Dual Stem Cell Therapy Improves the Myocardial Recovery Post-Infarction through Reciprocal Modulation of Cell Functions.

Mesenchymal stromal cells (MSC) are promising candidates for regenerative therapy of the infarcted heart. However, poor cell retention within the transplantation site limits their potential. We hypothesized that MSC benefits could be enhanced through a dual-cell approach using jointly endothelial colony forming cells (ECFC) and MSC. To assess this, we comparatively evaluated the effects of the therapy with MSC and ECFC versus MSC-only in a mouse model of myocardial infarction. Heart function was assessed by echocardiography, and the molecular crosstalk between MSC and ECFC was evaluated in vitro through direct or indirect co-culture systems. We found that dual-cell therapy improved cardiac function in terms of ejection fraction and stroke volume. In vitro experiments showed that ECFC augmented MSC effector properties by increasing Connexin 43 and Integrin alpha-5 and the secretion of healing-associated molecules. Moreover, MSC prompted the organization of ECFC into vascular networks. This indicated a reciprocal modulation in the functionality of MSC and ECFC. In conclusion, the crosstalk between MSC and ECFC augments the therapeutic properties of MSC and enhances the angiogenic properties of ECFC. Our data consolidate the dual-cell therapy as a step forward for the development of effective treatments for patients affected by myocardial infarction.

Evidence of mesenchymal stromal cell adaptation to local microenvironment following subcutaneous transplantation.

Subcutaneous transplantation of mesenchymal stromal cells (MSC) emerged as an alternative to intravenous administration because it avoids the pulmonary embolism and prolongs post-transplantation lifetime. The goal of this study was to investigate the mechanisms by which these cells could affect remote organs. To this aim, murine bone marrow-derived MSC were subcutaneously transplanted in different anatomical regions and the survival and behaviour have been followed. The results showed that upon subcutaneous transplantation in mice, MSC formed multicellular aggregates and did not migrate significantly from the site of injection. Our data suggest an important role of hypoxia-inducible signalling pathways in stimulating local angiogenesis and the ensuing modulation of the kinetics of circulating cytokines with putative protective effects at distant sites. These data expand the current understanding of cell behaviour after subcutaneous transplantation and contribute to the development of a non-invasive cell-based therapy for distant organ protection.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

A standard procedure for lentiviral-mediated labeling of murine mesenchymal stromal cells in vitro.

Tracking of stem cells after transplantation is effectively performed in vivo with imaging systems, assuming the cells are adequately labeled to facilitate their recognition. This study aimed to optimize a protocol for fluorescent labeling of mesenchymal stromal cells (MSCs) in vitro, by using a third-generation lentiviral system. Basically, 293T cells are seeded in high-glucose Dulbecco's modified Eagle medium with 10% FBS one day before transfection. Transfection is done for 24 h using a mix of transfer, packaging, regulatory, and envelope plasmids, in molar ratio of 4:2:1:1, respectively. After transfection, the cells are further cultured for two days. During this period, the viral medium is harvested two times, at 24-h intervals, with the first round being stored at 4°C until the second round is completed. The pooled viral medium is frozen in single-use aliquots. MSCs are transduced with 25 multiplicity of infection (MOI) and one day later the cells are passaged at standard seeding density and further grown for three days, when the fluorescence reach the maximum level. Our protocol provides particular experimental details for permanent MSC labeling that makes the procedure highly effective for therapeutic purposes, without affecting the functional properties of stem cells.

Association of intracellular lipid accumulation in subcutaneous adipocyte precursors and plasma adipokines in bariatric surgery candidates.

