Dose-dependent changes in cardiac function, strain and remodelling in a preclinical model of heart base irradiation.

BACKGROUND AND PURPOSE: Radiation induced cardiotoxicity (RICT) is as an important sequela of radiotherapy to the thorax for patients. In this study, we aim to investigate the dose and fractionation response of RICT. We propose global longitudinal strain (GLS) as an early indicator of RICT and investigate myocardial deformation following irradiation. METHODS: RICT was investigated in female C57BL/6J mice in which the base of the heart was irradiated under image-guidance using a small animal radiation research platform (SARRP). Mice were randomly assigned to a treatment group: single-fraction dose of 16 Gy or 20 Gy, 3 consecutive fractions of 8.66 Gy, or sham irradiation; biological effective doses (BED) used were 101.3 Gy, 153.3 Gy and 101.3 Gy respectively. Longitudinal transthoracic echocardiography (TTE) was performed from baseline up to 50 weeks post-irradiation to detect structural and functional effects. RESULTS: Irradiation of the heart base leads to BED-dependent changes in systolic and diastolic function 50 weeks post-irradiation. GLS showed significant decreases in a BED-dependent manner for all irradiated animals, as early as 10 weeks after irradiation. Early changes in GLS indicate late changes in cardiac function. BED-independent increases were observed in the left ventricle (LV) mass and volume and myocardial fibrosis. CONCLUSIONS: Functional features of RICT displayed a BED dependence in this study. GLS showed an early change at 10 weeks post-irradiation. Cardiac remodelling was observed as increases in mass and volume of the LV, further supporting our hypothesis that dose to the base of the heart drives the global heart toxicity.

A multimodality assessment of the protective capacity of statin therapy in a mouse model of radiation cardiotoxicity.

PURPOSE: Despite technological advances in radiotherapy (RT), cardiotoxicity remains a common complication in patients with lung, oesophageal and breast cancers. Statin therapy has been shown to have pleiotropic properties beyond its lipid-lowering effects. Previous murine models have shown statin therapy can reduce short-term functional effects of whole-heart irradiation. In this study, we assessed the efficacy of atorvastatin in protecting against the late effects of radiation exposure on systolic function, cardiac conduction, and atrial natriuretic peptide (ANP) following a clinically relevant partial-heart radiation exposure. MATERIALS AND METHODS: Female, 12-week old, C57BL/6j mice received an image-guided 16 Gy X-ray field to the base of the heart using a small animal radiotherapy research platform (SARRP), with or without atorvastatin from 1 week prior to irradiation until the end of the experiment. The animals were followed for 50 weeks with longitudinal transthoracic echocardiography (TTE) and electrocardiography (ECG) every 10 weeks, and plasma ANP every 20 weeks. RESULTS: At 30-50 weeks, mild left ventricular systolic function impairment observed in the RT control group was less apparent in animals receiving atorvastatin. ECG analysis demonstrated prolongation of components of cardiac conduction related to the heart base at 10 and 30 weeks in the RT control group but not in animals treated with atorvastatin. In contrast to systolic function, conduction disturbances resolved at later time-points with radiation alone. ANP reductions were lower in irradiated animals receiving atorvastatin at 30 and 50 weeks. CONCLUSIONS: Atorvastatin prevents left ventricular systolic dysfunction, and the perturbation of cardiac conduction following partial heart irradiation. If confirmed in clinical studies, these data would support the use of statin therapy for cardioprotection during thoracic radiotherapy.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Spatial Gene Expression Changes in the Mouse Heart After Base-Targeted Irradiation.

