Effects of non-coding RNAs and RNA-binding proteins on mitochondrial dysfunction in diabetic cardiomyopathy.

Vascular complications are the main cause of diabetes mellitus-associated morbidity and mortality. Oxidative stress and metabolic dysfunction underly injury to the vascular endothelium and myocardium, resulting in diabetic angiopathy and cardiomyopathy. Mitochondrial dysfunction has been shown to play an important role in cardiomyopathic disruptions of key cellular functions, including energy metabolism and oxidative balance. Both non-coding RNAs and RNA-binding proteins are implicated in diabetic cardiomyopathy, however, their impact on mitochondrial dysfunction in the context of this disease is largely unknown. Elucidating the effects of non-coding RNAs and RNA-binding proteins on mitochondrial pathways in diabetic cardiomyopathy would allow further insights into the pathophysiological mechanisms underlying diabetic vascular complications and could facilitate the development of new therapeutic strategies. Stem cell-based models can facilitate the study of non-coding RNAs and RNA-binding proteins and their unique characteristics make them a promising tool to improve our understanding of mitochondrial dysfunction and vascular complications in diabetes.

RNA-Binding Proteins: Emerging Therapeutics for Vascular Dysfunction.

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.

RNA-Binding Proteins Hold Key Roles in Function, Dysfunction, and Disease.

RNA-binding proteins (RBPs) are multi-faceted proteins in the regulation of RNA or its RNA splicing, localisation, stability, and translation. Amassing proof from many recent and dedicated studies reinforces the perception of RBPs exerting control through differing expression levels, cellular localization and post-transcriptional alterations. However, since the regulation of RBPs is reliant on the micro-environment and events like stress response and metabolism, their binding affinities and the resulting RNA-RBP networks may be affected. Therefore, any misregulation and disruption in the features of RNA and its related homeostasis can lead to a number of diseases that include diabetes, cardiovascular disease, and other disorders such as cancer and neurodegenerative diseases. As such, correct regulation of RNA and RBPs is crucial to good health as the effect RBPs exert through loss of function can cause pathogenesis. In this review, we will discuss the significance of RBPs and their typical function and how this can be disrupted in disease.

Diabetic endotheliopathy: RNA-binding proteins as new therapeutic targets.

Diabetic Endotheliopathy is widely regarded as a principal contributor to cardiovascular disease pathogenesis in individuals with Diabetes mellitus. The endothelium, the innermost lining of blood vessels, consists of an extensive monolayer of endothelial cells. Previously regarded as an interface, the endothelium is now accepted as an organ system with critical roles in vascular health; its dysfunction therefore is detrimental. Endothelial dysfunction induces blood vessel damage resulting in a restriction of blood and oxygen supply to tissues, the central pathology of cardiovascular disease. Hyperglycemic conditions have repeatedly been isolated as a pivotal inducer of endothelial cell dysfunction. Numerous studies have since proven hyperglycemic conditions to significantly alter the gene expression profile of endothelial cells, with this being largely attributable to the post-transcriptional regulation of RNA-binding proteins. In particular, the RBP Quaking-7 has recently emerged as a crucial mediator of diabetic endotheliopathy, with great potential to become a therapeutic target.

Author Correction: Targeting QKI-7 in vivo restores endothelial cell function in diabetes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Endothelial Cells Derived From Patients With Diabetic Macular Edema Recapitulate Clinical Evaluations of Anti-VEGF Responsiveness Through the Neuronal Pentraxin 2 Pathway.

