Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is believed this is the first report of the substantial expression of IIB mRNA in man as demonstrated in a subset of masseter fibres, but rarely in limb muscle, and in only a few fibres of the external oblique. These findings provide further evidence for the complexity of myosin gene expression, especially in jaw-closing muscles.
In situ hybridization; Myosin gene expression; ATPase histochemistry; Immunohistochemistry; External oblique muscle; Fibre types
Although the mammalian heart is one of the least regenerative organs in the body, recent evidence indicates that the myocardium undergoes a certain degree of renewal to maintain homeostasis during normal aging. However, the cellular origin of cardiomyocyte renewal has remained elusive due to lack of lineage tracing experiments focusing on putative adult cardiac precursor cells. We have generated triple-transgenic mice based on the tet-cre system to identify descendants of cells that have expressed the stem cell marker Sca1. We found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart. Our results reveal a source of cells for cardiomyocyte renewal and provide a possible explanation for the limited contribution of Sca1-derived cells to myocardial repair under pathological conditions.
•Sca1pos cells continuously generate cardiomyocytes during adult life•Some Sca1pos cells are tightly associated with cardiomyocytes•Sca1pos-derived cells show limited clonal expansion•Pressure overload moderately increases the number of Sca1-derived cardiomyocytes
To understand the renewal of cardiomyocytes, Uchida, Braun, and colleagues followed the fates of Sca1-positive cells in the heart. By utilizing triple-transgenic mice, they found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart.
The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.
Graft-versus-host disease (GVHD) causes most non-relapse mortality (NRM) following alternative donor (unrelated and mismatched related) hematopoietic cell transplant (HCT). We previously showed that increases in day +7 TNF-receptor-1 (TNFR1) ratios (post-transplant day +7/pre-transplant baseline) after myeloablative HCT correlate with outcomes including GVHD, NRM and survival. Therefore, we conducted a phase II trial at two centers testing whether the addition of the TNF-inhibitor etanercept (25 mg twice weekly from start of conditioning to day +56) to standard GVHD prophylaxis would lower TNFR1 levels, reduce GVHD rates, and improve NRM and survival.
Patients and Methods
Patients underwent myeloablative HCT from a matched unrelated donor (N=71), one-antigen mismatched unrelated donor (N=26) or one-antigen mismatched related donor (N=3) using either total body irradiation (TBI)-based conditioning (N=29) or non-TBI-based conditioning (N=71).
Compared to historical controls, the increase in post-transplant day +7 TNFR1 ratios was not altered in patients who received TBI-based conditioning, but was 40% lower in patients receiving non-TBI-based conditioning. The latter group experienced relatively low rates of severe grade 3-4 GVHD (14%), one-year NRM (16%), and high one-year survival (69%).
These findings suggest that (1) the effectiveness of TNF-inhibition with etanercept may depend on the conditioning regimen, and (2) attenuating the expected rise in TNFR1 levels early post-transplant correlates with good outcomes.
GVHD; hematopoietic cell transplantation; TNFα; TNFR1
miRNAs are small RNAs directing many developmental processes by posttranscriptional regulation of protein-coding genes. We uncovered a new role for miR-1-1/133a-2 and miR-1-2/133a-1 clusters in the specification of embryonic cardiomyocytes allowing transition from an immature state characterized by expression of smooth muscle (SM) genes to a more mature fetal phenotype. Concomitant knockout of miR-1-1/133a-2 and miR-1-2/133a-1 released suppression of the transcriptional co-activator myocardin, a major regulator of SM gene expression, but not of its binding partner SRF. Overexpression of myocardin in the embryonic heart essentially recapitulated the miR-1/133a mutant phenotype at the molecular level, arresting embryonic cardiomyocytes in an immature state. Interestingly, the majority of postulated miR-1/133a targets was not altered in double mutant mice, indicating that the ability of miR-1/133a to suppress target molecules strongly depends on the cellular context. Finally, we show that myocardin positively regulates expression of miR-1/133a, thus constituting a negative feedback loop that is essential for early cardiac development.
