The Role of Nitric Oxide Signalling on Direct Reprogramming of Adult Stem Cells and Fibroblasts

Nadeeka Bandara

Research output: ThesisDoctoral Thesis

64 Downloads (Pure)

Abstract

Degenerative diseases of the musculoskeletal, cardiovascular and gastrointestinal system are common health issues worldwide and current treatments are inefficient. In this context, cell therapy based approaches have attracted much interest, however, a number of improvements are required to enhance their therapeutic efficiency in particular promoting specific cellular differentiation events at the site of transplanted cells such as in bone fractures, myocardial infarction and chronic wounds. Genetic modification of adult stem cells is a promising approach to induce particular signalling pathways which may drive cellular differentiation to specific cell types. Nitric oxide (NO) is an important molecule which can interact with various alternative signalling pathways to promote cellular differentiation into a number of different cell types. NO is a pleiotropic diffusible gas and plays important roles, including maintenance of vascular tone, blood pressure regulation, bone and cardiovascular development and promotion of neo-angiogenesis. Endothelial nitric oxide synthase (eNOS/NOS3) is one three enzymes responsible for producing NO, and it is located in specialized cell membrane structures known as caveolae and is held in a tight conformation with caveolin-1 (CAV-1WT) protein. This interaction inhibits NO production and is relaxed during physiological shear stress allowing NO release. CAV-1WT acts as a scaffolding protein regulating eNOS activity, recent research has shown that mutations within the eNOS interacting scaffolding domain of CAV-1 (CSD; amino acids 82-101), in which substitution of phenylalanine (F) at the amino acid position 92 to alanine (CAV-1F92A) removes CSD mediated eNOS inhibition and increases eNOS mediated NO release. This thesis describes the effects of NO on a variety of cellular differentiation programs. eNOS-NO signalling was reconstituted in mesenchymal stem cells (MSCs) and fibroblasts using lentiviral vector technology. To increase eNOS derived NO release, eNOS and CAV-1F92A were co-expressed, which could significantly increase NO production in all modified cell types. Increasing NO availability in equine adipose derived stem cells through modification with lentiviral vectors to express eNOS alone (eASCeNOS) or eNOS+CAV-1F92A (eASCeNOS+CAV-1F92A) induced osteogenic differentiation (chapter 3) as evident by increased calcium deposition and osteogenic specific markers, Runx2 and Alp gene expression and the activity of a Runx2-eGFP reporter. Inhibition of eNOS through treatment of 2 mM L-NAME resulted in reduced Runx2, and Alp expression confirming that enhanced osteogenesis was NO mediated and this NO mediated osteogenesis was driven by endogenous regulation of Wnt/β-catenin signalling, by up-regulating canonical Wnt signalling and, inhibiting non-canonical Wnt signalling. This relationship between NO and enhanced osteogenic differentiation of adult stem cells is a novel finding. In chapter 4, the co-expression of eNOS and CAV-1F92A in rat bone marrow MSCs (rBMSCeNOS+CAV-1F92A) could significantly enhance endothelial differentiation through enhancing Wnt/β-catenin signalling, and the increased levels of NO could specifically direct rBMSC differentiation towards arterial endothelial cells (ECs), in which NO could promote arterial specific Notch1, DII4 and Hey2 gene expression whilst venous specific Coup-tfII and lymphatic specific Prox1 master transcription factors were down regulated. In addition, the expression of DNA methylatransferase (Dnmt1) and histone deacetylase (Sirt6) were down regulated in rBMSCeNOS+CAV-1F92A cells suggesting that NO may regulate epigenetic modification to promote endothelial differentiation. Demonstrating the vessel formation ability of genetically modified cells, subcutaneous transplantation of the rBMSCeNOS+CAV-1F92A cells seeded in polyurethane scaffolds in rats resulted in formation of blood vessels. Chapter 5 of this thesis analysed the effect of increased levels of NO through co expressing of eNOS and CAV-1F92A in human foreskin fibroblasts, BJ (BJeNOS+CAV-1F92A) on GMT mediated cardiac reprogramming and this concept has not been reported previously. Cardiac reprogramming of human fibroblasts, through expression of Gata4, Mef2C, and Tbx5 transcription factors (termed as GMT) has been shown to increase cardiomyocyte generation, however the efficiency is reported to be low. Reconstitution eNOS-NO signalling could significantly enhance GMT mediated cardiac reprogramming, possibly through induction of WNT3A expression. FACS analysis of cardiac specific cTNT expression, demonstrated that only BJGMT+eNOS+CAV-1F92A cells were positive for cTNT (0.1%) and treatment of BJGMT+eNOS+CAV-1F92A cells with the eNOS inhibitor, 2 mM L-NAME reduced cTNT expressing cells suggesting that cardiac reprogramming of BJ was NO mediated. Furthermore, BJGMT+eNOS+CAV-1F92A cells could significantly induce atrial specific, MYH6 and MYL7 gene expression whilst ventricular specific MYH7 and MYL2 genes were not detected. On the other hand, over expression of eNOS and CAV-1F92A together with GMT could reprogram BJ into cardiomyocytes-like cell with binuclear morphology suggesting that increased levels of NO may drive cardiac reprogramming of BJ into distinct type of cells. In chapter 6, a novel non-viral vector system capable of delivering a therapeutic gene to rBMSCs, was developed. A minicircle (MC) vector expressing eNOS (MC-eNOS) was constructed which increased gene delivery efficiency (21 ± 3 %) to rBMSCs was achieved compared to a conventional plasmid vector (9 ± 1 %) (P-eNOS) and also resulted in higher nitrite levels in MC-eNOS transfected cells. Furthermore, a therapeutic effect of eNOS delivery through the MC vector was seen through enhanced, in vitro capillary tubule formation in MC-eNOS transfected cells compared to P-eNOS and gene expression analysis showed, up regulation of angiogenic responsive genes, Vegfa and Fgf2 and their corresponding receptors Pdgfrα and Fgfr2. The endothelial-specific marker Cd31, also was significantly increased in MC-eNOS-cells indicating conversion of rBMSCs to endothelial lineage. In chapter 7, to investigate further potential new mutants of caveolin-1 which may contribute to enhance NO production, a series of mutants of the CAV-A subdomain were constructed. Using an Alanine scanning approach combined with bioinformatics analysis, IIe-84 substitution to alanine (I84A) were predicted to have lowest hydrophobicity, and mutants (D82A, G83A, and I84A) were generated using site directed mutagenesis approach. Lentiviral vectors expressing these mutant caveolin transgenes together with F92A (CAV-1F92A-D82A, CAV-1F92A-G83A, CAV-1F92A-I84A) or CAV-1I84A, and eNOS were delivered to HEK293T cells and nitrite levels was measured. No significant increase in nitrite levels was associated with these new CAV-A domain mutants highlighting the uniqueness of the F92A mutation in terms of eNOS mediated NO release.
In summary, the data presented in this thesis highlights the potential to promote cellular reprogramming through enhancing NO production using a mutant caveolin-1 transgene to reduce inhibition of eNOS activity. This approach may provide a novel platform for gene modified cell based regenerative medicine strategies.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
Supervisors/Advisors
  • Strappe, Padraig, Principal Supervisor
  • Wang, Lexin, Principal Supervisor
Publisher
Publication statusPublished - 2017

Fingerprint Dive into the research topics of 'The Role of Nitric Oxide Signalling on Direct Reprogramming of Adult Stem Cells and Fibroblasts'. Together they form a unique fingerprint.

Cite this