt kinase activity and apoptosis of TEC. A) Kinase reactions and Western blot analysis were performed on anti-Akt immunoprecipitates obtained from cell lysates of HMEC (row 1), TEC-25 (rows 2–4), and TEC-28 (rows 5–7). The fold increase over HMEC was evaluated by densitometric analysis. Treatment with 100 ng/mL sFlt-4 (rows 4 and 7) but not with 100 ng/mL sTie-2 (rows 3 and 6) reduced the Akt kinase activity. Data are representative of 3 experiments. B) Western blot analysis of expression of PTEN from cell lysates of HMEC (row 1), TEC-25 (rows 2–4), and TEC-28 (rows 5–7). Equal protein loading was verified by ß-actin detection. Treatment with 100 ng/mL sFlt-4 (rows 4 and 7) and, to a lesser extent, with 100 ng/mL sTie-2 (rows 3 and 6) increased PTEN. Fold variation over HMEC was evaluated by densitometric analysis. Data are representative of 3 experiments. C) Inhibitory effect of sFlt-4, but not of sTie-2, on the survival of TEC, as evaluated by TUNEL assay after 48 h serum starvation. Results are the mean ± SE of 5 experiments. ANOVA with Dunnett’s multicomparison test was performed between vehicle vs. sTie-2 or vs. sFlt-4 (*P<0.05).

CONCLUSIONS

Tumor angiogenesis is critical to the development and progression of cancer and is currently regarded as a potential therapeutic target. However, it is becoming clear from preliminary trials that some antiangiogenic drugs or strategies can lose their activity over time. Several potential mechanisms have been evoked, such as the great redundancy of tumor-secreted angiogenic growth factors and the antiapoptotic properties of TEC dependent on either direct cell interaction or epigenetic changes occurring after persistent cell activation.

We demonstrate here that the two TEC lines studied displayed a different phenotype from that of normal endothelial cells. In addition to the common vascular endothelial markers, TEC expressed most of the markers of endothelial activation and angiogenesis. Moreover, TEC expressed HLA-G, a nonclassical class I antigen is thought to protect the fetus from the maternal cellular immunity. Therefore, HLA-G expression by TEC may contribute to the immuno-escape of tumors.

Functional studies demonstrate that TEC exhibited altered proadhesive, survival, and angiogenic properties with respect to normal endothelial cells. The increased adhesion of renal tumor cells to TEC is consistent with the enhanced expression of adhesion molecules as detected by gene array and FACS analysis. This characteristic may be relevant not only for stimulation of angiogenesis, but also for the entry of tumor cells into the circulation. In vitro, TEC did not undergo senescence and showed resistance to apoptosis induced by serum starvation or by low doses of vincristine. At variance with normal endothelial cells, TEC were able to growth and to organize in the absence of serum in capillary-like structures, persisting for up to 1 wk. The ability of TEC to form vessels in vivo in immunodeficient mice may be related to their prosurvival phenotype, which was supported at the molecular level by the expression of several antiapoptotic genes. We found that the PI3 kinase/Akt pathway, a major player in cell survival, was up-regulated in TEC (Fig. 3 ). In parallel, tumor suppressor PTEN, a phosphatase that regulates the PI3 kinase-dependent activation of Akt, was reduced in TEC vs. normal endothelial cells. We observed that TEC expressed several proangiogenic growth factors, including Ang-1 and VEGF-D. The experiments performed with soluble recombinant inhibitors of VEGFR2–3-VEGF-D and tie-2-Ang-1 interaction indicated that autocrine production of these growth factors is relevant for Akt activation and for the proangiogenic and survival characteristics of TEC (Fig. 3) . These characteristics of TEC are similar to those of Kaposi’s sarcoma, a tumor of vascular origin, and independent of the presence of tumor cells, as they are maintained in cell culture, suggesting a persistent change in their phenotype. This could be due to epigenic mutations or to viral oncogenes. Alternatively, it can be speculated that TEC originate from endothelial precursors displaying an enhanced angiogenic potential, or from dedifferentiated tumor cells (Fig. 3) .

View larger version (51K): [in this window] [in a new window]   Figure 3. Tumor-derived endothelial cells (TEC) obtained from renal carcinomas show enhanced proangiogenic characteristics maintained in the absence of the original tumor. TEC display enhanced survival and stimuli-independent growth possibly due to the autocrine production of growth factors, including angiopoietin-1 and VEGF-D. These characteristics are similar to those of vascular tumor cells. TEC are proadhesive for tumor cells (T), showing expression of several adhesion molecules. Moreover, TEC express the HLA-G antigen, mainly involved in the fetal–maternal tolerance, suggesting a possible role in tumor protection from the immune system (L=lymphocyte). The question mark indicates a possible hypothesis to explain the altered characteristics of TEC.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0557fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0557fje

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