PURPOSE: Radiation cardiotoxicity (RC) is a clinically significant adverse effect of treatment for patients with thoracic malignancies. Clinical studies in lung cancer have indicated that heart substructures are not uniformly radiosensitive, and that dose to the heart base drives RC. In this study, we aimed to characterize late changes in gene expression using spatial transcriptomics in a mouse model of base regional radiosensitivity. METHODS AND MATERIALS: An aged female C57BL/6 mouse was irradiated with 16 Gy delivered to the cranial third of the heart using a 6 × 9 mm parallel opposed beam geometry on a small animal radiation research platform, and a second mouse was sham-irradiated. After echocardiography, whole hearts were collected at 30 weeks for spatial transcriptomic analysis to map gene expression changes occurring in different regions of the partially irradiated heart. Cardiac regions were manually annotated on the capture slides and the gene expression profiles compared across different regions. RESULTS: Ejection fraction was reduced at 30 weeks after a 16 Gy irradiation to the heart base, compared with the sham-irradiated controls. There were markedly more significant gene expression changes within the irradiated regions compared with nonirradiated regions. Variation was observed in the transcriptomic effects of radiation on different cardiac base structures (eg, between the right atrium [n = 86 dysregulated genes], left atrium [n = 96 dysregulated genes], and the vasculature [n = 129 dysregulated genes]). Disrupted biological processes spanned extracellular matrix as well as circulatory, neuronal, and contractility activities. CONCLUSIONS: This is the first study to report spatially resolved gene expression changes in irradiated tissues. Examination of the regional radiation response in the heart can help to further our understanding of the cardiac base's radiosensitivity and support the development of actionable targets for pharmacologic intervention and biologically relevant dose constraints.

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch­dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch­dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

FKBPL and SIRT-1 Are Downregulated by Diabetes in Pregnancy Impacting on Angiogenesis and Endothelial Function.

Diabetes in pregnancy is associated with adverse pregnancy outcomes including preterm birth. Although the mechanisms leading to these pregnancy complications are still poorly understood, aberrant angiogenesis and endothelial dysfunction play a key role. FKBPL and SIRT-1 are critical regulators of angiogenesis, however, their roles in pregnancies affected by diabetes have not been examined before in detail. Hence, this study aimed to investigate the role of FKBPL and SIRT-1 in pre-gestational (type 1 diabetes mellitus, T1D) and gestational diabetes mellitus (GDM). Placental protein expression of important angiogenesis proteins, FKBPL, SIRT-1, PlGF and VEGF-R1, was determined from pregnant women with GDM or T1D, and in the first trimester trophoblast cells exposed to high glucose (25 mM) and varying oxygen concentrations [21%, 6.5%, 2.5% (ACH-3Ps)]. Endothelial cell function was assessed in high glucose conditions (30 mM) and following FKBPL overexpression. Placental FKBPL protein expression was downregulated in T1D (FKBPL; p<0.05) whereas PlGF/VEGF-R1 were upregulated (p<0.05); correlations adjusted for gestational age were also significant. In the presence of GDM, only SIRT-1 was significantly downregulated (p<0.05) even when adjusted for gestational age (r=-0.92, p=0.001). Both FKBPL and SIRT-1 protein expression was reduced in ACH-3P cells in high glucose conditions associated with 6.5%/2.5% oxygen concentrations compared to experimental normoxia (21%; p<0.05). FKBPL overexpression in endothelial cells (HUVECs) exacerbated reduction in tubule formation compared to empty vector control, in high glucose conditions (junctions; p<0.01, branches; p<0.05). In conclusion, FKBPL and/or SIRT-1 downregulation in response to diabetic pregnancies may have a key role in the development of vascular dysfunction and associated complications affected by impaired placental angiogenesis.

ECSIT is a critical limiting factor for cardiac function.

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology.

GSTM1 Modulates Expression of Endothelial Adhesion Molecules in Uremic Milieu.

