Anca Chiriac

CXCR4+/FLK-1+ Biomarkers Select a Cardiopoietic Lineage from Embryonic Stem Cells

Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi‐lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue‐specified progenitor cells. From a comprehensive pool of 11,272 quality‐filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane‐associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over‐represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4+ progenitors were sorted from the embryonic stem cell pool into mesoderm‐restricted progeny according to co‐expression with the early mesoderm marker Flk‐1. In contrast to CXCR4−/Flk‐1− cells, the CXCR4+/Flk‐1+ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha‐fetoprotein markers were all depleted. In fact, the CXCR4+/Flk‐1+ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp‐1, GATA‐4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4+/Flk‐1+ transcriptome shared 97% identity with the CXCR4−/Flk‐1− counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4+/Flk‐1+ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk‐1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage.

Cardiogenic Induction of Pluripotent Stem Cells Streamlined Through a Conserved SDF-1/VEGF/BMP2 Integrated Network

Background Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. Methods and Results To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes). The streamlined pool of 288 genes organized into a core biological network that prioritized the “Cardiovascular Development” function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. Conclusions Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.

Transcriptome from circulating cells suggests dysregulated pathways associated with long-term recurrent events following first-time myocardial infarction

BACKGROUND Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for high-risk cardiovascular diseases. However, the feasibility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome. METHODS AND RESULTS Whole-genome expression microarray on blood samples from normal cardiac function controls (n=21) and first-time AMI patients (n=31) within 48-hours post-MI revealed expected differential gene expression profiles enriched for inflammation and immune-response pathways. To determine molecular signatures at the time of AMI associated with long-term outcomes, transcriptional profiles from sub-groups of AMI patients with (n=5) or without (n=22) any recurrent events over an 18-month follow-up were compared. This analysis identified 559 differentially-expressed genes. Bioinformatic analysis of this differential gene-set for associated pathways revealed 1) increasing disease severity in AMI patients is associated with a decreased expression of genes involved in the developmental epithelial-to-mesenchymal transition pathway, and 2) modulation of cholesterol transport genes that include ABCA1, CETP, APOA1, and LDLR is associated with clinical outcome. CONCLUSION Differentially regulated genes and modulated pathways were identified that were associated with recurrent cardiovascular outcomes in first-time AMI patients. This cell-based approach for risk stratification in AMI could represent a novel, non-invasive platform to anticipate modifiable pathways and therapeutic targets to optimize long-term outcome for AMI patients and warrants further study to determine the role of metabolic remodeling and regenerative processes required for optimal outcomes.

Decoded Calreticulin-Deficient Embryonic Stem Cell Transcriptome Resolves Latent Cardiophenotype

Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin‐deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin‐null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re‐prioritized essential cellular functions. Calreticulin‐calibrated signaling axes were uncovered, and network‐wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin‐deficient stem cell‐derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin‐dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin‐deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype. STEM CELLS 2010;28:1281–1291

Vascular-Directed Tissue Factor Pathway Inhibitor Overexpression Regulates Plasma Cholesterol and Reduces Atherosclerotic Plaque Development

Rationale: Tissue factor pathway inhibitor (TFPI) is a potent regulator of the tissue factor pathway and is found in plasma in association with lipoproteins. Objective: To determine the role of TFPI in the development of atherosclerosis, we bred mice which overexpress TFPI into the apolipoprotein E–deficient (apoE−/−) background. Methods and Results: On a high-fat diet, smooth muscle 22α (SM22α)-TFPI/apoE−/− mice were shown to have less aortic plaque burden compared to apoE−/− mice. Unexpectedly, SM22α-TFPI/apoE−/− had lower plasma cholesterol levels compared to apoE−/− mice. Furthermore, SM22α-TFPI mice fed a high-fat diet had lower cholesterol levels than did wild-type mice. Because TFPI is associated with lipoproteins and its carboxyl terminus (TFPIct) has been shown to be a ligand for the very-low-density lipoprotein (VLDL) receptor, we hypothesized that TFPI overexpression may regulate lipoprotein distribution. We quantified VLDL binding and uptake in vitro in mouse aortic smooth muscle cells from SM22α-TFPI and wild-type mice. Mouse aortic smooth muscle cells from SM22α-TFPI mice demonstrated higher VLDL binding and internalization compared to those from wild-type mice. Because SM22α-TFPI mice have increased circulating levels of TFPI antigen, we examined whether TFPIct may act to alter lipoprotein distribution. In vitro, TFPIct increased VLDL binding, uptake, and degradation in murine embryonic fibroblasts. Furthermore, this effect was blocked by heparinase treatment. In vivo, systemic administration of TFPIct reduced plasma cholesterol levels in apoE−/− mice. Conclusions: These studies suggest that overexpression of TFPI lowers plasma cholesterol through the interaction of its carboxyl terminus with lipoproteins and heparan sulfate proteoglycans.

