Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo.

Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton's jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17ß-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.

Mechanism of 17ß-estradiol stimulated integration of human mesenchymal stem cells in heart tissue.

Scarcity of gender specific donor hearts highlights the importance of mesenchymal stem cells (MSCs) as a therapeutic tool for heart repair. However, inefficient incorporation, retention, and activity of MSCs in cardiac tissue remain an obstacle. Since surges in follicular estradiol (E2; µmolar-range) trigger tissue remodeling (e.g. ovulation) and E2 exerts beneficial actions on the cardiovascular system, we hypothesized that E2 may promote/improve MSC-mediated cardiac repair processes. Using Wharton's jelly (WJ)-derived MSCs we assessed the effects of E2 on MSC proliferation, directed migration, and engraftment in murine heart slices (using xCELLigence real-time cell-impedance system, DNA quantification, and microscopy) and on MSC-induced angiogenesis in vivo (matrigel plug assay). Protein expression was assessed by Western blotting, ELISA/Luminex, and proteomic analysis; whereas mRNA expression was assessed by qRT-PCR. MSCs expressed estrogen receptors (ERs) -alpha and -beta. E2 promoted MSC proliferation and up-regulated mRNA and protein expression of ER-alpha, ER-beta, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase (MMP) -9, yet down-regulated MMP-2 expression. Moreover, E2 up-regulated expression of vascular endothelial growth factor (VEGF)-A, VEGFR-2, vascular cell adhesion protein-1 (VCAM-1), and angiogenin (ANG) and stimulated nitric oxide (NO) production via ER. Proteomic analysis of MSCs showed that E2 up-regulated 47 proteins, down-regulated 7 proteins, and increased the expression of key biochemical components/pathways involved in tissue repair. In MSCs co-cultured with murine heart-slices, E2 significantly induced MSC migration in an ER-alpha-dependent fashion and significantly increased the secretion of MMP-2, MMP-9, ANG, and VEGF. In an in vivo matrigel assay, E2-treated MSCs increased angiogenesis and hemoglobin content. In conclusion, E2-treatment increases the incorporation of MSCs in heart slices and promotes MSC-induced angiogenesis. These beneficial effects are mediated via increases in molecules/pathways involved in tissue remodeling and angiogenesis. We speculate that E2 may enhance MSC ability to repair/regenerate cardiac tissue.

Dihydrotestosterone induces pro-angiogenic factors and assists homing of MSC into the cardiac tissue.

The use of mesenchymal stem cells (MSC) as a therapeutic tool in cardiovascular diseases is promising. Since androgens exert some beneficial actions on the cardiovascular system, we tested our hypothesis that this hormone could promote MSC-mediated repair processes, also. Cultured MSCs isolated from Wharton's jelly were exposed to 30 nM dihydrotestosterone (DHT) for 1 or 4 days and the effects of the hormone on their growth/migration/adhesion and the underlying mechanisms were assessed. Results were obtained by real-time cell impedance measurements, and DNA quantification showed that DHT increased MSC proliferation by ~30%. As determined by xCELLigence system, DHT augmented (~2 folds) the migration of MSC toward cardiac tissue slices (at 12 h), and this effect was blocked by flutamide, an androgen receptor (AR) antagonist. Exposure of cells to DHT, upregulated the gene and protein expression of AR, EMMPRIN and MMP-9 and downregulated the expression of MMP-2 DHT significantly induced the release of nitric oxide by MSC (≥2-fold) and flutamide blocked this effect. When MSCs were co-cultured with cardiac slices, immunohistochemical analysis and qRT-PCR showed that the integration of DHT-stimulated MSC was significantly higher than that of in controls. In conclusion, our findings provide the first evidence that DHT promotes MSC growth, migration and integration into the cardiac slices. The modulating effects of DHT were associated with upregulation of ARs and of key molecules known to promote tissue remodeling and angiogenesis. Our findings suggest that priming of MSC with DHT may potentially increase their capability to regenerate cardiac tissue; in vivo studies are needed to confirm our in vitro findings.

Wilson's disease: a challenging diagnosis. Clinical manifestations and diagnostic procedures in 12 patients.

UNLABELLED: Wilson disease is a neurodegenerative disease of copper metabolism. The genetic defect, localized to chromosome arm 13q, has been shown to affect the copper-transporting adenosine triphosphatase (ATPase) gene (ATP7B) in the liver. Our aim was to study the clinical and laboratory characteristics of 12 children and young adults diagnosed with WD and point out the diagnostic difficulties. MATERIAL AND METHOD: We retrospectively analyzed the patients diagnosed with Wilson's disease between 2001 and 2009 diagnosed in Constanta County Emergency Hospital. Evaluation included detailed physical examination, conventional laboratory testing, genetic analysis, and liver biopsy. RESULTS: Patients with hepatic symptoms showed a considerably earlier onset of symptoms and a shorter diagnostic delay before definitive diagnosis than those with neuropsychiatrical symptoms. The mean age at diagnosis was 9.12 +/- 2.59 years (range 5 years-20 years). 10 patients were symptomatic, 6 were referred because of abnormal liver function test results and/or hepatomegaly, 4 had neuropsychiatrical symptoms and 2 received their diagnoses after family screening. Hepatic copper concentration was between 250 and 1200 micrograms/g. 4 patients had liver cirrhosis, five chronic hepatitis and one had massive hepatic necrosis on necropsy. Any person with recurrent hepatic disease and unexplained neurological symptoms should be investigated to have Wilson's disease. CONCLUSIONS: Detection of WD in children and young adults remains very difficult. The most important investigation is liver biopsy with the assessment of liver copper. Genetic analysis may help in doubtful cases.