Hieu T. Nim

View from the heart: cardiac fibroblasts in development, scarring and regeneration.

In the adult, tissue repair after injury is generally compromised by fibrosis, which maintains tissue integrity with scar formation but does not restore normal architecture and function. The process of regeneration is necessary to replace the scar and rebuild normal functioning tissue. Here, we address this problem in the context of heart disease, and discuss the origins and characteristics of cardiac fibroblasts, as well as the crucial role that they play in cardiac development and disease. We discuss the dual nature of cardiac fibroblasts, which can lead to scarring, pathological remodelling and functional deficit, but can also promote heart function in some contexts. Finally, we review current and proposed approaches whereby regeneration could be fostered by interventions that limit scar formation. Summary: This Review article looks at the role of cardiac fibroblasts in scarring and regeneration, as well as how these cells are specified during development and the unique characteristics that define them.

Microarray profiling to analyse adult cardiac fibroblast identity.

Heart failure is one of the leading causes of death worldwide [1–4]. Current therapeutic strategies are inefficient and cannot cure this chronic and debilitating condition [5]. Ultimately, heart transplants are required for patient survival, but donor organs are scarce in availability and only prolong the life-span of patients for a limited time. Fibrosis is one of the main pathological features of heart failure [6,7], caused by inappropriate stimulation of fibroblasts and excessive extracellular matrix production. Therefore, an in-depth understanding of the cardiac fibroblast is essential to underpin effective therapeutic treatments for heart failure [5]. Fibroblasts in general have been an underappreciated cell type, regarded as relatively inert and providing only basic functionality; they are usually referred to as the ‘biological glue’ of all tissues in the body. However, more recent literature suggests that they actively participate in organ homeostasis and disease [7,8]. We have recently uncovered a unique molecular identity for fibroblasts isolated from the heart [9], expressing a set of cardiogenic transcription factors that have been previously associated with cardiomyocyte ontogenesis. This signature suggests that cardiac fibroblasts may be ideal for use in stem cell replacement therapies, as they may retain the memory of where they derive from embryologically. Our data also revealed that about 90% of fibroblasts from both tail and heart origins share a cell surface signature that has previously been described for mesenchymal stem cells (MSCs), raising the possibility that fibroblasts and MSCs may in fact be the same cell type. Thus, our findings carry profound implications for the field of regenerative medicine. Here, we describe detailed methodology and quality controls related to the gene expression profiling of cardiac fibroblasts, deposited at the Gene Expression Omnibus (GEO) under the accession number GSE50531. We also provide the R code to easily reproduce the data quantification and analysis processes.

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.

Systems approaches in integrative cardiac biology: Illustrations from cardiac heterocellular signalling studies

Understanding the complexity of cardiac physiology requires system-level studies of multiple cardiac cell types. Frequently, however, the end result of published research lacks the detail of the collaborative and integrative experimental design process, and the underlying conceptual framework. We review the recent progress in systems modelling and omics analysis of the heterocellular heart environment through complementary forward and inverse approaches, illustrating these conceptual and experimental frameworks with case studies from our own research program. The forward approach begins by collecting curated information from the niche cardiac biology literature, and connecting the dots to form mechanistic network models that generate testable system-level predictions. The inverse approach starts from the vast pool of public omics data in recent cardiac biological research, and applies bioinformatics analysis to produce novel candidates for further investigation. We also discuss the possibility of combining these two approaches into a hybrid framework, together with the benefits and challenges. These interdisciplinary research frameworks illustrate the interplay between computational models, omics analysis, and wet lab experiments, which holds the key to making real progress in improving human cardiac wellbeing.

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

Enduring epigenetic landmarks define the cancer microenvironment

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

Point mutations in murine Nkx2-5 phenocopy human congenital heart disease and induce pathogenic Wnt signaling

Mutations in the Nkx2-5 gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the Nkx2-5 gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new Nkx2-5 point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of Nkx2-5-driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by Nkx2-5 in the heart and show that Nkx2-5-dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how Nkx2-5 regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by NKX2-5 mutations in patients.

VISIONET: intuitive visualisation of overlapping transcription factor networks, with applications in cardiogenic gene discovery

BackgroundExisting de novo software platforms have largely overlooked a valuable resource, the expertise of the intended biologist users. Typical data representations such as long gene lists, or highly dense and overlapping transcription factor networks often hinder biologists from relating these results to their expertise.ResultsVISIONET, a streamlined visualisation tool built from experimental needs, enables biologists to transform large and dense overlapping transcription factor networks into sparse human-readable graphs via numerically filtering. The VISIONET interface allows users without a computing background to interactively explore and filter their data, and empowers them to apply their specialist knowledge on far more complex and substantial data sets than is currently possible. Applying VISIONET to the Tbx20-Gata4 transcription factor network led to the discovery and validation of Aldh1a2, an essential developmental gene associated with various important cardiac disorders, as a healthy adult cardiac fibroblast gene co-regulated by cardiogenic transcription factors Gata4 and Tbx20.ConclusionsWe demonstrate with experimental validations the utility of VISIONET for expertise-driven gene discovery that opens new experimental directions that would not otherwise have been identified.

Interactive Geolocational and Coral Compositional Visualisation of Great Barrier Reef Heat Stress Data

The Great Barrier Reef is at critical risk of rapid decline, with mass coral bleaching following heatwave conditions constituting the most recent impact on the ecosystem. Here we describe an information-rich integrative visualisation suite of tools, tailored to ecologist users, for exploring a rich data set collected from the 2002 Great Barrier Reef heat stress event. Integration of data from multiple resources demonstrates how this visualisation is easily adaptable to any equivalent data, including data integration and visualisation from coral reef monitoring programs. The toolkit also makes such data accessible to any user for education, exploration and decision-making purposes.

Combinatorial Ranking of Gene Sets to Predict Disease Relapse: The Retinoic Acid Pathway in Early Prostate Cancer.

Background Quantitative high-throughput data deposited in consortia such as International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) present opportunities and challenges for computational analyses. Methods We present a computational strategy to systematically rank and investigate a large number (210–220) of clinically testable gene sets, using combinatorial gene subset generation and disease-free survival (DFS) analyses. This approach integrates protein–protein interaction networks, gene expression, DNA methylation, and copy number data, in association with DFS profiles from patient clinical records. Results As a case study, we applied this pipeline to systematically analyze the role of ALDH1A2 in prostate cancer (PCa). We have previously found this gene to have multiple roles in disease and homeostasis, and here we investigate the role of the associated ALDH1A2 gene/protein networks in PCa, using our methodology in combination with PCa patient clinical profiles from ICGC and TCGA databases. Relationships between gene signatures and relapse were analyzed using Kaplan–Meier (KM) log-rank analysis and multivariable Cox regression. Relative expression versus pooled mean from diploid population was used for z-statistics calculation. Gene/protein interaction network analyses generated 11 core genes associated with ALDH1A2; combinatorial ranking of the power set of these core genes identified two gene sets (out of 211 − 1 = 2,047 combinations) with significant correlation with disease relapse (KM log rank p < 0.05). For the more significant of these two sets, referred to as the optimal gene set (OGS), patients have median survival 62.7 months with OGS alterations compared to >150 months without OGS alterations (p = 0.0248, hazard ratio = 2.213, 95% confidence interval = 1.1–4.098). Two genes comprising OGS (CYP26A1 and RDH10) are strongly associated with ALDH1A2 in the retinoic acid (RA) pathways, suggesting a major role of RA signaling in early PCa progression. Our pipeline complements human expertise in the search for prognostic biomarkers in large-scale datasets.