Silvia Cantoni

Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro

BackgroundTerm Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).ResultsThe recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.ConclusionThe current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Drive Cardiac and Endothelial Fate in Term Placenta Human Mesenchymal Stem Cells and Enhance Cardiac Repair in Infarcted Rat Hearts

We have developed a mixed ester of hyaluronan with butyric and retinoic acid (HBR) that acted as a novel cardiogenic/vasculogenic agent in human mesenchymal stem cells isolated from bone marrow, dental pulp, and fetal membranes of term placenta (FMhMSCs). HBR remarkably enhanced vascular endothelial growth factor (VEGF), KDR, and hepatocyte growth factor (HGF) gene expression and the secretion of the angiogenic, mitogenic, and antiapoptotic factors VEGF and HGF, priming stem cell differentiation into endothelial cells. HBR also increased the transcription of the cardiac lineage-promoting genes GATA-4 and Nkx-2.5 and the yield of cardiac markerexpressing cells. These responses were notably more pronounced in FMhMSCs. FMhMSC transplantation into infarcted rat hearts was associated with increased capillary density, normalization of left ventricular function, and significant decrease in scar tissue. Transplantation of HBR-preconditioned FMhM-SCs further enhanced capillary density and the yield of human vWF-expressing cells, additionally decreasing the infarct size. Some engrafted, HBR-pretreated FMhMSCs were also positive for connexin 43 and cardiac troponin I. Thus, the beneficial effects of HBR-exposed FMhMSCs may be mediated by a large supply of angiogenic and antiapoptotic factors, and FMhMSC differentiation into vascular cells. These findings may contribute to further development in cell therapy of heart failure.

Inhibition of Hepatocellular Carcinomas in vitro and Hepatic Metastases in vivo in Mice by the Histone Deacetylase Inhibitor HA-But

Purpose: The purpose is to evaluate the CD44-mediated cellular targeting of HA-But, a hyaluronic acid esterified with butyric acid (But) residues, to hepatocellular carcinoma cell lines in vitro and to hepatic tumor metastases in vivo. Experimental Design: In vitro, the CD44-dependent cytotoxicity in two human hepatocellular carcinoma cell lines (HepB3 and HepG2) with high and low CD44 expression was investigated; in vivo, the effect on liver metastases originating from intrasplenic implants of Lewis lung carcinoma (LL3) or B16-F10 melanoma in mice was compared with the pharmacokinetics of organ and tissue distribution using different routes of administration. Results: HepB3 and HepG2 cell lines showed different expression of CD44 (78 and 18%, respectively), which resulted in a CD44-dependent HA-But inhibitory effect as demonstrated also by the uptake analysis performed using radiolabeled HA-But (99mTc-HA-But). Pharmacokinetic studies showed different rates of 99mTc-HA-But distribution according to the route of administration (i.v., i.p., or s.c.): very fast (a few minutes) after i.v. treatment, with substantial accumulation in the liver and spleen; relatively slow after i.p. or s.c. treatment, with marked persistence of the drug at the site of injection. The effect of s.c. and i.p. treatment with HA-But on liver metastases originating from intrasplenic implants of LL3 carcinoma or B16-F10 melanoma (both CD44-positive: 68 and 87%, respectively), resulted in 87 and 100% metastases-free animals, respectively (regardless of the route of administration), and a significant prolongation of the life expectancy compared with control groups. Conclusions: HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions.

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Affording Myocardial Survival and Repair without Stem Cell Transplantation

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [18F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Placental stem cells pre-treated with a hyaluronan mixed ester of butyric and retinoic acid to cure infarcted pig hearts: a multimodal study.

AIMS Pre-treating placenta-derived human mesenchymal stem cells (FMhMSCs) with a hyaluronan mixed ester of butyric and retinoic acid (HBR) potentiates their reparative capacity in rodent hearts. Our aim was to test FMhMSCs in a large-animal model by employing a novel combination of in vivo and ex vivo analyses. METHODS AND RESULTS Matched regional quantifications of myocardial function and viability were performed by magnetic resonance imaging (MRI) and positron emission tomography (PET) 4 weeks after myocardial infarction combined with intramyocardial injection of FMhMSCs (n = 7), or HBR-pre-treated FMhMSCs (HBR-FMhMSCs, n = 6), or saline solution (PBS, n = 7). Sham-operated pigs (n = 4) were used as control animals. Despite no differences in the ejection fraction and haemodynamics, regional MRI revealed, in pigs treated with HBR-FMhMSCs compared with the other infarcted groups, a 40% smaller infarct scar size and a significant improvement of the end-systolic wall thickening and circumferential shortening of the infarct border zone. Consistently, PET showed that myocardial perfusion and glucose uptake were, respectively, 35 and 23% higher in the border zone of pigs treated with HBR-FMhMSCs compared with the other infarcted groups. Histology supported in vivo imaging; the delivery of HBR-FMhMSCs significantly enhanced capillary density and decreased fibrous tissue by approximately 68%. Moreover, proteomic analysis of the border zone in the HBR-FMhMSCs group and the FMhMSCs group indicated, respectively, 45 and 30% phenotypic homology with healthy tissue, while this homology was only 26% in the border zone of the PBS group. CONCLUSION Our results support a more pronounced reparative potential of HBR-pre-treated FMhMSCs in a clinically relevant animal model of infarction and highlight the necessity of using combined diagnostic imaging to avoid underestimations of stem cell therapeutic effects in the heart.

