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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0693fje. |
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2
Departments of
* Cancer Biology and
Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA, and
SUGEN Inc., South San Francisco, California 94080, USA
2Correspondence: The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 444, Houston, TX 77030-4009, USA. E-Mail: lellis{at}mdanderson.org
SPECIFIC AIMS
We hypothesized that platelet-derived growth factor (PDGF-BB), a cytokine released from tumor and endothelial cells (ECs), may induce vascular endothelial growth factor (VEGF) expression in pericytes or human vascular smooth muscle cells (hVSMCs) via specific intracellular signaling pathways, which in turn may enhance EC survival in a paracrine manner. We also investigated the effect of conditioned medium (CM) from colon cancer cell lines and ECs on VEGF induction in perivascular cells and the ability of an inhibitor of the PDGF receptor (PDGFR) to block this effect.
PRINCIPAL FINDINGS
1. PDGF-BB induces VEGF in a time- and dose-dependent manner in
perivascular cells (pericyte-like cell lines)
The effect of PDGF-BB on VEGF induction was studied in murine
10T1/2 cells (as presumptive mural cell precursors) and hVSMCs by
Northern blot analysis. Both cell lines showed a maximal increase in
VEGF mRNA after 2 h incubation with PDGF-BB starting at a dose of
5 ng/ml. Treatment of PDGF-BB resulted in a threefold increase in VEGF
promoter activity as determined by transient transfections of 10T1/2
cells with VEGF promoter-reporter constructs. PDGF-BB further increased
the transcription of VEGF mRNA but did not alter VEGF mRNA stability.
2. PDGF-BB induces VEGF in 10T1/2 cells via the
phosphatidylinositol 3-kinase (PI3-K)-Akt pathway
Western blot analysis demonstrated that PDGF-BB (20 ng/ml)
increased the phosphorylation of Erk1/2 and Akt in 10T1/2 cells within
1 and 2 min of incubation, respectively. Whereas U0126 (10 µM) and
wortmannin (0.2 µM) effectively inhibited the phosphorylation of
Erk1/2 and Akt, respectively, only blockade of the Akt pathway
(wortmannin) led to a nearly complete suppression of VEGF mRNA
induction.
3. CM from colon cancer cells and ECs also induces VEGF in 10T1/2
cells, which can be blocked by the PDGFR tyrosine kinase inhibitor
SU6668
Since human umbilical vein endothelial cells (HUVECs)
and human colon cancer cell lines (SW620 and HT29) are known to produce
PDGF-BB, 10T1/2 cells were incubated with CM from these lines. Both
SW620 and HT29 CM increased VEGF mRNA expression above that produced by
PDGF-BB alone, whereas HUVEC-derived CM induced VEGF to a level
similar to PDGF-BB. As a control, CM derived from nonmalignant
human lung fibroblasts (MRC5) did not induce VEGF mRNA expression.
SU6668 (10 µM), a tyrosine kinase inhibitor of the receptors for
PDGF, VEGF (flk-1/KDR), and basic fibroblast growth factor (bFGF),
blocked intercellular signaling induced by PDGF-BB and prevented VEGF
mRNA up-regulation by colon cancer CM (Fig. 1
).
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4. PDGF-BB pretreated hVSMCs protect ECs from apoptosis in a
paracrine manner that is partly mediated by VEGF
HUVECs were incubated with ‘activated’ (pretreated with PDGF-BB)
or normal hVSMC-derived CM, and EC apoptosis was evaluated 24 h
later by TUNEL staining. Nonactivated hVSMC derived CM had no
significant effect on apoptosis in HUVECs (Fig. 2
)whereas CM derived from ‘activated hVSMCs significantly protected
HUVECs from apoptosis (P<0.001; two-tailed Student’s
t test). This effect was not attributable directly to
residual PDGF-BB in the activated CM since PDGF-BB added to CM did not
effect EC survival. Blocking VEGF in the activated CM using a specific
neutralizing antibody against VEGF165
significantly reversed the protective effect of activated CM on EC
apoptosis. However, activated CM treated with anti-VEGF antibody did
not completely reverse the protective effect of activated CM on EC,
suggesting that pericytes or hVSMCs protect ECs from apoptosis by
multiple mechanisms.
