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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 12, 2002 as doi:10.1096/fj.01-0594fje. |
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,3
*
Clinical Research Unit for Multiple Sclerosis and Neuroimmunology, Dept. of Neurology, Julius-Maximilians-University of Würzburg, Germany; and
Department of Neurosurgery, Julius-Maximilians-University of Würzburg, Germany
2Correspondence: Department of Neurology, Julius-Maximilians-University, Josef-Schneider-Str. 11, D-97080 Würzburg, Germany. E-mail: b.kallmann{at}mail.uni-wuerzburg.de
SPECIFIC AIMS
Cerebral endothelial cells as major constituents of the blood-brain barrier (BBB) form the active interphase between systemic circulation and highly specialized neuronal and glial tissue. We aimed to identify a gene expression profile characteristic for human cerebral endothelial cells (HCEC) in comparison to human umbilical vein endothelial cells (HUVEC).
PRINCIPAL FINDINGS
1. Identification of specific gene expression pattern in HCEC by cDNA microarray analysis
Highly purified mRNA derived from endothelial cell preparations of either human brain or umbilical vein was compared by cDNA microarray analysis of 375 genes. The genes investigated included cytokines and related factors, growth mediators, angiogenic factors, and their receptors. Under basal culture conditions, 35 gene transcripts were detected only in HCEC whereas 20 mRNAs were present only in HUVEC. Seventy-eight genes were constitutively expressed in both endothelial cell types with distinct expression levels. Several candidate genes with differential mRNA expression were further analyzed at the protein level in the in vitro culture systems and immunostaining was performed on human brain and umbilical cord specimens.
2. Neurotrophic and growth-supporting factors
Brain-derived neurotrophic factor (BDNF) production (Fig. 1
F) was significantly higher in unstimulated HCEC than HUVEC (97 pg/ml vs. 16 pg/ml). Similarly, stem cell factor (SCF) (Fig. 1C
) and transforming growth factor ß2 (TGF-ß2) (Fig. 1D
) release was enhanced in HCEC (27 pg/ml resp. 57 pg/ml) compared with HUVEC (5 pg/ml resp. 3.2 pg/ml). These results correlated well with mRNA findings. cDNA microarray analysis showed differential expression of pleiotrophin and insulin-like-growth factor binding proteins (IGF-BP) -1, -5, -6 in association with HCEC.
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3. Vasculo- and angiogenic factors
In accordance with the results of cDNA microarray analysis, VEGF (106 pg/ml, Fig. 1A
) and follistatin (12.5 ng/ml, Fig. 1B
) were released from HCEC under basal conditions, whereas in supernatants from HUVEC neither VEGF nor follistatin could be detected. The epidermal growth factor receptor erbB1 protein was expressed in HCEC but not HUVEC. This finding corresponded with in situ analysis showing erbB1 expression associated with endothelium of cerebral capillaries in normal human brain but not umbilical veins (Fig. 2
). For genes with potential angiogenic properties like FGF-1, -5, and chemokines with ELR-motif (e.g., GRO-
and ENA-78), mRNA expression was detected only in HCEC.
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4. Immunoregulatory factors
IL-6 and monocyte chemoattractant protein 1 (MCP-1) were released at significantly higher amounts from HCEC (1034 pg/ml resp. 5807 pg/ml, Fig. 1E, G
) vs. HUVEC (71 pg/ml resp. 1130 pg/ml, Fig. 1E, G
). Immunoblotting and -histochemistry demonstrated higher decorin expression in HCEC. In situ analysis showed decorin only in association with endothelium of human cerebral capillaries but not of umbilical veins. Oncostatin M-receptor ß subunit (OSM-Rß) protein was detected only in HCEC and endothelium of cerebral capillaries; no expression was found in either HUVEC or umbilical vein endothelium in situ, confirming data obtained by cDNA array analysis.
CONCLUSIONS AND SIGNIFICANCE
In this study, we could demonstrate a distinct gene expression pattern characteristic for primary HCEC. The gene products specifically associated with cerebral endothelial cells have neuroprotective and growth-supporting as well as immunoregulatory properties or are involved in vasculo- and angiogenesis (Fig. 3
). Differential gene expression was first identified by cDNA array analysis in cerebral endothelial cell cultures in comparison to HUVEC. For several candidate genes, association with cerebral endothelium was also confirmed at the protein level in vitro as well as in human brain tissue.
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Several factors known to protect neurons and to support neuronal growth and differentiation were expressed in and released spontaneously from HCEC (Fig. 3)
. The neurotrophin BDNF was released at substantially higher amounts by HCEC. This finding extends former reports about endothelial BDNF production demonstrating quantitative differences. Other mediators like TGF-ß2, IL-6, and pleiotrophin demonstrated to be associated with HCEC are known to have neurotrophic and -protective properties. These data suggest that cerebral endothelial cells have the capacity to modulate neuronal and glial cell differentiation within the central nervous system (CNS). They add to evidence from a recently published study suggesting that proliferation of neuronal and glial progenitor cells occurs within the vascular niche of specific brain regions and that HCEC may provide important signals for this process.
Another important feature of cerebral endothelial cell function is the production of vasculo- and angiogenic factors and their receptors suggesting auto- or paracrine loops of regulation (Fig. 3)
. VEGF, one of the most potent endothelial mitogens inducing angiogenesis, is released by HCEC under baseline conditions but was not detectable in HUVEC. mRNAs of other factors with known angiogenic properties like FGF-1 and -5, follistatin, and CXC chemokines with ELR-motif like ENA-78 and GRO- were expressed only in HCEC. Protein expression of erbB1, involved in growth regulation, endothelial cell differentiation, and angiogenesis, was shown for the first time on HCEC and endothelium of human brain capillaries (Fig. 2)
, whereas HUVEC did not express erbB1. Decorin, involved in the angiogenic cascade, was found in clear association with HCEC and endothelium of brain capillaries.
The third group of genes differentially expressed in HCEC is involved in immunoregulation (Fig. 3)
. Several chemokines including MCP-1, MPIF-1, ENA-78, GRO-, and Eotaxin revealed a predominant association with HCEC. OSM-Rß, the specific subunit of the receptor for oncostatin M, was expressed only by HCEC in vitro and in situ; no mRNA or protein expression could be detected in HUVEC. This has important implications for immune cell transmigration across the BBB, as expression of OSM by characteristic cellular infiltrates in inflammatory diseases of the CNS was recently demonstrated by our group. TGF-ß2 released to a significantly higher extent by HCEC has potent immunosuppressive effects. Finally, higher production of IL-6 was found for HCEC.
The results of this study were obtained from well-defined cerebral endothelial cell cultures of high purity. Differential protein expression for selected genes of interest was verified in brain tissue. Therefore, it appears relevant to enhance research linked to specific organ-related endothelial functions in primary cultures of endothelial cells originated from the organ of interest—in our case, from human brain.
In summary, we demonstrated that cerebral endothelial cells are characterized by a unique pattern of gene expression differing from HUVEC. Genes strongly associated with HCEC indicate that these cells are actively involved in neuroprotection and cell differentiation, angiogenesis, and immunoregulation. The results underscore the outstanding functions of cerebral endothelial cells in relation to their localization at the BBB as part of the specific architecture of cerebral vasculature. The genes identified by this study as characteristic for cerebral endothelial cells may contribute to a better understanding of endothelial cell function at the BBB.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0594fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0594fje 
3 Current address: Department of Neurosurgery, Ludwig-Maximilians-University of Munich, Germany.
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