BACKGROUND:The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material.OBJECTIVE:Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction.METHODS:Three μl of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays.RESULTS:Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies.CONCLUSION:We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.

The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogel-based tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT (R)) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.

Medtronic's INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenographic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls. (C) 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 401406, 2013

Cell-based tissue engineering strategies have shown tremendous promise for the repair of bone mass deficiencies, but the efficient and appropriate induction of stem cells down osteogenic pathways remains a significant roadblock to the effective implementation of cell-based therapies. When grown in culture, human Mesenchymal Stromal/Stem Cells (hMSCs) remain multipotent, requiring specific exogenous signals to induce osteogenic differentiation. hMSCs used in transplantations, therefore, must be presented with local signals, often provided by the host's own tissues, to be directed down bone-related lineages. This process is relatively inefficient and remains difficult to control. In an effort to enhance osteogenesis, hMSCs were transfected with specific miRNA mimics and inhibitors that had originally identified for their ability to increase Alkaline Phosphatase (ALP) activity. Transfection with miRNA reagents had the effect of sensitizing hMSCs to soluble osteogenic factors, resulting in a rapid and robust induction of bone-related markers, including ALP activity and calcium deposition. Synthetic 3D tissue constructs prepared with miRNA-transfected hMSCs demonstrated similar responses to soluble osteogenic signals, suggesting that controlling miRNA activity in hMSCs can be an effective tool for enhancing the induction of osteogenesis for tissue engineering purposes. Copyright (c) 2011 John Wiley & Sons, Ltd.

Thiol-ene-based poly(ethylene glycol) (PEG) hydrogels provide a unique functional platform for the sustained and localized delivery of bioactive small molecules like glucocorticoids. As a proof of concept, the synthetic glucocorticoid Dexamethasone (Dex) was conjugated to the N-terminus of a matrix metalloproteinase(MMP)-degradable peptide, which was then easily co-polymerized into PEG gel scaffolds by a thiol-ene polymerization mechanism. The conjugated Dex was locally sequestered until released by cleavage of the MMP-degradable peptide tether triggered by cell-secreted MMPs, and was only available for uptake by local co-encapsulated cells. Elevated alkaline phosphatase (ALP) activities and calcium deposition levels were observed for human mesenchymal stem cells (hMSCs) that were encapsulated in PEG hydrogels functionalized with 10 μM of a Dexamethasone-conjugated peptide (Dex-peptide). The cellular responses stimulated by the tethered Dex lasted for over 21 days. Using co-culture experiments, hMSCs encapsulated in hydrogels with the MMP-degradable Dex-peptides had elevated levels of ALP activity and calcium deposition, whereas no elevated cellular responses were observed in co-cultured hMSCs surrounding the gel. Moreover, modifying the peptide sequence to alter its susceptibility to cleavage and/or changing the Dex-peptide loading further regulated the hMSC response to Dex at different levels and on different time scales. Collectively, these results demonstrate a tunable system for the delivery of glucocorticoids in a localized and cell-dictated manner.

One of the primary goals for tissue engineering is to induce new tissue formation by stimulating specific cell function. Human mesenchymal stem cells (hMSCs) are a particularly important cell type that has been widely studied for differentiation down the osteogenic (bone) lineage, and we recently found that simple phosphate functional groups incorporated into poly(ethylene glycol) (PEG) hydrogels could induce osteogenesis without using differentiation medium by unknown mechanisms. Here, we aimed to determine whether direct or indirect cell/materials interactions were responsible for directing hMSCs down the osteogenic lineage on phosphate (PO(4))-functionalized PEG hydrogels. Our results indicated that serum components adsorbed onto PO(4)-PEG hydrogels from medium in a presoaking step were sufficient for attachment and spreading of hMSCs, even when seeded in serum-free conditions. Blocking antibodies for collagen and fibronectin (targeted to the hydrogel), as well as β1 and β3 integrin blocking antibodies (targeted to the cells), each reduced attachment of hMSCs to PO(4)-PEG hydrogels, suggesting that integrin-mediated interactions between cells and adsorbed matrix components facilitate attachment and spreading. Outside-in signaling, and not merely shape change, was found to be required for osteogenesis, as alkaline phosphatase activity and expression of CBFA1, osteopontin and collagen-1 were each significantly down regulated upon inhibition of focal adhesion kinase phosphorylation even though the focal adhesion structure or cell shape was unchanged. Our results demonstrate that complex function (i.e. osteogenic differentiation) can be controlled using simple functionalization strategies, such as incorporation of PO(4), but that the role of these materials may be due to more complex influences than has previously been appreciated.

Objective—Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2% to 3% of the U.S. population over the age of 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely because of their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (αSMA) expression, contractility, and collagen production. Methods and Results—In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Overexpression of αSMA increased VIC calcific nodule formation and contractility, whereas knockdown of αSMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of αSMA are essential to the formation of nodules. Statin treatment of VICs reduced αSMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure. Conclusions—Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore, we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.

Noncoding RNAs (ncRNAs) have recently been discovered to regulate mRNA transcription in trans, a role traditionally reserved for proteins. The breadth of ncRNAs as transacting transcriptional regulators and the diversity of signals to which they respond are only now becoming recognized. Here we show that human Alu RNA, transcribed from short interspersed elements (SINEs), is a transacting transcriptional repressor during the cellular heat shock response. Alu RNA blocks transcription by binding RNA polymerase II (Pol II) and entering complexes at promoters in vitro and in human cells. Transcriptional repression by Alu RNA involves two loosely structured domains that are modular, a property reminiscent of classical protein transcriptional regulators. Two other SINE RNAs, human scAlu RNA and mouse B1 RNA, also bind Pol II but do not repress transcription in vitro. These studies provide an explanation for why mouse cells harbor two major classes of SINEs, whereas human cells contain only one.

