Angiopoietin-1 promotes functional neovascularization that relieves ischemia by improving regional reperfusion in a swine chronic myocardial ischemia model

10.1007/s11373-006-9082-xJournal of Biomedical Science134579-59

Extracellular vesicle-carried Jagged-1 inhibits HUVEC sprouting in a 3D microenvironment

Abstract NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell–cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain (N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs

Enhancement of bone formation by genetically-engineered bone marrow stromal cells expressing BMP-2, VEGF and angiopoietin-1

10.1007/s10529-009-0007-4Biotechnology Letters3181183-1189BILE

Relative cell chemotaxis migration of MDA-MB-231 and UROtsa cells transformed by As+3 or Cd+2 compared to the parent UROtsa cells.

<p>The red horizontal line at 1, represents the UROtsa parent cells. <b>*</b>Denotes a statistically significant difference from the UROtsa parent cells (p < 0.05). (B). Relative cell migration of MDA-MB-231, non-transfected UROtsa, SPARC-transfected, and blank vector (DEST) cells lines compared to the parent UROtsa cells. The red horizontal line at 1, represents the UROtsa parent cells. <b>*</b>Denotes a statistically significant difference from the UROtsa parent cells; <b>**</b> denotes statistically significant difference from the non-transfected UROtsa counterpart (p < 0.05).</p

Histology and Immunohistochemistry of SPARC in Tumor Transplants Generated from Urospheres Isolated from As⁺³-and Cd⁺²-Transformed UROtsa Cell Lines and from As⁺³-and Cd⁺²-Transformed UROtsa Cell Lines Stably Transfected with SPARC.

<p>Tumor transplants were generated from urospheres isolated from four transformed UROtsa cell lines: As#3 (A, E); As#3-SPARC (B, F); Cd#4 (C, G); and Cd#4-SPARC (D, H). The histology of the tumors generated from the urospheres is shown in (A-D) while the SPARC immunolocalization (brown stain) is shown in (E-H). All images are shown with a 200X magnification.</p

Secretion of SPARC into culture media.

<p>Western analysis of SPARC protein secreted from confluent cultures of the parental UROtsa cells and SPARC-transfected cells. Conditioned media (M) was collected at 24 h (top panel) and 48 h (bottom panel) time points then concentrated as described in materials and methods and was compared to their respective cell lysates (L).</p

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As⁺³- and Cd⁺²-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

<div><p>Background</p><p>This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As⁺³) and cadmium (Cd⁺²)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd⁺²-and As⁺³-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.</p><p>Methods</p><p>Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As⁺³-and Cd⁺²-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.</p><p>Results</p><p>It was shown that the As⁺³-and Cd⁺²-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.</p><p>Conclusions</p><p>Tumor initiating cells isolated from SPARC-transfected As⁺³-and Cd⁺²-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.</p></div