miRNAs are small non-coding RNAs involved in posttranscriptional regulation of protein-coding genes. In the mammalian genome, two distinct gene clusters code for miR-1 and miR-133a. Primary sequences of mature miR-1 or miR-133a are identical and both gene clusters show similar expression in the heart and skeletal muscle. We have generated compound mutant mice of both miR-1/133a gene clusters resulting in early arrest of heart development while single cluster mutants showed normal morphology but reacted differently to pressure overload. Compound mutant cardiomyocytes were characterized by an immature, mixed smooth muscle-heart muscle phenotype, indicating that miR1-/133a are responsible for specification of the cardiomyogenic lineage. Our search for miR1-/133a targets identified myocardin, which was strongly up-regulated in mutant hearts, while several other putative miR-1/133a targets that have been described before were not altered, indicating that miR-1/133a target control strongly depends on the cellular context. Overexpression of myocardin in embryonic hearts recapitulated major aspects of the miR-1/133a mutant phenotype, suggesting that loss of myocardin suppression is the primary reason for incorrect heart muscle specification in the mutants. In addition, we found that myocardin overexpression stimulated expression of miR-1/133a, which argues for a negative feedback loop required for adjustment of myocardin concentrations in the heart.
Morphogenesis of the heart requires tight control of cardiac progenitor cell specification, expansion, and differentiation. Retinoic acid (RA) signaling restricts expansion of the second heart field (SHF), serving as an important morphogen in heart development. Here, we identify the LIM domain protein Ajuba as a crucial regulator of the SHF progenitor cell specification and expansion. Ajuba-deficient zebra-fish embryos show an increased pool of Isl1+ cardiac progenitors and, subsequently, dramatically increased numbers of cardiomyocytes at the arterial and venous poles. Furthermore, we show that Ajuba binds Isl1, represses its transcriptional activity, and is also required for autorepression of Isl1 expression in an RA-dependent manner. Lack of Ajuba abrogates the RA-dependent restriction of Isl1+ cardiac cells. We conclude that Ajuba plays a central role in regulating the SHF during heart development by linking RA signaling to the function of Isl1, a key transcription factor in cardiac progenitor cells.
Regeneration of the lens in newts is quite a unique process. The lens is removed in its entirety and regeneration ensues from the pigment epithelial cells of the dorsal iris via transdifferentiation. The same type of cells from the ventral iris are not capable of regenerating a lens. It is, thus, expected that differences between dorsal and ventral iris during the process of regeneration might provide important clues pertaining to the mechanism of regeneration. In this paper, we employed next generation RNA-seq to determine gene expression patterns during lens regeneration in Notophthalmus viridescens. The expression of more than 38,000 transcripts was compared between dorsal and ventral iris. Although very few genes were found to be dorsal- or ventral-specific, certain groups of genes were up-regulated specifically in the dorsal iris. These genes are involved in cell cycle, gene regulation, cytoskeleton and immune response. In addition, the expression of six highly regulated genes, TBX5, FGF10, UNC5B, VAX2, NR2F5, and NTN1, was verified using qRT-PCR. These graded gene expression patterns provide insight into the mechanism of lens regeneration, the markers that are specific to dorsal or ventral iris, and layout a map for future studies in the field.
Resampling algorithms provide an empirical, non-parametric approach to determine the statistical significance of annotations in different experimental settings. ResA3 (Resampling Analysis of Arbitrary Annotations, short: ResA) is a novel tool to facilitate the analysis of enrichment and regulation of annotations deposited in various online resources such as KEGG, Gene Ontology and Pfam or any kind of classification. Results are presented in readily accessible navigable table views together with relevant information for statistical inference. The tool is able to analyze multiple types of annotations in a single run and includes a Gene Ontology annotation feature. We successfully tested ResA using a dataset obtained by measuring incorporation rates of stable isotopes into proteins in intact animals. ResA complements existing tools and will help to evaluate the increasing number of large-scale transcriptomics and proteomics datasets (resa.mpi-bn.mpg.de).
Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called ‘noncoder’. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.
The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer metastasis. It is highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. The nuclear transcript MALAT-1 has been functionally associated with gene regulation and alternative splicing and its regulation has been shown to impact proliferation, apoptosis, migration and invasion.