We have found that mouse B2 RNA and human Alu RNA are transacting transcriptional repressors of RNA polymerase II. In response to heat shock, these ncRNAs are transcribed by RNA polymerase III from short interspersed elements (SINEs) that are abundant in the genomes and prototypically considered ‘junk’ DNA. B2 RNA and Alu RNA bind to core RNA polymerase II with high affinity and enter stable complexes at promoters of specific genes, where they repress transcription. Structure/function studies revealed that B2 RNA and Alu RNA contain two types of functionally distinct domains: Pol II binding domains and transcriptional repression domains. Our studies lead to a model in which Pol II binding domains in ncRNA transcriptional repressors bind a high affinity docking site on RNA polymerase II, thereby tethering distinct repression domains to the polymerase in order to block transcription. Surprisingly, the functional domains in SINE ncRNAs, much like those of classical protein transcriptional regulators, are modular and can be mixed and matched to create chimeric ncRNA transcriptional repressors. Thus, SINE‐encoded ncRNAs resemble prototypical protein regulators of eukaryotic transcription. Our work provides an explanation for how RNA polymerase II transcription is repressed during heat shock in mouse and human cells, and provides an evolutionary rationale for the maintenance of SINEs in the mouse and human genomes.

This study aimed to identify signaling pathways that oppose connective tissue fibrosis in the aortic valve. Using valvular interstitial cells (VICs) isolated from porcine aortic valve leaflets, we show that basic fibroblast growth factor (FGF-2) effectively blocks transforming growth factor-beta1 (TGF-beta1)-mediated myofibroblast activation. FGF-2 prevents the induction of alpha-smooth muscle actin (alphaSMA) expression and the exit of VICs from the cell cycle, both of which are hallmarks of myofibroblast activation. By blocking the activity of the Smad transcription factors that serve as the downstream nuclear effectors of TGF-beta1, FGF-2 treatment inhibits fibrosis in VICs. Using an exogenous Smad-responsive transcriptional promoter reporter, we show that Smad activity is repressed by FGF-2, likely an effect of the fact that FGF-2 treatment prevents the nuclear localization of Smads in these cells. This appears to be a direct effect of FGF signaling through mitogen-activated protein kinase (MAPK) cascades as the treatment of VICs with the MAPK/extracellular regulated kinase (MEK) inhibitor U0126 acted to induce fibrosis and blocked the ability of FGF-2 to inhibit TGF-beta1 signaling. Furthermore, FGF-2 treatment of VICs blocks the development of pathological contractile and calcifying phenotypes, suggesting that these pathways may be utilized in the engineering of effective treatments for valvular disease.

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

In the work presented here, we elucidate a mechanism for the repression of alpha-myosin heavy chain (MyHC) during pathological cardiac hypertrophy. We demonstrate that the transcription factor Yin Yang 1 (YY1) significantly decreases endogenous alpha-MyHC mRNA and protein expression in neonatal rat ventricular myocytes. Furthermore, mutation of the YY1 binding sites in the proximal rat alpha-MyHC promoter increases promoter activity and alleviates YY1-mediated repression of the promoter. Despite the presence of 5 sites that bind YY1, only one site, located at -94bp of the rat alpha-MyHC promoter, is both necessary and sufficient for pathological repression of the promoter by phorbol esters, revealing a unique mechanism for the repression of alpha-MyHC expression during cardiac hypertrophy.

YY1 is a transcription factor that can repress or activate transcription of the genes with which it interacts. In this report we show that YY1 is a negative regulator of the αMyHC gene, which, with βMyHC are the molecular motors of the heart. αMyHC mRNA and protein levels are down regulated in hypertrophy and heart failure, and this is thought to be detrimental for heart contractility. We show that YY1 specifically interacts with the αMyHC promoter and that over-expression of YY1 in cardiac cells repress the activity of the αMyHC promoter. We also show that the 170–200 aa region of YY1, important for its interaction with HATs and HDACs, is important for its repressive activity and that YY1 deleted for this region is an activator of the αMyHC promoter. Moreover, we show that YY1 levels and DNA binding activity are increased in failing human left ventricules and in a mouse model of hypertrophic cardiomiopathy (HCM), where αMyHC levels are decreased. The results suggest that YY1 is a negative regulator of the αMyHC gene expression.

Yin Yang 1 (YY1) is a transcription factor that can repress or activate transcription of the genes with which it interacts. In this report we show that YY1 is a negative regulator of the alpha-myosin heavy chain (alphaMyHC) gene, which, with betaMyHC are the molecular motors of the heart. alphaMyHC mRNA and protein levels are down-regulated in hypertrophy and heart failure, and this is thought to be detrimental for cardiac contractility. We show that YY1 specifically interacts with the alphaMyHC promoter and that overexpression of YY1 in cardiac cells represses the activity of the alphaMyHC promoter. We also show that the 170-200-amino acid region of YY1, important for its interaction with histone acetyl transferases and histone deacetylases, is important for its repressive activity and that YY1 deleted in this region is an activator of the alphaMyHC promoter. Moreover, we show that YY1 levels and DNA binding activity are increased in failing human left ventricles and in a mouse model of hypertrophic cardiomyopathy, where alphaMyHC levels are decreased. These results suggest that YY1 is a negative regulator of alphaMyHC gene expression.