Deletion polymorphism of glutathione S-transferase M1 (GSTM1), a phase II detoxification and antioxidant enzyme, increases susceptibility to end-stage renal disease (ESRD) as well as the development of cardiovascular diseases (CVD) among ESRD patients and leads to their shorter cardiovascular survival. The mechanisms by which GSTM1 downregulation contributes to oxidative stress and inflammation in endothelial cells in uremic conditions have not been investigated so far. Therefore, the aim of the present study was to elucidate the effects of GSTM1 knockdown on oxidative stress and expression of a panel of inflammatory markers in human umbilical vein endothelial cells (HUVECs) exposed to uremic serum. Additionally, we aimed to discern whether GSTM1-null genotype is associated with serum levels of adhesion molecules in ESRD patients. HUVECs treated with uremic serum exhibited impaired redox balance characterized by enhanced lipid peroxidation and decreased antioxidant enzyme activities, independently of the GSTM1 knockdown. In response to uremic injury, HUVECs exhibited alteration in the expression of a series of inflammatory cytokines including retinol-binding protein 4 (RBP4), regulated on activation, normal T cell expressed and secreted (RANTES), C-reactive protein (CRP), angiogenin, dickkopf-1 (Dkk-1), and platelet factor 4 (PF4). GSTM1 knockdown in HUVECs showed upregulation of monocyte chemoattractant protein-1 (MCP-1), a cytokine involved in the regulation of monocyte migration and adhesion. These cells also have shown upregulated intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). In accordance with these findings, the levels of serum ICAM-1 and VCAM-1 (sICAM-1 and sVCAM-1) were increased in ESRD patients lacking GSTM1, in comparison with patients with the GSTM1-active genotype. Based on these results, it may be concluded that incubation of endothelial cells in uremic serum induces redox imbalance accompanied with altered expression of a series of cytokines involved in arteriosclerosis and atherosclerosis. The association of GSTM1 downregulation with the altered expression of adhesion molecules might be at least partly responsible for the increased susceptibility of ESRD patients to CVD.

Epigenetic Regulation of Endothelial Cell Function by Nucleic Acid Methylation in Cardiac Homeostasis and Disease.

Pathological remodelling of the myocardium, including inflammation, fibrosis and hypertrophy, in response to acute or chronic injury is central in the development and progression of heart failure (HF). While both resident and infiltrating cardiac cells are implicated in these pathophysiological processes, recent evidence has suggested that endothelial cells (ECs) may be the principal cell type responsible for orchestrating pathological changes in the failing heart. Epigenetic modification of nucleic acids, including DNA, and more recently RNA, by methylation is essential for physiological development due to their critical regulation of cellular gene expression. As accumulating evidence has highlighted altered patterns of DNA and RNA methylation in HF at both the global and individual gene levels, much effort has been directed towards defining the precise role of such cell-specific epigenetic changes in the context of HF. Considering the increasingly apparent crucial role that ECs play in cardiac homeostasis and disease, this article will specifically focus on nucleic acid methylation (both DNA and RNA) in the failing heart, emphasising the key influence of these epigenetic mechanisms in governing EC function. This review summarises current understanding of DNA and RNA methylation alterations in HF, along with their specific role in regulating EC function in response to stress (e.g. hyperglycaemia, hypoxia). Improved appreciation of this important research area will aid in further implicating dysfunctional ECs in HF pathogenesis, whilst informing development of EC-targeted strategies and advancing potential translation of epigenetic-based therapies for specific targeting of pathological cardiac remodelling in HF.

Role of A Novel Angiogenesis FKBPL-CD44 Pathway in Preeclampsia Risk Stratification and Mesenchymal Stem Cell Treatment.

CONTEXT: Preeclampsia is a leading cardiovascular complication in pregnancy lacking effective diagnostic and treatment strategies. OBJECTIVE: To investigate the diagnostic and therapeutic target potential of the angiogenesis proteins, FK506-binding protein like (FKBPL) and CD44. DESIGN AND INTERVENTION: FKBPL and CD44 plasma concentration or placental expression were determined in women pre- or postdiagnosis of preeclampsia. Trophoblast and endothelial cell function was assessed following mesenchymal stem cell (MSC) treatment and in the context of FKBPL signaling. SETTINGS AND PARTICIPANTS: Human samples prediagnosis (15 and 20 weeks of gestation; n ≥ 57), or postdiagnosis (n = 18 for plasma; n = 4 for placenta) of preeclampsia were used to determine FKBPL and CD44 levels, compared to healthy controls. Trophoblast or endothelial cells were exposed to low/high oxygen, and treated with MSC-conditioned media (MSC-CM) or a FKBPL overexpression plasmid. MAIN OUTCOME MEASURES: Preeclampsia risk stratification and diagnostic potential of FKBPL and CD44 were investigated. MSC treatment effects and FKBPL-CD44 signaling in trophoblast and endothelial cells were assessed. RESULTS: The CD44/FKBPL ratio was reduced in placenta and plasma following clinical diagnosis of preeclampsia. At 20 weeks of gestation, a high plasma CD44/FKBPL ratio was independently associated with the 2.3-fold increased risk of preeclampsia (odds ratio = 2.3, 95% confidence interval [CI] 1.03-5.23, P = 0.04). In combination with high mean arterial blood pressure (>82.5 mmHg), the risk further increased to 3.9-fold (95% CI 1.30-11.84, P = 0.016). Both hypoxia and MSC-based therapy inhibited FKBPL-CD44 signaling, enhancing cell angiogenesis. CONCLUSIONS: The FKBPL-CD44 pathway appears to have a central role in the pathogenesis of preeclampsia, showing promising utilities for early diagnostic and therapeutic purposes.

