Effect of Recombinant Human Lubricin on Model Tear Film Stability.

Purpose: To investigate and quantify the effect of recombinant human lubricin (rh-lubricin) on model tear film stability. Methods: A custom-built, interferometry-based instrument called the Interfacial Dewetting and Drainage Optical Platform was used to create and record the spatiotemporal evolution of model acellular tear films. Image segmentation and analysis was performed in MATLAB to extract the most essential features from the wet area fraction versus time curve, namely the evaporative break-up time and the final wet area fraction (A10). These two parameters indicate the tear film stability in the presence of rh-lubricin in its unstressed and stressed forms. Results: Our parameters successfully captured the trend of increasing tear film stability with increasing rh-lubricin concentration, and captured differences in rh-lubricin efficacy after various industrially relevant stresses. Specifically, aggregation and fragmentation caused by a 4-week, high temperature stress condition negatively impacted rh-lubricin's ability to maintain model tear film stability. Adsorbed rh-lubricin alone was not sufficient to resist break-up and maintain full area coverage of the model tear film surface. Conclusions: Our results demonstrate that fragmentation and aggregation can negatively impact rh-lubricin's ability to maintain a stable tear film. In addition, the ability of rh-lubricin to maintain wetted area coverage is due to both freely dispersed and adsorbed rh-lubricin. Translational Relevance: Our platform and analysis method provide a facile, intuitive, and clinically relevant means to quantify the effect of ophthalmic drugs and formulations intended for improving tear film stability, as well as capture differences between variants related to drug stability and efficacy.

A Mucin-Deficient Ocular Surface Mimetic Platform for Interrogating Drug Effects on Biolubrication, Antiadhesion Properties, and Barrier Functionality.

Dry eye disease (DED) affects more than 100 million people worldwide, causing significant patient discomfort and imposing a multi-billion-dollar burden on global health care systems. In DED patients, the natural biolubrication process that facilitates pain-free blinking goes awry due to an imbalance of lipids, aqueous medium, and mucins in the tear film, resulting in ocular surface damage. Identifying strategies to reduce adhesion and shear stresses between the ocular surface and the conjunctival cells lining the inside of the eyelid during blink cycles is a promising approach to improve the signs and symptoms of DED. However, current preclinical models for screening ocular lubricants rely on scarce, heterogeneous tissue samples or model substrates that do not capture the complex biochemical and biophysical cues present at the ocular surface. To recapitulate the hierarchical architecture and phenotype of the ocular interface for preclinical drug screening, we developed an in vitro mucin-deficient DED model platform that mimics the complexity of the ocular interface and investigated its utility in biolubrication, antiadhesion, and barrier protection studies using recombinant human lubricin, a promising investigational therapy for DED. The biomimetic platform recapitulated the pathological changes in biolubrication, adhesion, and barrier functionality often observed in mucin-deficient DED patients and demonstrated that recombinant human lubricin can reverse the damage induced by mucin loss in a dose- and conformation-dependent manner. Taken together, these results highlight the potential of the platform─and recombinant human lubricin─in advancing the standard of care for mucin-deficient DED patients.

Mucin-Like Glycoproteins Modulate Interfacial Properties of a Mimetic Ocular Epithelial Surface.

Dry eye disease (DED) has high personal and societal costs, but its pathology remains elusive due to intertwined biophysical and biochemical processes at the ocular surface. Specifically, mucin deficiency is reported in a subset of DED patients, but its effects on ocular interfacial properties remain unclear. Herein a novel in vitro mucin-deficient mimetic ocular surface (Mu-DeMOS) with a controllable amount of membrane-tethered mucin molecules is developed to represent the diseased ocular surfaces. Contact angle goniometry on mimetic ocular surfaces reveals that high surface roughness, but not the presence of hydrophilic mucin molecules, delivers constant hydration over native ocular surface epithelia. Live-cell rheometry confirms that the presence of mucin-like glycoproteins on ocular epithelial cells reduces shear adhesive strength at cellular interfaces. Together, optimal surface roughness and surface chemistry facilitate sustainable lubrication for healthy ocular surfaces, while an imbalance between them contributes to lubrication-related dysfunction at diseased ocular epithelial surfaces. Furthermore, the restoration of low adhesive strength at Mu-DeMOS interfaces through a mucin-like glycoprotein, recombinant human lubricin, suggests that increased frictional damage at mucin-deficient cellular surfaces may be reversible. More broadly, these results demonstrate that Mu-DeMOS is a promising platform for drug screening assays and fundamental studies on ocular physiology.

Argonaute 2 Is Required for Extra-embryonic Endoderm Differentiation of Mouse Embryonic Stem Cells.

