Up-Regulation of Endothelial Delta-like 4 Expression Correlates with Vessel Maturation in Bladder Cancer

Abstract

Purpose: Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer.To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. Experimental Design: The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and a-smooth muscle actin (a-SMA) using conventional immunohistochemistry.Results: The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of a-SMA, 98.7% of DLL4-positive tumor vessels coexpressed a-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. Conclusion: DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

“…Normal kidney did not express Dll4, but endothelial cells in renal cell carcinoma showed endothelial expression of Dll4, consistent with previous in situ hybridisation results (Supplementary Figures 1E and F) (Patel et al, 2005). Serial sections of bladder cancer showed endothelial colocalisation of Dll4 mRNA using in situ hybridisation (Patel et al, 2006), and protein using immunohistochemistry (Supplementary Figures 1D -F). …”

“…This is consistent with data from human xenografts (Noguera-Troise et al, 2006) and in situ hybridisation data for Dll4 in human bladder cancer (Patel et al, 2006) and human renal cell carcinoma (Mailhos et al, 2001;Patel et al, 2005). The lack of expression of Dll4 in the endothelial cells and epithelium of normal colonic crypts is consistent with the lack of VEGF expression by normal colorectal mucosa (Jubb et al, 2003) and the lack of intestinal toxicity and goblet cell hyperplasia observed in anti-Dll4-treated mice (Ridgway et al, 2006).…”

“…The probe used was a 741-bp cloned fragment of human Dll4 (position 1775 to 2516 bp, relative to the adenosine of the start codon ATG; Patel et al, 2006). In situ hybridisation for b-actin was used as a positive control for mRNA integrity.…”

Regulation of vascular morphogenesis by Notch signaling

“…Suggesting that the important role of Dll4 and Notch signaling is not restricted to only developmental angiogenesis, Dll4 is prominently expressed in tumor blood vessels in a VEGF-dependent fashion (Patel et al 2005(Patel et al , 2006Hainaud et al 2006;Noguera-Troise et al 2006). Indeed, blockade of Dll4-Notch signaling with the Dll4/Fc antagonist, anti-Dll4 antibodies, or ␥-secretase inhibitor promotes endothelial sprouting and thereby increases vascular density in xenograft tumor models in mice (Noguera-Troise et al 2006;Ridgway et al 2006;Scehnet et al 2007).…”

Tips, Stalks, Tubes: Notch-Mediated Cell Fate Determination and Mechanisms of Tubulogenesis during Angiogenesis

“…The Notch pathway was implicated in tumor angiogenesis when the human Notch ligand Delta-like 4 (DLL4) was found to be up-regulated in tumor blood vessels in several types of cancer (Mailhos et al 2001;Patel et al 2005;Patel et al 2006). Given the Notch pathway's close association with the VEGF pathway, it was thought that this up-regulation might somehow contribute to VEGF-independent angiogenesis, and several groups subsequently investigated the effects of Dll4 inhibition on physiological angiogenesis in mouse models.…”