Here, we have developed a human and a mouse knockout system to study the loss-of-function phenotypes of this important ncRNA. In human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases. Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation nor cell cycle progression nor nuclear architecture in human lung or liver cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not give rise to any obvious phenotype or histological abnormalities in Malat1-null compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA MALAT1 is compatible with cell viability and normal development.
MALAT1; human knockout model; knockout mouse; long non-coding RNA
Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signaling. Besides neuroectodermal malformations and tumors, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia) demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here, we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1Prx1) resulted in muscle dystrophy characterized by fibrosis, reduced number of muscle fibers and reduced muscle force. This was caused by an early defect in myogenesis affecting the terminal differentiation of myoblasts between E12.5 and E14.5. In parallel, the muscle connective tissue cells exhibited increased proliferation at E14.5 and an increase in the amount of connective tissue as early as E16.5. These changes were accompanied by excessive mitogen-activated protein kinase pathway activation. Satellite cells isolated from Nf1Prx1 mice showed normal self-renewal, but their differentiation was impaired as indicated by diminished myotube formation. Our results demonstrate a requirement of neurofibromin for muscle formation and maintenance. This previously unrecognized function of neurofibromin may contribute to the musculoskeletal problems in NF1 patients.
Cardiomyocyte development in mammals is characterized by a transition from hyperplastic to hypertrophic growth soon after birth. The rise of cardiomyocyte cell mass in postnatal life goes along with a proportionally bigger increase in the mitochondrial mass in response to growing energy requirements. Relatively little is known about the molecular processes regulating mitochondrial biogenesis and mitochondrial DNA (mtDNA) maintenance during developmental cardiac hypertrophy. Genome-wide transcriptional profiling revealed the activation of transcriptional regulatory circuits controlling mitochondrial biogenesis in growing rat hearts. In particular, we detected a specific upregulation of factors involved in mtDNA expression and translation. More surprisingly, we found a specific upregulation of DNA repair proteins directly linked to increased oxidative damage during heart mitochondrial biogenesis, but only relatively minor changes in the mtDNA replication machinery. Our study paves the way for improved understanding of mitochondrial biogenesis, mtDNA maintenance and physiological adaptation processes in the heart and provides the first evidence for the recruitment of nucleotide excision repair proteins to mtDNA in cardiomyocytes upon DNA damage.
Exon arrays are regularly used to analyze differential splicing events. GeneChip Gene 1.0 ST Arrays (gene arrays) manufactured by Affymetrix, Inc. are primarily used to determine expression levels of transcripts, although their basic design is rather similar to GeneChip Exon 1.0 ST Arrays (exon arrays). Here, we show that the newly developed Gene Array Analyzer (GAA), which evolved from our previously published Exon Array Analyzer (EAA), enables economic and user-friendly analysis of alternative splicing events using gene arrays. To demonstrate the applicability of GAA, we profiled alternative splicing events during embryonic heart development. In addition, we found that numerous developmental splicing events are also activated under pathological conditions. We reason that the usage of GAA considerably expands the analysis of gene expression based on gene arrays and supplies an additional level of information without further costs and with only little effort.
Notophthalmus viridescens, a member of the salamander family is an excellent model organism to study regenerative processes due to its unique ability to replace lost appendages and to repair internal organs. Molecular insights into regenerative events have been severely hampered by the lack of genomic, transcriptomic and proteomic data, as well as an appropriate database to store such novel information. Here, we describe ‘Newt-omics’ (http://newt-omics.mpi-bn.mpg.de), a database, which enables researchers to locate, retrieve and store data sets dedicated to the molecular characterization of newts. Newt-omics is a transcript-centred database, based on an Expressed Sequence Tag (EST) data set from the newt, covering ∼50 000 Sanger sequenced transcripts and a set of high-density microarray data, generated from regenerating hearts. Newt-omics also contains a large set of peptides identified by mass spectrometry, which was used to validate 13 810 ESTs as true protein coding. Newt-omics is open to implement additional high-throughput data sets without changing the database structure. Via a user-friendly interface Newt-omics allows access to a huge set of molecular data without the need for prior bioinformatical expertise.