Sodium butyrate inhibits platelet‐derived growth factor‐induced proliferation and migration in pulmonary artery smooth muscle cells through Akt inhibition

Sodium butyrate (BU) is a molecule that acts as a histone deacetylase inhibitor. As compared with its well‐known antineoplastic/antiproliferative effects, little is known about BU action on vascular cell dynamics. An imbalance of proliferation and migration in pulmonary arterial smooth muscle cells (PASMCs) is essential in the onset and progression of pulmonary arterial hypertension (PAH), a disease that is characterized by vascular lung derangement and that frequently has an unfavorable outcome. Here, we show that, in PASMCs of PAH rats, BU counteracted platelet‐derived growth factor (PDGF)‐induced Ki67 expression, and arrested the cell cycle, mainly at G0/G1. BU decreased proliferating cell nuclear antigen, c‐Myc and cyclin D1 transcription and protein expression, while increasing p21 expression. BU reduced the transcription of PDGF receptor‐β, and that of Ednra and Ednrb, two major receptors in PAH progression. Wound healing, migration and pulmonary artery ring assays indicated that BU inhibited PDGF‐induced PASMC migration. BU strongly inhibited PDGF‐induced Akt phosphorylation, an effect reversed by the phosphatase inhibitor calyculin A. BU‐treated cells showed a remarkable increase in acetylated Akt, indicating an inverse relationship between the levels of acetylated Akt and phospho‐Akt. These findings may provide novel perspectives on the use of histone deacetylase inhibitors in PAH.

Hyaluronan esters drive Smad gene expression and signaling enhancing cardiogenesis in mouse embryonic and human mesenchymal stem cells.

Background Development of molecules chemically modifying the expression of crucial orchestrator(s) of stem cell commitment may have significant biomedical impact. We have recently developed hyaluronan mixed esters of butyric and retinoic acids (HBR), turning cardiovascular stem cell fate into a high-yield process. The HBR mechanism(s) remain still largely undefined. Methodology/Principal Findings We show that in both mouse embryonic stem (ES) cells and human mesenchymal stem cells from fetal membranes of term placenta (FMhMSCs), HBR differentially affected the patterning of Smad proteins, one of the major conductors of stem cell cardiogenesis. Real-time RT-PCR and Western blot analyses revealed that in both cell types HBR enhanced gene and protein expression of Smad1,3, and 4, while down-regulating Smad7. HBR acted at the transcriptional level, as shown by nuclear run-off experiments in isolated nuclei. Immunofluorescence analysis indicated that HBR increased the fluorescent staining for Smad1,3, and 4, confirming that the transcriptional action of HBR encompassed the upregulation of the encoded Smad proteins. Chromatin immune precipitation and transcriptional analyses showed that HBR increased the transcription of the cardiogenic gene Nkx-2.5 through Smad4 binding to its own consensus Smad site. Treatment of mouse ES cells and FMhMSCs with HBR led to the concomitant overexpression of both Smad4 and α-sarcomeric actinin. Smad4 silencing by the aid of lentiviral-mediated Smad4 shRNA confirmed a dominant role of Smad4 in HBR-induced cardiogenesis. Conclusions/Significance The use of HBR may pave the way to novel combinatorial strategies of molecular and stem cell therapy based on fine tuning of targeted Smad transciption and signaling leading to a high-throughput of cardiogenesis without the needs of gene transfer technologies.

Dissecting histone deacetylase role in pulmonary arterial smooth muscle cell proliferation and migration.