BACKGROUND: The adipocyte expansion is a critical process with implications in the pathogenesis of obesity associated metabolic syndrome. Impaired adipogenesis leads to dysfunctional, hypertrophic adipocytes, local inflammation and peripheric insulin resistance. METHODS: We assessed the relationship between the adipogenic differentiation capacity of the subcutaneous adipose derived stem cells (ASCs), evaluated by total lipid accumulation, and the metabolic and hormonal profile in a group of obese female patients proposed for bariatric surgery (N = 20) versus normal weight female controls (N = 7). RESULTS: The lipid accumulation (measured as optical density at 492 nm) of ASCs during their differentiation to adipocytes was significantly lower in ASCs isolated from obese patients as compared to ASCs isolated from normal weight patients (0.49 ± 0.1 vs. 0.71 ± 0.1, p < 0.001). Significant negative correlations between lipid accumulation in adipogenic differentiated ASCs and plasma concentrations of triglycerides (p < 0.01), insulin (p < 0.001), HOMA-IR (p < 0.01), adiponectin (p < 0.05) and leptin/adiponectin ratio (p < 0.05) were found in obese group. CONCLUSIONS: In severely obese female patients, the abnormal adipogenesis is related to insulin resistance and leptin/adiponectin ratio. The abnormal lipid accumulation in the mature adipocyte derived from obese ASCs could possible predict the further development of type 2 diabetes mellitus in severely obese patients and influence the selection of patients for bariatric surgery.

Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR-377/VE-PTP axis.

The possibility to employ stem/progenitor cells in the cardiovascular remodelling after myocardial infarction is one of the main queries of regenerative medicine. To investigate whether endothelial progenitor cells (EPCs) participate in the restoration of hypoxia-affected myocardium, we used a co-culture model that allowed the intimate interaction between EPCs and myocardial slices, mimicking stem cell transplantation into the ischaemic heart. On this model, we showed that EPCs engrafted to some extent and only transiently survived into the host tissue, yet produced visible protective effects, in terms of angiogenesis and protection against apoptosis and identified miR-377-VE-PTP axis as being involved in the protective effects of EPCs in hypoxic myocardium. We also showed that collagen, the main component of the myocardial scar, was important for these protective effects by preserving VE-PTP levels, which were otherwise diminished by miR-377. By this, a good face of the scar is revealed, which was so far perceived as having only detrimental impact on the exogenously delivered stem/progenitor cells by affecting not only the engraftment, but also the general protective effects of stem cells.

Noninvasive molecular ultrasound monitoring of vessel healing after intravascular surgical procedures in a preclinical setup.

OBJECTIVE: Cardiovascular interventions induce damage to the vessel wall making antithrombotic therapy inevitable until complete endothelial recovery. Without a method to accurately determine the endothelial status, many patients undergo prolonged anticoagulation therapy, denying them any invasive medical procedures, such as surgical operations and dental interventions. Therefore, we aim to introduce molecular ultrasound imaging of the vascular cell adhesion molecule (VCAM)-1 using targeted poly-n-butylcyanoacrylate microbubbles (MB(VCAM-1)) as an easy accessible method to monitor accurately the reendothelialization of vessels. APPROACH AND RESULTS: ApoE(-/-) mice were fed with an atherogenic diet for 1 and 12 weeks and subsequently, endothelial denudation was performed in the carotid arteries using a guidewire. Molecular ultrasound imaging was performed at different time points after denudation (1, 3, 7, and 14 days). An increased MB(VCAM-1) binding after 1 day, a peak after 3 days, and a decrease after 7 days was found. After 12 weeks of diet, MB(VCAM-1) binding also peaked after 3 days but remained high until 7 days, indicating a delay in endothelial recovery. Two-photon laser scanning microscopy imaging of double fluorescence staining confirmed the exposure of VCAM-1 on the superficial layer after arterial injury only during the healing phase. After complete reendothelialization, VCAM-1 expression persisted in the subendothelial layer but was not reachable for the MBV(CAM-1) anymore. CONCLUSION: Molecular ultrasound imaging with MB(VCAM-1) is promising to assess vascular damage and to monitor endothelial recovery after arterial interventions. Thus, it may become an important diagnostic tool supporting the development of adequate therapeutic strategies to personalize anticoagulant and anti-inflammatory therapy after cardiovascular intervention.

Pre-stimulation with FGF-2 increases in vitro functional coupling of mesenchymal stem cells with cardiac cells.

