1. Endostatin's Antiangiogenic Signaling Network
Amir Abdollahi, Philip Hahnfeldt, Christian Maercker, Hermann-Josef Gröne, Juergen Debus, Wilhelm Ansorge, Judah Folkman, Lynn Hlatky, Peter E Huber 
Molecular Cell 
Volume 13, Issue 5, Pages (March 2004)
DOI: /S (04)

2. Figure 1
Differential Expression Pattern in Different Functional Groups
The distributions of gene expression induction/suppression (>2-fold upregulated [red]/>2-fold downregulated [blue]) among functional groups observed after 4 hr endostatin treatment of HDMVEC. Prior to the experiment we assigned 6635 Unigene clusters (“genes”) to 24 different functional groups. Within the regulated genes, “G protein coupled receptors,” “angiogenesis induction,” and “transcription factors” were more often found to be underexpressed after endostatin treatment (p < 0.05). In contrast, “stress” genes were more often found to be upregulated (p < 0.04). Transcripts encoding cytokines, cytokine receptors, cellular adhesion molecules, G proteins, and gene families associated with cancer tend to be underexpressed after endostatin treatment, while apoptosis and cardiovascular genes tend to be overexpressed.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 Real-Time Quantitative RT-PCR
Time course of mRNA expression of selected genes in HDMVEC treated with 200 ng/ml endostatin: untreated control, 0.5, 2, 4, 8, 12, 24, and 48 hr after endostatin treatment. The bars represent the mean ± SD (n = 4) normalized to control (A and B). Dose response of mRNA expression of selected genes in HDMVEC following 4 hr treatment with endostatin (C). Cells were treated with 0 (control), 100, 200, 500, and 1000 ng/ml endostatin. Bars represent mean ± SD (n = 4) normalized to control.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 Regulation on Protein Level
(A) Protein Phosphorylation on Antibody Array. Protein lysates from HDMVEC after 30 min incubation with 500 ng/ml endostatin were hybridized on an antibody array containing 400 immobilized antibodies against signaling proteins. The phosphorylation status of the tyrosine residues from the cellular proteins bound to the array was detected using a horseradish peroxidase-conjugated anti-phosphotyrosine antibody. 19 proteins were differentially-phosphorylated in response to endostatin involved in at least 5 distinct pathways (Table 2). Part of Control array (left panel) and endostatin treated array (right panel).
(B) Confirmation of phospho-JNK (p-JNK), and Id-1 protein regulation after endostatin treament by Western analysis. Id-1 protein level is decreased in endothelial cells after endostatin. In accordance with the antibody array data, a time-dependent JNK dephosphorylation is demonstrated.
(C) In vivo confirmation of Id-1 protein regulation by immunohistochemistry. A431 xenograft tumors were grown subcutaneously in nude mice, Id1 expression is markedly reduced by endostatin, in both tumor cells and the tumor endothelium (→).
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4 Protein Expression
Confocal images of immunocytochemistry of HDMVEC with and without 6 hr incubation with 500 ng/ml endostatin. After fixation, cells were washed and incubated with a monoclonal anti-Id-1 and anti-NF-κB primary antibody, washed, and then incubated with the Alexa-488 conjugated anti-rabbit secondary antibody (green). Id-1 (A and B) and NF-κB (C and D) protein are localized to the nuclear (red) and perinuclear cellular area. In accordance with the genomic data, Id-1, NF-κB protein are at a reduced level following endostatin treatment (B and D).
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5 Endostatin Signaling Network
Integration of endostatin signaling network in endothelial cells with emphasis on the downregulation of proangiogenic pathways. Effects of endostatin include the RNA downregulation of key pathways involved in angiogenesis such as Ids, HIF1-α, Ephrins, NF-κB, AP-1 (and MAPK), Stats, Ets, and thrombinreceptors (the coagulation cascade) (orange ovals). The orange ovals represent major pathways that we focused on because of their ability to regulate several genes and their importance for elucidation of the network. Upstream and downstream of these key regulatory elements, endostatin downregulates a cascade of interdependent genes including genes of the VEGF family, Bcl-2, LDH-A, MMPs, TNF-α, COX-2, αVβ3 (blue ovals). In addition, endostatin also dephosphorylates many proteins involved in angiogenic cell signaling including Id1, JNK, NF-κB, or Bcl-2 (small circle P-) or phosphorylates proteins such as cyclin D (small circle P+). This network of inter-pathway communications shows that endostatin influences a large number of signaling pathways involved in angiogenesis.
Molecular Cell  , DOI: ( /S (04) )