The relationship between regulatory T cells (Tregs) and acute graft-versus-host disease (GVHD) in clinical allogeneic bone marrow transplantation (BMT) recipients is not well established. We conducted a prospective analysis of peripheral blood Tregs as determined by the frequency of CD4+CD25hiFOXP3+ lymphocytes in 215 BMT patients. Autologous BMT patients (N=90) and allogeneic BMT patients without GVHD (N=65) had similar Treg frequencies, whereas allogeneic patients with GVHD (N=60) had Treg frequencies that were 40% less than those without GVHD. Treg frequencies decreased linearly with increasing grades of GVHD at onset and correlated with eventual maximum grade of GVHD (p<0.001). In addition, frequency of Tregs at onset of GVHD predicted the response to GVHD treatment (p=0.003). Patients with Treg frequencies less than the median had higher non-relapse mortality than patients with Tregs greater than the median, but experienced equivalent relapse mortality, resulting in an inferior survival at two years (38% vs. 63%, p=0.03). Treg frequency may therefore have important prognostic value as a biomarker of acute GVHD.
Allogeneic BMT; Acute graft-versus-host-disease; Regulatory T cells; Biomarker
We propose a phase I clinical trial design that seeks to determine the cumulative safety of a series of administrations of a fixed dose of an investigational agent. In contrast with traditional phase I trials that are designed solely to find the maximum tolerated dose of the agent, our design instead identifies a maximum tolerated schedule that includes a maximum tolerated dose as well as a vector of recommended administration times. Our model is based on a non-mixture cure model that constrains the probability of dose limiting toxicity for all patients to increase monotonically with both dose and the number of administrations received. We assume a specific parametric hazard function for each administration and compute the total hazard of dose limiting toxicity for a schedule as a sum of individual administration hazards. Throughout a variety of settings motivated by an actual study in allogeneic bone marrow transplant recipients, we demonstrate that our approach has excellent operating characteristics and performs as well as the only other currently published design for schedule finding studies. We also present arguments for the preference of our non-mixture cure model over the existing model.
Adaptive design; Bayesian statistics; Dose finding study; Phase I trial; Weibull distribution
Polymers are often used to modify surface properties to control interfacial processes. Their sensitivity to solvent conditions and ability to undergo conformational transitions makes polymers attractive in tailoring surface properties with specific functionalities leading to applications in diverse areas ranging from tribology to colloidal stability and medicine. A key example is polyethylene glycol (PEG), which is widely used as a protein-resistant coating given its low toxicity and biocompatibility. We report here a microcantilever-based sensor for the in situ characterization of PEG monolayer formation on Au using the “grafting to” approach. Moreover, we demonstrate how microcantilevers can be used to monitor conformational changes in the grafted PEG layer in different solvent conditions. This is supported by atomic force microscope (AFM) images and force–distance curve measurements of the microcantilever chip surface, which show that the grafted PEG undergoes a reversible collapse when switching between good and poor solvent conditions, respectively.
AFM; cantilever sensor; polyethylene glycol; polymer brush; reversible collapse; static mode
We propose a hierarchical model for the probability of dose-limiting toxicity (DLT) for combinations of doses of two therapeutic agents. We apply this model to an adaptive Bayesian trial algorithm whose goal is to identify combinations with DLT rates close to a pre-specified target rate. We describe methods for generating prior distributions for the parameters in our model from a basic set of information elicited from clinical investigators. We survey the performance of our algorithm in a series of simulations of a hypothetical trial that examines combinations of four doses of two agents. We also compare the performance of our approach to two existing methods and assess the sensitivity of our approach to the chosen prior distribution.
dose-finding study; dose-escalation study; two-dimensional; adaptive design; Bayesian statistics
Graft-versus-host-disease (GVHD), the major complication of allogeneic bone marrow transplantation (BMT), affects the skin, liver and gastrointestinal (GI) tract. There are no plasma biomarkers specific for any acute GVHD target organ. We used a large scale, quantitative proteomic discovery procedure to identify biomarker candidates of skin GVHD and validated the lead candidate, elafin, by ELISA in samples from 492 patients. Elafin was overexpressed in GVHD skin biopsies. Plasma levels of elafin were significantly higher at the onset of skin GVHD, correlated with the eventual maximum grade of GVHD, and were associated with a greater risk of death relative to other known risk factors (hazard ratio of 1.78). We conclude that elafin has significant diagnostic and prognostic value as a biomarker of skin GVHD.
