Calpain-mediated vimentin cleavage occurs upstream of MT1-MMP membrane translocation to facilitate endothelial sprout initiation.

Endothelial cells normally line the vasculature and remain quiescent. However, these cells can be rapidly stimulated to undergo morphogenesis and initiate new blood vessel formation given the proper cues. This study reports a new mechanism for initiating angiogenic sprout formation that involves vimentin, the major intermediate filament protein in endothelial cells.

Fluid shear stress and sphingosine 1-phosphate activate calpain to promote membrane type 1 matrix metalloproteinase (MT1-MMP) membrane translocation and endothelial invasion into three-dimensional collagen matrices.

The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K.

The sphingosine 1-phosphate (S1P) signaling pathway is regulated during pregnancy in sheep.

Because sphingosine 1-phosphate (S1P) is a potent stimulator of angiogenesis, we hypothesized that the S1P pathway is activated to stimulate endometrial/placental angiogenesis during pregnancy. We initially localized S1P signaling pathway members in the gravid and nongravid uterine horns of unilaterally pregnant ewes. Sphingosine kinase-1 expression was greater in gravid compared to nongravid horns.

Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices.

Seeding a monolayer of primary human endothelial cells on the surface of a polymerized three-dimensional collagen matrix in the presence of pro-angiogenic stimuli allows manipulation and analysis of rapid sprouting responses. This protocol is useful for elucidating incompletely defined intracellular mechanisms downstream of pro-angiogenic factors that regulate sprout formation and initiation, and can also be used to test the efficacy of pro-and anti-angiogenic compounds. We present protocols to culture endothelial cells, prepare three-dimensional collagen matrices and quantify and image rapid endothelial sprouting responses (24 h).

ADAM17 co-purifies with TIMP-3 and modulates endothelial invasion responses in three-dimensional collagen matrices.

In this study, we investigated potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. As a strategy to identify TIMP-3 binding proteins, we used tandem affinity purification, employing recombinant adenoviruses constructed to deliver TIMP-3 fused to C-terminal S and His tags (TIMP-3-S-His) or TIMP-1-S-His control to endothelial cells prior to extraction. Western blotting of final eluates revealed robust binding of A Disintegrin and Metalloproteinase (ADAM) 17 and a slight association of ADAM15 to TIMP-3, but not TIMP-1 control.

Molecular profile of endothelial invasion of three-dimensional collagen matrices: insights into angiogenic sprout induction in wound healing.

Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate, basic FGF, and VEGF.

Loss of singleminded-2s in the mouse mammary gland induces an epithelial-mesenchymal transition associated with up-regulation of slug and matrix metalloprotease 2.

The short splice variant of the basic helix-loop-helix Per-Arnt-Sim transcription factor Singleminded-2, SIM2s, has been implicated in development and is frequently lost or reduced in primary breast tumors. Here, we show that loss of Sim2s causes aberrant mouse mammary gland ductal development with features suggestive of malignant transformation, including increased proliferation, loss of polarity, down-regulation of E-cadherin, and invasion of the surrounding stroma. Additionally, knockdown of SIM2s in MCF-7 breast cancer cells contributed to an epithelial-mesenchymal transition (EMT) and increased tumorigenesis.

Inhibition of breast cancer growth and invasion by single-minded 2s.

Single-minded 2 (SIM2) is a member of the bHLH-PAS family of transcription factors. SIM2 was initially identified by positional cloning on chromosome 21 and is thought to contribute to the etiology of trisomy-21 [Down syndrome (DS)]. In addition to the physical and mental deficiencies associated with this genetic disease, it has become apparent that women with DS are 10-25 times less likely to die from breast cancer in comparison with age-matched normal populations.

Differential transcriptional regulation by mouse single-minded 2s.

Single-minded 1 and 2 are unique members of the basic helix-loop-helix Per-Arnt-Sim family as they are transcriptional repressors. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Sim2s is expressed at high levels in kidney and skeletal muscle; however, the ratio of Sim2 to Sim2s mRNA differs between these tissues.