Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture

Abstract

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (−8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as −5 to −3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased over time. QRT-PCR analysis of the anti-CEACAM1 treated ES cells revealed a significant decrease in the expression of Ceacam1, Pecam1, Tie-1, and Flk-1, while VE-Cad and Tie-2 expression were unaffected. These results suggest that the expression and signaling of CEACAM1 may affect the expression of other factors known to play critical roles in vasculogenesis. Furthermore this 3D model of vasculogenesis in an environment of extracellular matrix may be a useful model for comparison to existing models of angiogenesis.

Tissue vascularization with entothelial-like mesenchymal stromal cells

“…Culture on Matrigel also induced KDR expression in MSCs which can further stimulate endothelial differentiation by increasing MSC sensitivity to VEGF present in culture medium. These results are similar to the ones obtained by Gu et al [75] while studying murine embryonic stem cells. They showed that increased ex-pression of endothelial markers is induced by extracellular matrix via CEACAM1, a glycoprotein involved in cell-cell adhesion.…”

“…Recent studies demonstrate that vasculogenesis can also occur in adults, when endothelial progenitor and bone marrow-derived cells are recruited [74]. Alternatively, angiogenesis is the formation of new vessels by sprouting of existing capillaries and is dominant post-natally [75]. Being able to control and direct both processes opens the possibility to create capillary networks both within tissues as well as in in vitro engineered constructs.…”

Clinical and experimental studies regarding the expression and diagnostic value of carcinoembryonic antigen-related cell adhesion molecule 1 in non-small-cell lung cancer

“…This protein is widely expressed in a variety of proliferating and quiescent epithelial, endothelial, and haematopoietic cells [2]. CEACAM1 is involved in a variety of cell biological events, such as morphogenesis [3], vasculogenesis [4], cell motility [5], cell proliferation [6,7], infection, and inflammation [2]. CEACAM1 exists in 11 known isoforms, resulting from differential splicing and proteolytic processing.…”

6″-Debromohamacanthin A, a Bis (Indole) Alkaloid, Inhibits Angiogenesis by Targeting the VEGFR2-Mediated PI3K/AKT/mTOR Signaling Pathways

“…In the present study, we found that DBHA inhibited neovascularization by suppressing microvessel sprouting in mES cell-derived EBs. The anti-angiogenic activity of DBHA was confirmed by an in vitro angiogenesis sprouting model system using the mES/EB model [44]. Previously, we have found that mES cell-derived EBs matured in a type-I collagen gel and formed capillary-like structures, as observed by morphological changes (3-D culture) and the expression of PECAM (2-D culture).…”

“…Therefore, the mES/EB-derived 3D model is recognized as an excellent system to examine anti-angiogenic activity [41,42,43,44]. Recently, we demonstrated the anti-angiogenic activity of honokiol, a natural angiogenesis inhibitor, with the mES cell-derived EB system [13].…”