The functional coupling of transplanted cells with host myocardial cells is a significant challenge in mesenchymal stem cell (MSC) cardiomyoplasty being related to cell survival and therapeutic outcomes. Priming of MSCs with growth factors has been reported to improve their therapeutic efficacy through gap junction-mediated mechanisms. However, the expression pattern of Connexin43 (Cx43) in growth factor-stimulated MSC was not previously addressed. In this study we investigated how the pre-treatment with growth factors modulates MSC ability to integrate into the host tissue after transplantation, with particular focus on the expression of Cx43 and its cellular distribution. Our results showed that stimulation of MSCs with IGF-1, FGF-2, but not TGFß, increased the level of Cx43 at both mRNA and protein levels. IGF-1 stimulation resulted in a shift of the fibroblast morphology into an epithelial morphology in several well-defined areas of stimulated cells. Confocal microscopy examination revealed that the increase of Cx43 was restricted to the epithelial-like cells and did not occur in other cells. In variance, FGF-2 induced a rod-shape morphology of every single cell, which achieved an extremely low cell index. FGF-2 stimulation also induced a time-dependent increase in Cx43, with a regular distribution pattern in all cells. Dye transfer assay coupled with confocal microscopy and flow cytometry analysis demonstrated functional in vitro cell coupling between FGF-2-stimulated MSCs as well as between FGF-2-stimulated cells and H9c2 cardiomyoblasts, a scenery that mimick MSC transplantation into the myocardium. We conclude that the stimulation of MSCs with FGF-2 prior to transplantation may facilitate their access among the myocardial cells and increase the functional coupling between transplanted and host cells.

Remote transplantation of mesenchymal stem cells protects the heart against ischemia-reperfusion injury.

Cardioprotection can be evoked through extracardiac approaches. This prompted us to investigate whether remote transplantation of stem cells confers protection of the heart against ischemic injury. The cardioprotective effect of subcutaneous transplantation of naïve versus heme oxygenase-1 (HMOX-1)-overexpressing mouse mesenchymal stem cells (MSC) to mice was investigated in hearts subjected to ischemia-reperfusion in a Langendorff perfusion system. Mice were transplanted into the interscapular region with naïve or HMOX-1 transfected MSC isolated from transgenic luciferase reporter mice and compared to sham-treated animals. The fate of transplanted cells was followed by in vivo bioluminescence imaging, revealing that MSC proliferated, but did not migrate detectably from the injection site. Ex vivo analysis of the hearts showed that remote transplantation of mouse adipose-derived MSC (mASC) resulted in smaller infarcts and improved cardiac function after ischemia-reperfusion compared to sham-treated mice. Although HMOX-1 overexpression conferred cytoprotective effects on mASC against oxidative stress in vitro, no additive beneficial effect of HMOX-1 transfection was noted on the ischemic heart. Subcutaneous transplantation of MSC also improved left ventricular function when transplanted in vivo after myocardial infarction. Plasma analysis and gene expression profile of naïve- and HMOX-1-mASC after transplantation pointed toward pentraxin 3 as a possible factor involved in the remote cardioprotective effect of mASC. These results have significant implications for understanding the behavior of stem cells after transplantation and development of safe and noninvasive cellular therapies with clinical applications. Remote transplantation of MSC can be considered as an alternative procedure to induce cardioprotection.

Controlled intramyocardial release of engineered chemokines by biodegradable hydrogels as a treatment approach of myocardial infarction.

Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time-span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time-span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met-CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease-resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time-controlled release using Met-CCL5-FDH and CXCL12 (S4V)-SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time-controlled, biopolymer-mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell-based therapies.

Cardiomyocyte apoptosis in ischaemia-reperfusion due to the exogenous oxidants at the time of reperfusion.

Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic-reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia-reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25-OH-cholesterol (25-hydroxycholesterol) for 1-3 days. Both ischaemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25-OH-cholesterol promoted CMC apoptosis by a caspase 3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein, Bax and post-translational degradation of the anti-apoptotic protein, Bcl-2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored.

A procedure for in vitro evaluation of the immunosuppressive effect of mouse mesenchymal stem cells on activated T cell proliferation.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) represent adult cells with multipotent capacity. Besides their capacity to differentiate into multiple lineages in vitro and in vivo, increasing evidence points towards the immunomodulatory capacity of these cells, as an important feature for their therapeutic power. Although not included in the minimal criteria established by the International Society for Cellular Therapy as a defining MSC attribute, demonstration of the immunomodulatory capacity of MSC can be useful for the characterization of these cells before being considered MSC. METHODS: Here we present a simple and reliable protocol by which the immunosuppressive effect of mouse bone marrow-derived MSC can be evaluated in vitro. It is based on the measuring of the proliferation of activated T cells cultured in direct contact with irradiated MSC. RESULTS: Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation, which can be quantified as a percentage of maximum proliferation. Our data shows that batch-to-batch variability can be determined within one or multiple experiments, by extracting the area under curve of T cell proliferation plotted against the absolute number of MSC in co-culture. CONCLUSIONS: The validation of the immunosupressive capacity of MSC could be added to the characterization of the cells before being used in various MSC-based approaches to treat immunological diseases. Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation. The immunosuppressive properties of MSC vary between batches, but not between different passages of the same batch.

miRNAs generated from Meg3-Mirg locus are downregulated during aging.