In mouse, although four Argonaute (AGO) proteins with partly overlapping functions in small-RNA pathways exist, only Ago2 deficiency causes embryonic lethality. To investigate the role of AGO2 during mouse early development, we generated Ago2-deficient mouse embryonic stem cells (mESCs) and performed a detailed characterization of their differentiation potential. Ago2 disruption caused a global reduction of microRNAs, which resulted in the misregulation of only a limited number of transcripts. We demonstrated, both in vivo and in vitro, that AGO2 is dispensable for the embryonic germ-layer formation. However, Ago2-deficient mESCs showed a specific defect during conversion into extra-embryonic endoderm cells. We proved that this defect is cell autonomous and can be rescued by both a catalytically active and an inactive Ago2, but not by Ago2 deprived of its RNA binding capacity or by Ago1 overexpression. Overall, our results suggest a role for AGO2 in stem cell differentiation.

Dicer, a new regulator of pluripotency exit and LINE-1 elements in mouse embryonic stem cells.

A gene regulation network orchestrates processes ensuring the maintenance of cellular identity and genome integrity. Small RNAs generated by the RNAse III DICER have emerged as central players in this network. Moreover, deletion of Dicer in mice leads to early embryonic lethality. To better understand the underlying mechanisms leading to this phenotype, we generated Dicer-deficient mouse embryonic stem cells (mESCs). Their detailed characterization revealed an impaired differentiation potential, and incapacity to exit from the pluripotency state. We also observed a strong accumulation of LINE-1 (L1s) transcripts, which was translated at protein level and led to an increased L1s retrotransposition. Our findings reveal Dicer as a new essential player that sustains mESCs self-renewal and genome integrity by controlling L1s regulation.

Noncanonical function of DGCR8 controls mESC exit from pluripotency.

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. The analysis of newly generated mutant mESCs revealed that DGCR8 is essential for the exit from the pluripotency state. To dissociate canonical versus noncanonical functions of DGCR8, we complemented the mutant mESCs with a phosphomutant DGCR8, which restored microRNA levels but did not rescue the exit from pluripotency defect. Integration of omics data and RNA immunoprecipitation experiments established DGCR8 as a direct interactor of Tcf7l1 mRNA, a core component of the pluripotency network. Finally, we found that DGCR8 facilitated the splicing of Tcf7l1, an event necessary for the differentiation of mESCs. Our data reveal a new noncanonical function of DGCR8 in the modulation of the alternative splicing of Tcf7l1 mRNA in addition to its established function in microRNA biogenesis.

The Role of RNA Interference in Stem Cell Biology: Beyond the Mutant Phenotypes.

Complex gene regulation systems ensure the maintenance of cellular identity during early development in mammals. Eukaryotic small RNAs have emerged as critical players in RNA interference (RNAi) by mediating gene silencing during embryonic stem cell self-renewal. Most of the proteins involved in the biogenesis of small RNAs are essential for proliferation and differentiation into the three germ layers of mouse embryonic stem cells. In the last decade, new functions for some RNAi proteins, independent of their roles in RNAi pathways, have been demonstrated in different biological systems. In parallel, new concepts in stem cell biology have emerged. Here, we review and integrate the current understanding of how RNAi proteins regulate stem cell identity with the new advances in the stem cell field and the recent non-canonical functions of the RNAi proteins. Finally, we propose a reevaluation of all RNAi mutant phenotypes, as non-canonical (small non-coding RNA independent) functions may contribute to the molecular mechanisms governing mouse embryonic stem cells commitment.

Exit from Pluripotency Assay of Mouse Embryonic Stem Cells.

A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed ( Ying et al., 2003 ; Betschinger et al., 2013 ). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this 'exit from pluripotency assay' ( Bodak et al., 2017 ; Cirera-Salinas et al., 2017 ). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.

Autoregulation of glypican-1 by intronic microRNA-149 fine tunes the angiogenic response to FGF2 in human endothelial cells.

MicroRNA-149 (miR-149) is located within the first intron of the glypican-1 (GPC1) gene. GPC1 is a low affinity receptor for fibroblast growth factor (FGF2) that enhances FGF2 binding to its receptor (FGFR1), subsequently promoting FGF2-FGFR1 activation and signaling. Using bioinformatic approaches, both GPC1 and FGFR1 were identified and subsequently validated as targets for miR-149 (both the mature strand, miR-149, and the passenger strand, miR-149*) in endothelial cells (ECs). As a consequence of their targeting activity towards GPC1 and FGFR1, both miR-149 and miR-149* regulated FGF2 signaling and FGF2-induced responses in ECs, namely proliferation, migration and cord formation. Moreover, lentiviral overexpression of miR-149 reduced in vivo tumor-induced neovascularization. Importantly, FGF2 transcriptionally stimulated the expression of miR-149 independently of its host gene, therefore assuring the steady state of FGF2-induced responses through the regulation of the GPC1-FGFR1 binary complex in ECs.

