Regular Article
Semaphorin 5A promotes angiogenesis by increasing endothelial cell proliferation, migration, and decreasing apoptosis

https://doi.org/10.1016/j.mvr.2009.10.005Get rights and content

Abstract

Semaphorin 5A (mouse, Sema5A; human, SEMA5A), is an axon regulator molecule and plays major roles during neuronal and vascular development. The importance of Sema5A during vasculogenesis, however, is unclear. The fact that Sema5A deficient mice display a defective branching of cranial vasculature supports its participation in blood vessel formation. In this study, we tested our hypothesis that Sema5A regulates angiogenesis by modulating various steps during angiogenesis. Accordingly, we demonstrated that the treatment of immortalized endothelial cells with recombinant extracellular domain of mouse Sema5A significantly increased endothelial cell proliferation and migration and decreased apoptosis. We also observed a relative increase of endothelial expression of anti-apoptotic genes relative to pro-apoptotic genes in Sema5A-treated endothelial cells suggesting its role in inhibition of apoptosis. In addition, our data suggest that Sema5A decreases apoptosis through activation of Akt, increases migration through activating Met tyrosine kinases and extracellular matrix degradation through matrix metalloproteinase 9. Moreover, in vivo Matrigel plug assays demonstrated that Sema5A induces endothelial cell migration from pre-existing vessels. In conclusion, the present work shows the pro-angiogenic role of Sema5A and provides clues on the signaling pathways that underlie them.

Introduction

Angiogenesis plays a key role in normal development as well as several pathological conditions including cancer. The process of angiogenesis is a complex multistep process, and one of the mechanisms by which angiogenesis occurs is through sprouting and remodeling of new blood vessels from existing blood vessels into a complex network. The process of angiogenesis is initiated by destabilization of existing matured vessels through vascular permeability, relaxation of intraendothelial cell contacts and alleviation of periendothelial support. Endothelial cells from destabilized matured vessels undergo proliferation followed by lumen formation, migration to an area of new blood vessel formation, attenuation of cells and fusion to pre-existing vessels. Increased survival and differentiation allow further development depending on the tissue origin. Finally, endothelial cells remodel and reorganize into mature blood vessels by formation of three dimensional networks (Carmeliet, 2000, Folkman, 2006). Multiple key molecular regulators direct single or multiple steps of this process (Carmeliet, 2000). Recent reports suggest that members of semaphorin family of proteins mediate several steps during angiogenesis (Eichmann, 2005, Serini, 2003, Weinstein, 2005).

Semaphorins are members of a large family of highly conserved, secreted glycosylphospatidylinositol (GPI)-anchored and transmembrane signaling proteins that share a common sema domain of ∼ 500 amino acids at their amino-terminal region. They are classified into eight sub-families based on their structural similarity, species of origin, and presence of class-specific carboxy-terminal domains (Goodman, 1999, Tamagnone, 1999, Tamagnone and Comoglio, 2000). The functions of semaphorins are exerted by binding to a family of transmembrane proteins called plexins that share the sema domain with semaphorins as well as with the neuropilins (Ellis, 2006) and receptor tyrosine kinases, Met and Ron (Kruger, 2005, Tamagnone, 1999). Recent reports demonstrate the non-neuronal role of semaphorins in the immune response (Walzer, 2005, Yamada, 1999), cardiac (Fiore, 2005, Gu, 2005, Torres-Vazquez, 2004) and skeletal development (Behar et al., 1996) and tumor formation (Sekido, 1996, Xiang, 2002). Although the most thoroughly studied sub-families of semaphorins in angiogenesis are sub-families III and IV, the role of the other sub-families of semaphorins remains largely unknown. It has already been shown that Sema3A and Sema3F have been shown to inhibit the motility of endothelial cells as well as angiogenesis (Bielenberg, 2004, Favier, 2006, Kessler, 2004, Miao, 1999). In contrast, other members of the Semaphorin family such as Sema4D has been found to increase angiogenesis (Banu, 2006, Basile, 2004, Conrotto, 2005). However, the roles of the Semaphorin V sub-family of proteins in angiogenesis remain unclear.