Aging determines a multilevel functional decline and increases the risk for cardiovascular pathologies. MicroRNAs are recognized as fine tuners of all cellular functions, being involved in various cardiac diseases. The heart is one of the most affected organs in aged individuals, however little is known about the extent and robustness to which miRNA profiles are modulated in cardiac cells during aging. This paper provides a comprehensive characterization of the aging-associated miRNA profile in the murine cardiac fibroblasts, which are increasingly recognized for their active involvement in the cardiac physiology and pathology. Next-generation sequencing of cardiac fibroblasts isolated from young and old mice revealed that an important fraction of the miRNAs generated by the Meg3-Mirg locus was downregulated during aging. To address the specificity of this repression, four miRNAs selected as representative for this locus were further assessed in other cells and organs isolated from aged mice. The results suggested that the repression of miRNAs generated by the Meg3-Mirg locus was a general feature of aging in multiple organs. Bioinformatic analysis of the predicted target genes identified Integrin Beta-2 as an aged-upregulated gene, which was thereafter confirmed in multiple mouse organs. In conclusion, our study provides new data concerning the mechanisms of natural aging and highlights the robustness of the miRNA modulation during this process.

Short lifespan of syngeneic transplanted MSC is a consequence of in vivo apoptosis and immune cell recruitment in mice.

Mesenchymal stromal cells (MSC) are attractive tools for cell-based therapy, yet the mechanisms underlying their migration and survival post-transplantation are unclear. Accumulating evidence indicates that MSC apoptosis modulates both innate and adaptive immune responses which impact on MSC therapeutic effects. Using a dual tracking system, namely the Luciferase expression and VivoTrack680 labelling, and in vivo optical imaging, we investigated the survival and migration of MSC transplanted by various routes (intravenous, subcutaneous, intrapancreatic and intrasplenic) in order to identify the best delivery approach that provides an accumulation of therapeutic cells to the injured pancreas in the non-obese diabetic (NOD) mouse. The results showed that transplanted MSC had limited migration capacity, irrespective of the administration route, and were short-lived with almost total disappearance at 7 days after transplantation. Within one day after transplantation, cells activated hypoxia signalling pathways, followed by Caspase 3-mediated apoptosis. These were subsequently followed by local recruitment of immune cells at the transplantation site, and the engulfment of apoptotic MSC by macrophages. Our results argue for a "hit and die" mechanism of transplanted MSC. Further investigations will elucidate the molecular crosstalk between the inoculated and the host-immune cells.

Heart function assessment during aging in apolipoprotein E knock-out mice.

BACKGROUND: Apolipoprotein (apo) E isoforms have strong correlations with metabolic and cardiovascular diseases. However, it is not clear if apoE has a role in development of non-ischemic cardiomyopathy. Our study aims to analyze the involvement of apoE in non-ischemic cardiomyopathy. METHODS AND RESULTS: Serial echo-cardiographic measurements were performed in old wildtype and apoE deficient (apoE-/-) mice. Morphological and functional cardiac parameters were in normal range in both groups at the age of 12 month. At the age of 18 months, both groups had shown ventricular dilation and increased heart rates. However, the apoE-/- mice presented signs of diastolic dysfunction by hypertrophic changes in left ventricle, due probably to arterial hypertension. The right ventricle was not affected by age or genotype.  CONCLUSION: Even in the absence of high fat diet, apoE deficiency in mice induces mild changes in the cardiac function of the left ventricle during aging, by developing diastolic dysfunction, which leads to heart failure with preserved ejection fraction. However, further studies are necessary to conclude over the role of apoE in cardiac physiology and its involvement in development of heart failure.

Identification of a Hematopoietic Cell Population Emerging From Mouse Bone Marrow With Proliferative Potential In Vitro and Immunomodulatory Capacity.