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CONCLUSIONS
EC survival mechanisms are essential for embryologic
vasculogenesis and tumor angiogenesis. VEGF is known as an EC survival
and angiogenic factor. However, a significant percentage of the tumor
vasculature is also composed of pericytes. Mature developing blood
vessels are defined as those with colocalized ECs and pericytes. It has
recently been recognized that pericytes are also associated with tumor
vessels. Pericytes are believed to be generated from mesenchymal cells
by in situ differentiation or by migration and dedifferentiation of
arterial smooth muscle cells. In the developing mouse, EC-derived
PDGF-BB mediates migration of pericytes toward the sprouting
capillaries. Collectively, pericytes are believed to stabilize the
vasculature; however, the detailed mechanism of this phenomenon remains
to be elucidated. Besides protection of EC apoptosis by cell–cell
contact, our studies have led us to hypothesize that there is an
additional paracrine loop by which pericytes enhance EC survival. Since
there are no true human pericyte cell lines, we used two pericyte-like
cell lines for our investigations (10T1/2 cells, hVSMCs). We
demonstrated that PDGF-BB induced VEGF mRNA expression in both 10T1/2
cells and hVSMCs, which in turn affected EC survival (Fig. 3
). The induction of VEGF mRNA by PDGF-BB was secondary
to enhanced transcription primarily via the PI3-K signaling pathway.
|
The majority of investigations demonstrating the effect of pericytes on EC survival have been done in developmental embryology and maturation of the retinal vascular network. Relatively little work has been done investigating pericyte function in the tumor vasculature. In addition to ECs, a variety of malignant cell lines including colon cancer cell lines secrete PDGF and PDGF-like proteins. Both colon cancer cell lines used in our experiments have been shown to secrete PDGF-BB into culture medium. In contrast, bFGF is not expressed in HT-29 colon cancer cells. HUVEC and colon cancer cell CM led to 4- and 10-fold induction of VEGF mRNA in pericyte-like cells, respectively, in contrast to CM derived from lung fibroblasts. The extent of VEGF induction in 10T1/2 cells by CM derived from colon cancer cell lines was even greater than that achieved by PDGF-BB, suggesting that their CM contains additional factors that contribute to VEGF induction in pericyte-like cells.
VEGF is known to protect ECs from apoptosis. We therefore determined whether hVSMC-derived VEGF has a biological effect on ECs. hVSMCs exposed to PDGF-BB secreted biologically active VEGF into the medium; it in turn significantly protected HUVECs from apoptosis caused by serum starvation. PDGF-BB treatment alone had no effect on EC survival. Neutralizing VEGF165 activity in activated CM using specific antibodies significantly reduced its anti-apoptotic effect. This implies that perivascular cells activated by PDGF-BB can exert a stabilizing function on ECs by inducing VEGF. However, it is clear that pericytes enhance EC survival by other means as well, e.g., possibly by direct cell–cell contact.
Previous studies from our laboratory have demonstrated that SU6668 markedly inhibits growth and vascularity of colon cancer liver metastases in vivo and induces EC apoptosis in tumors. SU6668-treated mice survive significantly longer than untreated mice with hepatic metastasis. The effect of SU6668 as an anti-angiogenic agent was previously ascribed primarily to its blockade of the VEGF, bFGF, and PDGF receptors on ECs. Here we hypothesize another major mechanism for SU6668 induction of EC apoptosis: blockade of the PDGF-receptor on pericytes, inhibiting the pericyte’s ability to enhance EC survival. In this study, pretreatment of pericyte-like cells with SU6668 completely inhibited VEGF induction by both PDGF-BB and colon cancer cell-derived CM. Further, SU6668 blocked PDGF-BB induction of phosphorylation of Akt, thereby inhibiting this downstream pathway. As a result, we hypothesize that in our previous in vivo studies the perivascular cells produced less VEGF, possibly contributing to apoptosis of the tumor ECs. Therefore, therapy with SU6668 may render the vasculature more vulnerable to anti-angiogenic therapy due to inhibition of pericyte function.
In conclusion, our data demonstrate that treatment of pericyte-like cells with PDGF-BB leads to VEGF induction, which in turn enhances EC survival in a paracrine manner. CM-derived from activated hVSMCs protected ECs from apoptosis, which was partially mediated by VEGF. Colon cancer cells and ECs may both contribute to this model by secreting PDGF-BB, which in turn induces VEGF mRNA in pericyte-like cells. Therefore, in order to disrupt this complex cross-talk between colon cancer cells, pericytes and the vessel endothelium, anti-angiogenic therapy might be more efficient targeting several tyrosine kinase receptors simultaneously, including the VEGF and PDGF receptors. Treatment of pericyte-like cells with SU6668 completely inhibited VEGF mRNA induction by PDGF-BB or colon cancer CM. Additional studies are necessary to further elucidate the function of perivascular cells in tumor angiogenesis and develop a better understanding of how cancer cells module EC and pericyte function and survival. With a more detailed understanding of angiogenesis in cancer biology, targeting of selected growth factors or their receptors on various cell types involved in angiogenesis may lead to improved therapeutic strategies for cancer patients.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0693fje ; to cite this
article, use FASEB J. (March 5, 2001)
10.1096/fj.00-0693fje 
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