Pulmonary Arterial Hypertension (PAH) is a rare and devasting condition characterized by elevated pulmonary vascular resistance and pulmonary artery pressure leading to right-heart failure and premature death. Pathologic alterations in proliferation, migration and survival of all cell types composing the vascular tissue play a key role in the occlusion of the vascular lumen. In the current study, we initially investigated the action of selective class I and class II HDAC inhibitors on the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) after exposure to Platelet Derived Growth Factor (PDGF). Class I HDAC inhibitors were able to counteract the hyperproliferative response to PDGF, reducing both proliferation and migration in PASMCs, while class II were ineffective. Selective silencing with siRNAs targeted against different HDACs revealed a major role of class I, and within this class, of HDAC1 in mediating PDGF-induced Akt Phosphorylation and Cyclin D1 (CycD1) expression. These results from these combinatorial approaches were further confirmed by the ability of a specific HDAC1 inhibitor to antagonize the PDGF action. The finding that HDAC1 is a major conductor of PDGF-induced patterning in PAH-PASMCs prompts the development of novel selective inhibitors of this member of class I HDACs as a potential tool to control lung vascular homeostasis in PAH.

Rosuvastatin elicits KDR-dependent vasculogenic response of human placental stem cells through PI3K/AKT pathway.

The growth and plasticity of engrafted human mesenchymal stem cells is regulated by external stimuli. Rosuvastatin (RSV) promotes myocardial neovascularization and limits myocardial remodeling in patients with chronic heart failure (CHF). While these non-lipid benefits may in part depend on the activation of stem cells, experimental evidence that RSV directly elicits vasculogenic differentiation of human mesenchymal stem cells is still lacking. We assessed whether RSV may drive a gene program of vascular commitment and the secretion of trophic mediators with antiapoptotic, angiogenic and antifibrotic activities in human mesenchymal stem cells from full-term placentas (FMhMSCs). With real-time RT-PCR, immunofluorescence, chemiluminescence, Western blot analysis, and in vitro vasculogenesis assays, we show that RSV enhanced expression of vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), encoding a major VEGF receptor, hepatocyte growth factor (HGF), and platelet-derived growth factor-BB (PDGF-BB) in a time- and dose-dependent manner. GATA-4 and Nkx-2.5 transcription was not affected. RSV enhanced capillary-like formation in vitro, but capillary-embedded FMhMSCs lacked endothelial marker expression, suggesting a role of pericyte-like elements in tube formation. In HUVEC/FMhMSC cocultures, RSV increases PDGFRβ expression in FMhMSCs, and enhanced capillary density and organizational efficiency, promoting a long-lasting survival of tubular networks. RSV also activated PI3K-Akt pathway; the vasculogenic effects of the statin were abrogated following PI3K inhibition by LY294002. In conclusion, RSV-induced increase in capillary formation was dependent on VEGF and KDR. RSV promotes the activation of paracrine signals for vascular commitment of FMhMSCs through PI3K-Akt pathway. This observation may pave the way to the use of RSV as a pharmacological enhancer of stem cell potential for cardiovascular cell therapy.

Inhibition of Human Pancreatic Cell Line Mia Paca2 Proliferation by Ha-but, a Hyaluronic Butyric Ester: A Preliminary Report

Objectives: Inhibition of histone deacetylase activity is one of the epigenetic mechanisms in the regulation of the cellular gene expression. We investigated the antitumor effect of HA-But, a new histone deacetylase inhibitor, in which hyaluronic acid is esterified with butyric acid residues and selectively bind to CD44, overexpressed in most human cancers, including pancreatic cancer. Methods: We analyzed the effect of HA-But on the expression level of some cell cycle (p21waf1/cip1, p27kip1, p53, and cyclin D1), apoptosis (BAX, caspase-7, Bcl-2, and survivin), and angiogenesis-related (vascular endothelial growth factor [VEGF] A165, VEGF-C, and VEGF-D) proteins on MIA PaCa-2, a pancreas carcinoma cell line that expressed CD44 in a high percentage (99%) of cells. Results: HA-But was 7-fold more effective than sodium butyrate in inhibiting cell proliferation; it induced p21waf1/cip1, p27kip1, p53, and cyclin D1 modulation, resulting in a block of the cell cycle at G0/G1 and G2/M phases. Moreover, Ha-But induced apoptosis, affecting theexpression level of either proapoptotic or antiapoptotic factors, reduced the expression level of VEGF-A165 and VEGF-D, and inhibited the angiogenesis process in vitro. Conclusions: On the basis of these results, which demonstrated aninteresting antiproliferative, proapoptotic, and antiangiogenic activity, Ha-But could be a promising candidate for the treatment of pancreatic cancer.