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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 3, 2004 as doi:10.1096/fj.04-1604fje. |
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* Divisions of Nephrology and Hematology-Oncology and Center for Study of the Tumor Microenvironment, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA;
Division of Nephrology, Yale University School of Medicine, New Haven, Connecticut, USA; and The
Department of Cell Biology and Anatomy, University of Miami, Florida, USA
1Correspondence: Renal Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston 02215, MA, USA. E-mail: vsukhatm{at}bidmc.harvard.edu
SPECIFIC AIMS
In the vasculature, pericytes and endothelial cells are closely apposed to each other. Communication between these two cell types is essential for normal blood vessel development. In this study, we aimed to identify genes that are differentially expressed when 10T1/2 cells differentiate to pericytes in vitro.
PRINCIPAL FINDINGS
1. Identification of genes expressed during the differentiation of 10T1/2 cells to pericytes using cDNA microarray analysis
We used the well-characterized in vitro model for differentiation of 10T1/2 cells to pericytes. Cells were induced to differentiate in the presence of TGF-ß1. Microarray analysis revealed an entire angiogenic program of genes being expressed during this differentiation process.
2. Validation of protein expression corresponding to genes identified by microarray analysis
Several gene transcripts that were differentially expressed in the pericytes viz., VEGF-A, IL-6, integrin 
5, EphA2 were further analyzed in cultures grown in vitro by immunocytochemistry as well as by Western blot.
3. Functional significance of genes identified by cDNA microarray analysis
a. Effect on HUVE cells
Under specific conditions in 2-dimensional cocultures of 10T1/2 and HUVE cells, HUVE cells formed cord-like structures.10T1/2 cells that were in contact with the HUVE cells differentiated to pericytes. This was not observed when both cell types were cultured individually. This cord like structure formation by ECs could be disrupted when neutralizing antibodies to VEGF, IL-6 were added. Anti integrin 5 did not have any effect (Fig. 1
)
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b. Effect on 10T1/2 cells
In cocultures of 10T1/2 and HUVE cells, HUVE cells induced the differentiation of 10T1/2 cells that were in contact with them. In the presence of neutralizing antibodies to VEGF and IL-6, the % age of differentiated 10T1/2 cells decreased by 50–70%. (Fig. 2
)
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4. Role of the identified genes in 10T1/2 cell differentiation
10T1/2 cells were induced to undergo differentiation in the presence of TGF-ß1. In the presence of neutralizing antibodies to VEGF, the differentiation of 10T1/2 cells as determined by SMA was considerably reduced.
CONCLUSIONS AND SIGNIFICANCE
This is the first study to show changes in gene expression during pericyte differentiation. Our microarray analysis revealed an entire "angiogenic program" of gene expression. We validated the expression of some of these genes at the protein level in in vitro cultures of induced vs. uninduced 10T1/2 cells, viz., HB-EGF, VEGF-A, VEGF-C, IL-6, integrin 5, tenascin. In addition, we examined expression of CTGF, an ECM protein known to be specifically induced by TGF-ß. Several of these proteins have been implicated in angiogenesis.
Production of angiogenic factors suggest an auto- and/or paracrine loops of regulation.
Our data suggest that VEGF-A plays an autocrine role in differentiation of 10T1/2 cells to pericytes.
Cocultures of 10T1/2 cells and ECs were established where a role for some of these factors was studied. In these cocultures, each cell type influenced the other, both via factors produced as well as cell-cell contact. HUVEC cells formed cord-like structures and 10T1/2 cells differentiated to pericytes. Both these effects were significantly reduced when neutralizing antibodies to anti-VEGF or anti-IL-6 were added.
The results of this study suggest that pericytes may play a much greater role than previously anticipated in inducing angiogenesis (Fig. 3
). Pericytes may initiate angiogenesis by secreting factors that cause ECs to migrate and proliferate. Pericytes would then line the blood vessels and stabilize them. So far, anti-VEGF therapy has been used to regress immature blood vessels but has had no effect on stable pericyte lined blood vessels. Knowing which genes are expressed during the differentiation of mesenchymal precursors to pericytes may provide yet another level of interrupting the process of angiogenesis, especially with respect to a stable, pericyte-lined blood vessel.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1604fje;
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