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ADAM17 mediates endothelial invasion in three‐dimensional collagen matrices
Author(s) -
Bayless Kayla J.,
Kwak HyeongIl,
Mendoza E. Adriana,
Maxwell Steve A.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a194-b
Subject(s) - angiogenesis , disintegrin , gene knockdown , gene silencing , blot , microbiology and biotechnology , biology , metalloproteinase , endothelial stem cell , matrix metalloproteinase , transfection , recombinant dna , green fluorescent protein , small interfering rna , cell culture , chemistry , biochemistry , cancer research , gene , in vitro , genetics
The objective of this study was to determine the potential mechanisms through which the known anti‐angiogenic factor, tissue inhibitor of metalloproteinase‐3 (TIMP‐3) blocks angiogenesis. Endothelial monolayers were infected with recombinant adenoviruses constructed to deliver TIMP‐3 fused to carboxy‐terminal S and His tags (TIMP‐3‐SHis) along with TIMP‐1‐S‐His and green fluorescent protein (GFP) control. Primary endothelial cells were allowed to express constructs for 36–48 hours prior to extraction and tandem affinity purification. Silver staining of final eluates revealed distinct protein interactions. Samples were analyzed by Western blotting and revealed binding of A Disintegrin and Metalloproteinase‐17 (ADAM17) and ADAM15, to TIMP‐3, but not TIMP‐1 or GFP controls (n=4). Analyses of samples using mass spectrometry are underway in large‐scale experiments to identify distinct binding partners of TIMP‐3. To confirm the involvement of ADAM17 or ADAM15 in mediating angiogenic events, models of primary human endothelial cell invasion into three‐dimensional matrices were utilized. Silencing expression of ADAM17 and ADAM15 using small interfering RNA (siRNA) successfully prevented protein expression in invading cell cultures. Knockdown of ADAM17, but not ADAM15 significantly interfered with invasion, resulting in decreased density of invasion and decreased sprout length. Taken together, these data implicate a role for ADAM17 in mediating endothelial sprouting events during angiogenesis. The authors would like to acknowledge the support of American Heart Association Scientist Development Grant # 0530020N to KJB.