Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature

Cdna Cloning of a Rat Small-Intestinal Na+/so42- Cotransporter

We have isolated a cDNA (ileal NaSi-1) from rat small intestine by homology screening with a cDNA (renal NaSi-1) encoding rat kidney cortex Na+-SO42- cotransport. Ileal NaSi-1 cRNA specifically stimulates Na+-dependent SO42- uptake in a time- and dose-depen dent manner in Xenopus laevis oocytes, with kinetic parameters almost identical to those of the renal NaSi-1. Ileal NaSi-1 cDNA contains 2722 base pairs (bp), almost 500bp more than the renal NaSi-1 cDNA; however, it encodes a protein of 595 amino acids identical to the renal NaSi-1 protein. Northern blot analysis shows strong signals in rat lower small intestine and kidney cortex (2.9 x 10(3) and 2.3 x 10(3) bases), with the ileal NaSi-1 corresponding to the longer transcript. We conclude that we have identified a rat ileal cDNA that encodes a membrane protein most likely involved in brush-border Na+-SO42- cotransport. It differs to the renal NaSi-1 only in the length of the 3' untranslated region, suggesting that the major difference lies in the differential use of polyadenylation signal

Expression of Rat Ileal Na+-Sulfate Cotransport in Xenopus-Laevis Oocytes - Functional-Characterization

Small-intestinal sulphate absorption is a Na+-dependent process having its highest rate in the ileum; it involves brush-border membrane Na+-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na+-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na+-independent sulphate uptake. For mRNA-induced transport, the apparent K-m value for sulphate interaction was 0.6 +/- 0.2 mM and that for sodium interaction was 25 +/- 2 mM (Hill coefficient: 2.3 +/- 0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Using a rat renal Nac-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073 - 8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na+-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na+-sulphate cotransport is closely related to rat proximal-tubular brush-border membrane Na+-sulphate cotranspor