Several tools have been developed to explore and search Gene Ontology (GO) databases allowing efficient GO enrichment analysis and GO tree visualization. Nevertheless, identification of highly specific GO-terms in complex data sets is relatively complicated and the display of GO term assignments and GO enrichment analysis by simple tables or pie charts is not optimal. Valuable information such as the hierarchical position of a single GO term within the GO tree (topological ordering), or enrichment within a complex set of biological experiments is not displayed. Pie charts based on GO tree levels are, themselves, one-dimensional graphs, which cannot properly or efficiently represent the hierarchical specificity for the biological system being studied.
Here we present a new method, which we name PCA2GO, capable of GO analysis using complex multidimensional experimental settings. We employed principal component analysis (PCA) and developed a new score, which takes into account the relative frequency of certain GO terms and their specificity (hierarchical position) within the GO graph. We evaluated the correlation between our representation score R and a standard measure of enrichment, namely p-values to convey the versatility of our approach to other methods and point out differences between our method and commonly used enrichment analyses. Although p values and the R score formally measure different quantities they should be correlated, because relative frequencies of GO terms occurrences within a dataset are an indirect measure of protein numbers related to this term. Therefore they are also related to enrichment. We showed that our score enables us to identify more specific GO-terms i.e. those positioned further down the GO-graph than other common tools used for this purpose. PCA2GO allows visualization and detection of multidimensional dependencies both within the acyclic graph (GO tree) and the experimental settings. Our method is intended for the analysis of several experimental sets, not for one set, like standard enrichment tools. To demonstrate the usefulness of our approach we performed a PCA2GO analysis of a fractionated cardiomyocyte protein dataset, which was identified by enhanced liquid chromatography-mass spectrometry (GeLC-MS). The analysis enabled us to detect distinct groups of proteins, which accurately reflect properties of biochemical cell fractions.
We conclude that PCA2GO is an alternative efficient GO analysis tool with unique features for detection and visualization of multidimensional dependencies within the dataset under study. PCA2GO reveals strongly correlated GO terms within the experimental setting (in this case different fractions) by PCA group formation and improves detection of more specific GO terms within experiment dependent GO term groups than standard p value calculations.
The newt Notophthalmus viridescens possesses the remarkable ability to respond to cardiac damage by formation of new myocardial tissue. Surprisingly little is known about changes in gene activities that occur during the course of regeneration. To begin to decipher the molecular processes, that underlie restoration of functional cardiac tissue, we generated an EST database from regenerating newt hearts and compared the transcriptional profile of selected candidates with genes deregulated during zebrafish heart regeneration.
A cDNA library of 100,000 cDNA clones was generated from newt hearts 14 days after ventricular injury. Sequencing of 11520 cDNA clones resulted in 2894 assembled contigs. BLAST searches revealed 1695 sequences with potential homology to sequences from the NCBI database. BLAST searches to TrEMBL and Swiss-Prot databases assigned 1116 proteins to Gene Ontology terms. We also identified a relatively large set of 174 ORFs, which are likely to be unique for urodele amphibians. Expression analysis of newt-zebrafish homologues confirmed the deregulation of selected genes during heart regeneration. Sequences, BLAST results and GO annotations were visualized in a relational web based database followed by grouping of identified proteins into clusters of GO Terms. Comparison of data from regenerating zebrafish hearts identified biological processes, which were uniformly overrepresented during cardiac regeneration in newt and zebrafish.
We concluded that heart regeneration in newts and zebrafish led to the activation of similar sets of genes, which suggests that heart regeneration in both species might follow similar principles. The design of the newly established newt EST database allows identification of molecular pathways important for heart regeneration.
VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145–deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry–based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT1 receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145–deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.