MicroRNA 33 regulates glucose metabolism.

Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small noncoding RNAs (microRNAs [miRNAs]) in the posttranscriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a and miR33b, intronic miRNAs located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression, leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression of PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.

A regulatory role for microRNA 33* in controlling lipid metabolism gene expression.

hsa-miR-33a and hsa-miR-33b, intronic microRNAs (miRNAs) located within the sterol regulatory element-binding protein 2 and 1 genes (Srebp-2 and -1), respectively, have recently been shown to regulate lipid homeostasis in concert with their host genes. Although the functional role of miR-33a and -b has been highly investigated, the role of their passenger strands, miR-33a* and -b*, remains unclear. Here, we demonstrate that miR-33a* and -b* accumulate to steady-state levels in human, mouse, and nonhuman primate tissues and share a similar lipid metabolism target gene network as their sister strands. Analogous to miR-33, miR-33* represses key enzymes involved in cholesterol efflux (ABCA1 and NPC1), fatty acid metabolism (CROT and CPT1a), and insulin signaling (IRS2). Moreover, miR-33* also targets key transcriptional regulators of lipid metabolism, including SRC1, SRC3, NFYC, and RIP140. Importantly, inhibition of either miR-33 or miR-33* rescues target gene expression in cells overexpressing pre-miR-33. Consistent with this, overexpression of miR-33* reduces fatty acid oxidation in human hepatic cells. Altogether, these data support a regulatory role for the miRNA* species and suggest that miR-33 regulates lipid metabolism through both arms of the miR-33/miR-33* duplex.

Control of cholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144.

RATIONALE: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including adenosine triphosphate-binding cassette transporter A1 (ABCA1) and adenosine triphosphate-binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. OBJECTIVE: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. METHODS AND RESULTS: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed an miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to apolipoproteinA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 seems to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. CONCLUSIONS: miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression and modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease.

Mir-33 regulates cell proliferation and cell cycle progression.

Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. microRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression, acting predominantly at posttranscriptional level. Recent work from our group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the Srebp genes, regulate cholesterol and fatty acid metabolism in concert with their host genes. Here, we show that hsa-miR-33 family members modulate the expression of genes involved in cell cycle regulation and cell proliferation. MiR-33 inhibits the expression of the cyclin-dependent kinase 6 (CDK6) and cyclin D1 (CCND1), thereby reducing cell proliferation and cell cycle progression. Overexpression of miR-33 induces a significant G 1 cell cycle arrest in Huh7 and A549 cell lines. Most importantly, inhibition of miR-33 expression using 2'fluoro/methoxyethyl-modified (2'F/MOE-modified) phosphorothioate backbone antisense oligonucleotides improves liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these results suggest that Srebp/miR-33 locus may cooperate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

MicroRNA-758 regulates cholesterol efflux through posttranscriptional repression of ATP-binding cassette transporter A1.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of macrophage cholesterol efflux and protects cells from excess intracellular cholesterol accumulation; however, the mechanism involved in posttranscriptional regulation of ABCA1 is poorly understood. We previously showed that microRNA-33 (miR-33) is 1 regulator. Here, we investigated the potential contribution of other microRNAs (miRNAs) to posttranscriptional regulation of ABCA1 and macrophage cholesterol efflux. METHODS AND RESULTS: We performed a bioinformatic analysis for identifying miRNA target prediction sites in ABCA1 gene and an unbiased genome-wide screen to identify miRNAs modulated by cholesterol excess in mouse peritoneal macrophages. Quantitative real-time reverse transcription-polymerase chain reaction confirmed that miR-758 is repressed in cholesterol-loaded macrophages. Under physiological conditions, high dietary fat excess in mice repressed miR-758 both in peritoneal macrophages and, to a lesser extent, in the liver. In mouse and human cells in vitro, miR-758 repressed the expression of ABCA1, and conversely, the inhibition of this miRNA by using anti-miR-758 increased ABCA1 expression. In mouse cells, miR-758 reduced cellular cholesterol efflux to apolipoprotein A1 (apoA1), and anti-miR-758 increased it. miR-758 directly targets the 3'-untranslated region of Abca1 as assessed by 3'-untranslated region luciferase reporter assays. Interestingly, miR-758 is highly expressed in the brain, where it also targets several genes involved in neurological functions, including Slc38a1, Ntm, Epha7, and Mytl1. CONCLUSION: We identified miR-758 as a novel miRNA that posttranscriptionally controls ABCA1 levels in different cells and regulates macrophage cellular cholesterol efflux to apoA1, opening new avenues to increase apoA1 and raise high-density lipoprotein levels.

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling.

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.