Sema5A belongs to sub-family V of semaphorins, and is an integral membrane protein with characteristic seven thrombospondin specific repeats (TSP-1) (He, 2002, Kruger, 2005). Neuroepithelial cells ensheathing retinal axons express SEMA5A, which functions as an inhibitory cue for neurite outgrowth as well as a stimulatory cue for growth cone collapse, thus leading to the proper guidance of retinal projections to their targets (Goldberg, 2004, Oster, 2003). Sema5A was reported to be a bi-functional molecule, exerting its repelling or attracting signals depending on the type of receptor it binds (Kantor et al., 2004). A recent report (Artigiani et al., 2004) and our previous study (Sadanandam et al., 2008) demonstrated that Sema5A interacts with Plexin B3.

In spite of the promiscuous function of Sema5A inactivation leads to embryonic lethality, which can be attributed to defects in the regional patterning of the cranial vasculature (Fiore et al., 2005). In addition, a recent report demonstrates that the migration of cells (including human umbilical vein endothelial cells) depends on the signaling of Sema5A through the hepatocyte growth factor receptor (Met) (Artigiani et al., 2004). Together, these studies suggest a role for Sema5A in physiological angiogenesis during embryonic development. However, the mechanism(s) by which SEMA5A mediates angiogenesis is not known.

In this study, we hypothesized that Sema5A regulates multiple events during angiogenesis by modulating endothelial cell proliferation, apoptosis and migration. Our data suggest that Sema5A decreases apoptosis through activation of Akt, increases migration through activation of Met tyrosine kinases and extracellular matrix degradation through matrix metalloproteinase 9 (MMP9). The present work shows the pro-angiogenic role of Sema5A and provides clues on the underlying signaling pathways.

Section snippets

Cell lines and reagents

Immortalized human dermal microvascular endothelial cells (HMEC-1) were obtained from the Center for Disease Control and Prevention (Atlanta, GA) (Ades et al., 1992). The cells were maintained in culture as an adherent monolayer in RPMI-1640 at 37 °C temperature and 5% CO2 supplemented with 5% fetal calf serum (FCS), 1X nonessential amino acids, 2 mM l-glutamine, 1X vitamin solution (Catalogue #MT-25-020-CI) and 40 μg/ml gentamycin (Mediatech, Herndon, VA). The cultures were free of mycoplasma

Sema5A enhances proliferation and inhibits apoptosis of endothelial cells

We examined whether recombinant Sema5A (extracellular domain) modulates endothelial cell proliferation, an important step during the angiogenic process. HMEC-1 cells were incubated with media alone or media containing different concentrations of recombinant Sema5A or VEGF-A (positive control) and cell proliferation was determined. We observed an increase in endothelial cell proliferation at different concentrations following treatment with Sema5A compared to media alone (Fig. 1A), indicating

Discussion

Although angiogenesis has been widely studied, the understanding of the molecular events involved in this complex process is still in its infancy. Recent reports suggest that in addition to known first generation angiogenic growth factors (VEGF, fibroblast growth factor (FGF)-2), other molecules implicated in axonal guidance may also play critical roles in blood vessel guidance, endothelial cell proliferation, migration, cell collapse and endothelial cell progenitor homing during pathological

Acknowledgments

This work was supported in part by grants CA72781 (R.K.S.), from the National Cancer Institute, National Institutes of Health and Nebraska Research Initiative Molecular Therapeutics Program (R.K.S.). We thank Dr. Ajay P Singh, Shyamali Mandal and Thomas J. Wilson, the University of Nebraska Medical Center, NE; for the careful reading and critiques of this manuscript. We thank the Monoclonal Antibody Facility for their help in the purification of recombinant Sema5A.

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    Current address: Life Sciences Division, Ernest O. Lawrence Berkeley National Lab, Mail stop 977R225A, One Cyclotron Road, Berkeley, CA 94720, USA.

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