Principal Investigator: Lianchun Wang
Grant Title: Functional Analysis Of Endothelial Heparan Sulfate In Angiogenesis In VivoAngiogenesis is a process of forming new blood vessels and is essential for development, tissue repair andpathological processes such as tumorigenesis. Understanding the mechanisms that control angiogenesis isessential for development of new therapeutics to treat angiogenesis-related human diseases. Heparan sulfate(HS) is abundantly expressed in developing and mature vasculature. Biochemical and in vitro cell-based
studies have shown that HS interacts with both proangiogenic and anti-antiangiogenic factors, raising thequestion - what is the function of HS in angiogenesis in vivo ? Examination of HS mutant mice has
demonstrated that HS is cell-autonomously required for mural cell (MC) recruitment in angiogenesis and theenhancement of MC recruitment function correlates with the facilitation of both platelet-derived growth
factor-B (PDGF-B) and transforming growth factor-β (TGF-β) signaling. However, the in vivo role of HS
expressed by endothelial cells (EC), the leading and central cell type in angiogenesis in development,
remains largely unknown (1, 2). In very recent studies, we examined EC-specific N-deaceylase/N-sulfatase-1
knockout (Ndst1ECKO) mice in which the N-, 2- and 6-O-sulfation of EC-HS is reduced by 20-60%, andobserved that the mutant mice developed a vascular branch defect uniquely localized in diaphragm, thus
documenting the first in vivo evidence showing that EC-HS functions to facilitate developmental
angiogenesis (3). Our molecular mechanism studies uncovered that the EC-Ndst1 ablation uniquely disrupts
the Slit3-Robo4 signaling, a novel angiogenic pathway which we recently elucidated, that leads to thelocalized diaphragm vascular development defect in the Ndst1ECKO mice (3, 4). Taken together, these studies
established that EC-HS essentially facilitates Slit3-Robo4 signaling in developmental angiogenesis indiaphragm. However, the role of EC-HS in angiogenesis in other organs remains unknown. Since the Ndst1
ablation only partially reduces EC-HS sulfation modification, we postulate that the residual EC-HS structure
is functionally sufficient to sustain the angiogenic function of EC-HS in non-diaphragm tissue/organs andthis was tested in current study. We generated EC-specific Ext1 knockout (Ext1ECKO) mice in which theexpression of EC-HS is completely abolished. The Ext1ECKO mice exhibit profound and severe
developmental angiogenesis defects, including disruptions of EC migration and MC attachment in
vasculature, and are embryonic lethal, showing that EC-HS is essentially required for developmental
angiogenesis in various organs. Meanwhile, the much severe developmental angiogenesis defects, comparedto the Slit3 knockout (Slit3-/-) and Robo4 knockout (Robo4-/-) mice, also suggest that EC-HS modulates otherangiogenic signaling to essentially promote developmental angiogenesis. Mouse phenotype further observedthat EC-HS deficiency leads to excessive vessel regression while lymph angiogenesis appears normal. Now
we are further testing if EC-HS interacts with key angiogenic signaling, such as VEGF and PDGF signaling,
to facilitate vascular development. We anticipate that our further studies will yield significant new insights
into mechanisms that regulate angiogenesis and may also open a new direction to develop the urgently
needed therapeutics that block multiple-angiogenic signaling by targeting only HS to effectively treat
angiogenesis-related human diseases.
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Wang L, Fuster M, Sriramarao P, and Esko JD. Endothelial heparan sulfate deficiency impairs Lselectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. NatImmunol. 2005;6(9):902-10.
Fuster MM, and Wang L. In: Lijuan Z ed. Progress in Molecular Biology and Translational Science.
Academic Press; 2010:179-212.
Zhang B, Xiao W, Qiu H, Zhang F, Moniz HA, Jaworski A, Condac E, Gutierrez-Sanchez G, HeissC, Clugston RD, et al. Heparan sulfate deficiency disrupts developmental angiogenesis and causes
congenital diaphragmatic hernia. J Clin Invest. 2014;124(1):209-21.
Qiu H, Xiao W, Yue J, and Wang L. Heparan sulfate modulates slit3-induced endothelial cellmigration. Methods Mol Biol. 2015;1229(549-55.