Hyaluronan Esters Drive Smad Gene Expression and Signaling Enhancing Cardiogenesis in Mouse Embryonic and Human Mesenchymal Stem Cells

Abstract

BackgroundDevelopment 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 FindingsWe 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/SignificanceThe 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.

Seeing Cell Biology with the Eyes of Physics

“…This finding points at the relevant pleiotropic role of hyaluronic acid (HA) and glycosaminoglycans in the intracrine regulation of cell polarity and at the chance of using physical energies to preserve and direct this fundamental attribute of Life. HA has been used as a component to promote cardiogenesis in mouse embryonic [42] and human adult stem cells of different origin in vitro and in vivo [43][44][45] and to induce cardiac repair in vivo without stem cell transplantation in a rat model of myocardial infarction [46]. Accordingly, HAS2 suppression abolished the capability of human ES cells to differentiate in vitro along the cardiogenic and vasculogenic lineages [47].…”

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“…BA and RA have also been grafted within a synthetic molecule in the form of HA mixed esters (HBR) [33]. As a result, HBR has been shown to remarkably enhance the differentiation potential of stem cells, affording a high throughput of cardiogenesis in both mouse embryonic stem cells (mESCs) and human adult mesenchymal stem cells (hMSCs) of different origin, including the bone marrow (BMhMSCs), the dental pulp (DPhMSCs) and fetal membrane of term placenta (FMhMSCs) [33][34][35]. HBR, owing to the HA moiety, uses the CD44 hyaluronan receptor, present in both mESCs and hMSCs, to obtain a specific internalization of BA and RA followed by hydrolysis of HBR itself, operated by ubiquitous intracellular esterases [33].…”

“…In fact, depending upon the intracellular concentration, retinoic acid can produce either neurogenesis or cardiogenesis [49][50][51][52]. The fine tuning of the degree of substitution of RA within HBR, obtained through the development of the process of synthesis and esterification, has allowed us to obtain a mixed ester releasing intracellular nanomolar concentrations of RA, which mostly results into cardiogenesis [33][34][35], with neurogenesis prevailing at micromolar concentrations [52]. Following the treatment with HBR of the different populations of hMSCs we were able to enhance by several orders of magnitude the expression of genes and proteins involved in cardiogenesis, such as GATA4, NKX-2.5 and different isoforms of Smad proteins [34].…”

“…The fine tuning of the degree of substitution of RA within HBR, obtained through the development of the process of synthesis and esterification, has allowed us to obtain a mixed ester releasing intracellular nanomolar concentrations of RA, which mostly results into cardiogenesis [33][34][35], with neurogenesis prevailing at micromolar concentrations [52]. Following the treatment with HBR of the different populations of hMSCs we were able to enhance by several orders of magnitude the expression of genes and proteins involved in cardiogenesis, such as GATA4, NKX-2.5 and different isoforms of Smad proteins [34]. The treated stem cells consistently expressed markers for terminal cardiac differentiation, including alpha-myosin heavy chain, alpha-sarcomeric actinin and troponin I (Tn-I).…”

“…Of note, the exposure of both mESCs or hMSCs to HBR or a mixture of HA, BA, and RA resulted in the overexpression of the prodynorphin gene [33][34], which was shown to act as a master regulator of cardiogenesis [55,56]. To this end, dynorphin B, a biologically active end-product of the prodynorphin gene was also found to promote the terminal differentiation of embryonal carcinoma (EC) cells into spontaneously beating cardiac myocytes [57].…”