Diabetic macular edema (DME) remains a leading cause of vision loss worldwide. DME is commonly treated with intravitreal injections of vascular endothelial growth factor (VEGF)-neutralizing antibodies. VEGF inhibitors (anti-VEGFs) are effective, but not all patients fully respond to them. Given the potential side effects, inconvenience, and high cost of anti-VEGFs, identifying who may not respond appropriately to them and why is essential. Herein we determine first the response to anti-VEGFs, using spectral-domain optical coherence tomography scans obtained from a cohort of patients with DME throughout the 1st year of treatment. We found that fluid fully cleared at some time during the 1st year in 28% of eyes ("full responders"); fluid cleared only partly in 66% ("partial responders"); and fluid remained unchanged in 6% ("nonresponders"). To understand this differential response, we generated induced pluripotent stem cells (iPSCs) from full responders and nonresponders, from subjects with diabetes but no DME, and from age-matched volunteers without diabetes. We differentiated these iPSCs into endothelial cells (iPSC-ECs). Monolayers of iPSC-ECs derived from patients with diabetes showed a marked and prolonged increase in permeability upon exposure to VEGF; the response was significantly exaggerated in iPSC-ECs from nonresponders. Moreover, phosphorylation of key cellular proteins in response to VEGF, including VEGFR2, and gene expression profiles, such as that of neuronal pentraxin 2, differed between full responders and nonresponders. In this study, iPSCs were used in order to predict patients' responses to anti-VEGFs and to identify key mechanisms that underpin the differential outcomes observed in the clinic. This approach identified NPTX2 as playing a significant role in patient-linked responses and as having potential as a new therapeutic target for DME.

Targeting QKI-7 in vivo restores endothelial cell function in diabetes.

Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications.

The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

Dysfunction of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) leads to ischaemia, the central pathology of cardiovascular disease. Stem cell technology will revolutionise regenerative medicine, but a need remains to understand key mechanisms of vascular differentiation. RNA-binding proteins have emerged as novel post-transcriptional regulators of alternative splicing and we have previously shown that the RNA-binding protein Quaking (QKI) plays roles in EC differentiation. In this study, we decipher the role of the alternative splicing isoform Quaking 6 (QKI-6) to induce VSMC differentiation from induced pluripotent stem cells (iPSCs). PDGF-BB stimulation induced QKI-6, which bound to HDAC7 intron 1 via the QKI-binding motif, promoting HDAC7 splicing and iPS-VSMC differentiation. Overexpression of QKI-6 transcriptionally activated SM22 (also known as TAGLN), while QKI-6 knockdown diminished differentiation capability. VSMCs overexpressing QKI-6 demonstrated greater contractile ability, and upon combination with iPS-ECs-overexpressing the alternative splicing isoform Quaking 5 (QKI-5), exhibited higher angiogenic potential in vivo than control cells alone. This study demonstrates that QKI-6 is critical for modulation of HDAC7 splicing, regulating phenotypically and functionally robust iPS-VSMCs. These findings also highlight that the QKI isoforms hold key roles in alternative splicing, giving rise to cells which can be used in vascular therapy or for disease modelling.This article has an associated First Person interview with the first author of the paper.

Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling.

The mortality rate for (cardio)-vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS-ECs) with typical EC characteristics. This research combined iPS cell technologies and next-generation sequencing to acquire an insight into the transcriptional regulation of iPS-ECs. We identified endothelial cell-specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS-ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signaling, opening the horizon for drug screening and cell-based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell-derived ECs from a very small amount of blood through ESM1 signaling, which greatly enhances their functionality and increases their therapeutic potential. Stem Cells 2019;37:226-239.

RBPs Play Important Roles in Vascular Endothelial Dysfunction Under Diabetic Conditions.

Diabetes is one of the major health care problems worldwide leading to huge suffering and burden to patients and society. Diabetes is also considered as a cardiovascular disorder because of the correlation between diabetes and an increased incidence of cardiovascular disease. Vascular endothelial cell dysfunction is a major mediator of diabetic vascular complications. It has been established that diabetes contributes to significant alteration of the gene expression profile of vascular endothelial cells. Post-transcriptional regulation by RNA binding proteins (RBPs) plays an important role in the alteration of gene expression profile under diabetic conditions. The review focuses on the roles and mechanisms of critical RBPs toward diabetic vascular endothelial dysfunction. Deeper understanding of the post- transcriptional regulation by RBPs could lead to new therapeutic strategies against diabetic manifestation in the future.

Regenerating the Cardiovascular System Through Cell Reprogramming; Current Approaches and a Look Into the Future.