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.

Cardiac sub-volume targeting demonstrates regional radiosensitivity in the mouse heart.

BACKGROUND AND PURPOSE: Radiation-induced cardiac toxicity (RICT) remains one of the most critical dose limiting constraints in radiotherapy. Recent studies have shown higher doses to the base of the heart are associated with worse overall survival in lung cancer patients receiving radiotherapy. This work aimed to investigate the impact of sub-volume heart irradiation in a mouse model using small animal image-guided radiotherapy. MATERIALS AND METHODS: C57BL/6 mice were irradiated with a single fraction of 16 Gy to the base, middle or apex of the heart using a small animal radiotherapy research platform. Cone beam CT and echocardiography were performed at baseline and at 10 week intervals until 50 weeks post-treatment. Structural and functional parameters were correlated with mean heart dose (MHD) and volume of heart receiving 5 Gy (V5). RESULTS: All irradiated mice showed a time dependent increase in left ventricle wall thickness in diastole of ~0.2 mm detected at 10 weeks post-treatment, with the most significant and persistent changes occurring in the heart base-irradiated animals. Similarly, statistically different functional effects (p < 0.01) were observed in base-irradiated animals which showed the most significant decreases compared to controls. The observed functional changes did not correlate with MHD and V5 (R2 < 0.1), indicating that whole heart dosimetry parameters do not predict physiological changes resulting from cardiac sub-volume irradiation. CONCLUSIONS: This is the first report demonstrating the structural and functional consequences of sub-volume targeting in the mouse heart and reverse translates clinical observations indicating the heart base as a critical radiosensitive region.

Downregulation of PPARα during Experimental Left Ventricular Hypertrophy Is Critically Dependent on Nox2 NADPH Oxidase Signalling.

Pressure overload-induced left ventricular hypertrophy (LVH) is initially adaptive but ultimately promotes systolic dysfunction and chronic heart failure. Whilst underlying pathways are incompletely understood, increased reactive oxygen species generation from Nox2 NADPH oxidases, and metabolic remodelling, largely driven by PPARα downregulation, are separately implicated. Here, we investigated interaction between the two as a key regulator of LVH using in vitro, in vivo and transcriptomic approaches. Phenylephrine-induced H9c2 cardiomyoblast hypertrophy was associated with reduced PPARα expression and increased Nox2 expression and activity. Pressure overload-induced LVH and systolic dysfunction induced in wild-type mice by transverse aortic constriction (TAC) for 7 days, in association with Nox2 upregulation and PPARα downregulation, was enhanced in PPARα-/- mice and prevented in Nox2-/- mice. Detailed transcriptomic analysis revealed significantly altered expression of genes relating to PPARα, oxidative stress and hypertrophy pathways in wild-type hearts, which were unaltered in Nox2-/- hearts, whilst oxidative stress pathways remained dysregulated in PPARα-/- hearts following TAC. Network analysis indicated that Nox2 was essential for PPARα downregulation in this setting and identified preferential inflammatory pathway modulation and candidate cytokines as upstream Nox2-sensitive regulators of PPARα signalling. Together, these data suggest that Nox2 is a critical driver of PPARα downregulation leading to maladaptive LVH.

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization.

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

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.