There is continuing interest in therapeutic applications of bone marrow-derived mesenchymal stromal cells (MSC). Unlike human counterparts, mouse MSC are difficult to propagate in vitro due to their contamination with adherent hematopoietic cells that overgrow the cultures. Here we investigated the properties of these contaminating cells, referred to as bone marrow-derived proliferating hematopoietic cells (BM-PHC). The results showed that both BM-PHC and MSC had strong immunomodulatory properties on T cells in vitro, with PGE2 and NO involved in this mechanism. However, BM-PHC were stronger immunomodulators than MSC, with CCL-6 identified as putative molecule responsible for superior effects. In vivo studies showed that, in contrast to BM-PHC, MSC endorsed a more rapid xenograft tumor rejection, thus indicating a particular context in which only MSC therapy would produce positive outcomes. In conclusion, bone marrow contains two cell populations with immunomodulatory properties, which are valuable sources for therapeutic studies in specific disease-relevant contexts.

MiR-29a Increase in Aging May Function as a Compensatory Mechanism Against Cardiac Fibrosis Through SERPINH1 Downregulation.

Deregulation of microRNA (miRNA) profile has been reportedly linked to the aging process, which is a dominant risk factor for many pathologies. Among the miRNAs with documented roles in aging-related cardiac diseases, miR-18a, -21a, -22, and -29a were mainly associated with hypertrophy and/or fibrosis; however, their relationship to aging was not fully addressed before. The purpose of this paper was to evaluate the variations in the expression levels of these miRNAs in the aging process. To this aim, multiple organs were harvested from young (2-3-months-old), old (16-18-months-old), and very old (24-25-months-old) mice, and the abundance of the miRNAs was evaluated by quantitative real-time (RT)-PCR. Our studies demonstrated that miR-21a, miR-22, and miR-29a were upregulated in the aged heart. Among them, miR-29a was highly expressed in many other organs, i.e., the brain, the skeletal muscle, the pancreas, and the kidney, and its expression was further upregulated during the natural aging process. Western blot, immunofluorescence, and xCELLigence analyses concurrently indicated that overexpression of miR-29a in the muscle cells decreased the collagen levels as well as cell migration and proliferation. Computational prediction analysis and overexpression studies identified SERPINH1, a specific chaperone of procollagens, as a potential miR-29a target. Corroborating to this, significantly downregulated SERPINH1 levels were found in the skeletal muscle, the heart, the brain, the kidney, and the pancreas harvested from very old animals, thereby indicating the role of the miR-29a-SERPINH1 axis in the aging process. In vitro analysis of miR-29a effects on fibroblast and cardiac muscle cells pointed toward a protective role of miR-29a on aging-related fibrosis, by reducing cell migration and proliferation. In conclusion, our study indicates an adaptive increase of miR-29 in the natural aging process and suggests its role as a transcriptional repressor of SERPINH1, with a potential therapeutic value against adverse matrix remodeling and aging-associated tissue fibrosis.

Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll gradient.

Given the complex composition of bone marrow, a cell separation technique that results in populations enriched in progenitor cells is required for cellular differentiation and transplantation studies. In the present study, we designed a method that allows for the isolation of a progenitor-enriched population of bone marrow by exploiting the physical properties of these cells. Bone marrow aspirate was separated on a discontinuous Percoll gradient (ranging from 1.050 to 1.083 g/cm3) that resulted in the recovery of six cell fractions. The fractions were characterized by FACS and RT-PCR (reverse transcription-PCR) analyses and evaluated for their capacity to differentiate into haematopoietic and mesenchymal cells. Fraction IV, including cells with a density of 1.070-1.076 g/ml, contained 11.68% of total bone marrow cells and was enriched in c-kit+ and Sca-1+ (stem cell antigen-1) progenitor cells as compared with total bone marrow. This fraction demonstrated an increase in clonogenic capacity under specific conditions as well as a potential to generate a mesenchymal stem cell culture in a shorter period than that using bone marrow aspirate. Furthermore, this fraction lacked differentiated cell types and contained cells positive for endothelial markers, which further increases its value in cellular transplant. In conclusion, a bone marrow subpopulation that is enriched in progenitor cells and may be valuable in cellular transplant therapy can be isolated by exploiting the physical properties of these cells.

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.

Publisher Correction: Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

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