Cardiovascular disease (CVD), despite the advances of the medical field, remains one of the leading causes of mortality worldwide. Discovering novel treatments based on cell therapy or drugs is critical, and induced pluripotent stem cells (iPS Cells) technology has made it possible to design extensive disease-specific in vitro models. Elucidating the differentiation process challenged our previous knowledge of cell plasticity and capabilities and allows the concept of cell reprogramming technology to be established, which has inspired the creation of both in vitro and in vivo techniques. Patient-specific cell lines provide the opportunity of studying their pathophysiology in vitro, which can lead to novel drug development. At the same time, in vivo models have been designed where in situ transdifferentiation of cell populations into cardiomyocytes or endothelial cells (ECs) give hope toward effective cell therapies. Unfortunately, the efficiency as well as the concerns about the safety of all these methods make it exceedingly difficult to pass to the clinical trial phase. It is our opinion that creating an ex vivo model out of patient-specific cells will be one of the most important goals in the future to help surpass all these hindrances. Thus, in this review we aim to present the current state of research in reprogramming toward the cardiovascular system's regeneration, and showcase how the development and study of a multicellular 3D ex vivo model will improve our fighting chances.

Follistatin-Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating ß-Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase-3ß Activity.

The fight against vascular disease requires functional endothelial cells (ECs) which could be provided by differentiation of induced Pluripotent Stem Cells (iPS Cells) in great numbers for use in the clinic. However, the great promise of the generated ECs (iPS-ECs) in therapy is often restricted due to the challenge in iPS-ECs preserving their phenotype and function. We identified that Follistatin-Like 3 (FSTL3) is highly expressed in iPS-ECs, and, as such, we sought to clarify its possible role in retaining and improving iPS-ECs function and phenotype, which are crucial in increasing the cells' potential as a therapeutic tool. We overexpressed FSTL3 in iPS-ECs and found that FSTL3 could induce and enhance endothelial features by facilitating ß-catenin nuclear translocation through inhibition of glycogen synthase kinase-3ß activity and induction of Endothelin-1. The angiogenic potential of FSTL3 was also confirmed both in vitro and in vivo. When iPS-ECs overexpressing FSTL3 were subcutaneously injected in in vivo angiogenic model or intramuscularly injected in a hind limb ischemia NOD.CB17-Prkdcscid/NcrCrl SCID mice model, FSTL3 significantly induced angiogenesis and blood flow recovery, respectively. This study, for the first time, demonstrates that FSTL3 can greatly enhance the function and maturity of iPS-ECs. It advances our understanding of iPS-ECs and identifies a novel pathway that can be applied in cell therapy. These findings could therefore help improve efficiency and generation of therapeutically relevant numbers of ECs for use in patient-specific cell-based therapies. In addition, it can be particularly useful toward the treatment of vascular diseases instigated by EC dysfunction. Stem Cells 2018;36:1033-1044.

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis.

The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA-binding protein Quaking isoform 5 (QKI-5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA-binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient-specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI-5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI-5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3' UTR of STAT3. Importantly, mouse iPS-ECs overexpressing QKI-5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI-5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI-5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI-5 is induced during EC differentiation from iPSCs. RNA binding protein QKI-5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI-5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). Stem Cells 2017 Stem Cells 2017;35:952-966.

MicroRNA-199b Modulates Vascular Cell Fate During iPS Cell Differentiation by Targeting the Notch Ligand Jagged1 and Enhancing VEGF Signaling.

AIMS: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. METHODS AND RESULTS: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis. CONCLUSIONS: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405-1418.

Direct reprogramming of adult cells: avoiding the pluripotent state.

The procedure of using mature, fully differentiated cells and inducing them toward other cell types while bypassing an intermediate pluripotent state is termed direct reprogramming. Avoiding the pluripotent stage during cellular conversions can be achieved either through ectopic expression of lineage-specific factors (transdifferentiation) or a direct reprogramming process that involves partial reprogramming toward the pluripotent stage. Latest advances in the field seek to alleviate concerns that include teratoma formation or retroviral usage when it comes to delivering reprogramming factors to cells. They also seek to improve efficacy and efficiency of cellular conversion, both in vitro and in vivo. The final products of this reprogramming approach could be then directly implemented in regenerative and personalized medicine.