Adipose-Derived Stem Cells Promote Lymphangiogenesis in Response to VEGF-C Stimulation or TGF-β1 Inhibition

Abstract

Aims Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. Materials & methods ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-β1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-β1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed. Results Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-β1 signaling was inhibited using the dominant-negative TGF-β1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not. Conclusion Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-β1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.

In VitroInduction of Human Adipose-Derived Stem Cells into Lymphatic Endothelial-Like Cells

“…Many studies have demonstrated that MSCs can be induced into vascular endothelial-like cell types. However, few studies have been published showing the differentiation of MSCs into lymphatic endothelial-like cells, and selective differentiation has remained a technical challenge (Yan et al, 2011). To address this question, we modified the vascular endothelial-like cell induction protocol.…”

“…Stimulation of BM-MSCs with vascular endothelial growth factor-C (VEGF-C), a ligand of VEGFR2 and VEGFR3, induces expression of the lymphangiogenic markers podoplanin and lymphatic vessel endothelial hyaluronan receptor (LYVE-1) (Lee et al, 2010); however, this expression was only temporary, suggesting that a different stimulus may be necessary. ADSCs pretreated with native VEGF-C and transplanted into a mouse model showed increased proliferation relative to untreated cells (Yan et al, 2011). However, the VEGF-C-treated ADSCs increased the frequency of angiogenic cells (5· ) and lymphangiogenic cells (3· ), demonstrating that native VEGF-C does not specifically induce lymphangiogenesis (Yan et al, 2011).…”

“…ADSCs pretreated with native VEGF-C and transplanted into a mouse model showed increased proliferation relative to untreated cells (Yan et al, 2011). However, the VEGF-C-treated ADSCs increased the frequency of angiogenic cells (5· ) and lymphangiogenic cells (3· ), demonstrating that native VEGF-C does not specifically induce lymphangiogenesis (Yan et al, 2011).…”

“…Second, we developed a protocol to differentiate hADSCs into lymphatic endothelial-like cells using a mutant VEGF-C (VEGF-C156s) that binds VEGFR3 but not VEGFR2. This binding specificity may allow for selective differentiation of hADSCs into lymphatic endothelial tissue, instead of producing both angiogenic and lymphangiogenic lineages (Yan et al, 2011). We analyzed in vitro differentiation potential using standard protocols for BM-MSC adipogenesis and osteogenesis.…”

Tissue Engineering and Regeneration of Lymphatic Structures

“…TGF-β1 has been shown to have potent antilymphangiogenic activity resulting in decreased LEC proliferation, downregulation of lymphatic specific gene expression, impaired lymphatic tubule formation and markedly decreased in vivo LEC migration [67]. This concept is supported by the findings of Yan et al who used an in vivo matrigel lymphangiogenesis assay to show that TGF-β1 inhibition synergistically increases the lymphangiogenic effects of VEGF-C [68]. In this study, matrigel was used as a matrix to provide cells with a stable infrastructure for cellular ingrowth and development.…”