SDF-1-enhanced cardiogenesis requires CXCR4 induction in pluripotent stem cells.

Transformation of pluripotent stem cells into cardiac tissue is the hallmark of cardiogenesis, yet pro-cardiogenic signals remain partially understood. Preceding cardiogenic induction, a surge in CXCR4 chemokine receptor expression in the early stages of stem cell lineage specification coincides with the acquisition of pre-cardiac profiles. Accordingly, CXCR4 selection, in conjunction with mesoderm-specific VEGF type II receptor FLK-1 co-expression, segregates cardiogenic populations. To assess the functionality of the CXCR4 biomarker, targeted activation and disruption were here exploited in the context of embryonic stem cell-derived cardiogenesis. Implicated as a cardiogenic hub through unbiased bioinformatics analysis, induction of the CXCR4/SDF-1 receptor–ligand axis triggered enhanced beating activity in stem cell progeny. Gene expression knockdown of CXCR4 disrupted spontaneous embryoid body differentiation and blunted the expression of cardiogenic markers MEF2C, Nkx2.5, MLC2a, MLC2v, and cardiac-MHC. Exogenous SDF-1 treatment failed to rescue cardiogenic-deficient phenotype, demonstrating a requirement for CXCR4 expression in mediating SDF-1 effects. Thus, a pro-cardiogenic signaling role for the CXCR4/SDF1 axis is herein revealed within pluripotent stem cell progenitors, exposing a functional target to promote lineage-specific differentiation.

Lineage specification of Flk-1+ progenitors is associated with divergent Sox7 expression in cardiopoiesis

Embryonic stem cell differentiation recapitulates the diverse phenotypes of a developing embryo, traceable according to markers of lineage specification. At gastrulation, the vascular endothelial growth factor (VEGF) receptor, Flk-1 (KDR), identifies a mesoderm-restricted potential of embryonic stem cells. The multi-lineage propensity of Flk-1(+) progenitors mandates the mapping of fate-modifying co-factors in order to stratify differentiating cytotypes and predict lineage competency. Here, Flk-1-based selection of early embryonic stem cell progeny separated a population depleted of pluripotent (Oct4, Sox2) and endoderm (Sox17) markers. The gene expression profile of the Flk-1(+) population was notable for a significant upregulation in the vasculogenic Sox7 transcription factor, which overlapped with the emergence of primordial cardiac transcription factors GATA-4, Myocardin and Nkx2.5. Sorting the parental Flk-1(+) pool with the chemokine receptor CXCR4 to enrich the cardiopoietic subpopulation uncovered divergent Sox7 expression, with a 7-fold induction in non-cardiac compared to cardiac progenitors. Bioinformatic resolution sequestered a framework of gene expression relationship between Sox transcription factor family members and the Flk-1/CXCR4 axes with significant integration of beta-catenin signaling. Thus, differential Sox7 gene expression presents a novel biomarker profile, and possible regulatory switch, to distinguish cardiovascular pedigrees within Flk-1(+) multi-lineage progenitors.

CXCR4+ and FLK-1+ Identify Circulating Cells Associated with Improved Cardiac Function in Patients Following Myocardial Infarction

The biomarkers CXCR4/FLK-1 select cardiac progenitors from a stem cell pool in experimental models. However, the translational value of these cells in human ischemic heart disease is unknown. Here, flow-cytometry identified CD45−/CXCR4+/FLK-1+ cells in 30 individuals without ischemic heart disease and 33 first-time acute myocardial infarction (AMI) patients. AMI patients had higher CD45−/CXCR4+/FLK-1+ cell-load at 48-h and 3- and 6-months post-AMI (p = 0.003,0.04,0.04, respectively) than controls. Cardiovascular risk factors and left ventricular (LV) ejection fraction were not associated with cell-load. 2D-speckle-tracking strain echocardiography assessment of LV systolic function showed improvement in longitudinal strain and dyssynchrony during follow-up associated with longitudinal increases in and higher 48-h post-AMI CD45−/CXCR4+/FLK-1+ cell-load (r = −0.525, p = 0.025; r = −0.457, p = 0.029, respectively). In conclusion, CD45−/CXCR4+/FLK-1+ cells are present in adult human circulation, increased in AMI and associated with improved LV systolic function. Thus, CD45−/CXCR4+/FLK-1+ cells may provide a diagnostic tool to follow cardiac regenerative capacity and potentially serve as a prognostic marker in AMI.

Bioinformatic Primer for Clinical and Translational Science

The advent of high‐throughput technologies has accelerated generation and expansion of genomic, transcriptomic, and proteomic data. Acquisition of high‐dimensional datasets requires archival systems that permit efficiency of storage and retrieval, and so, multiple electronic repositories have been initiated and maintained to meet this demand. Bioinformatic science has evolved, from these intricate bodies of dynamically updated information and the tools to manage them, as a necessity to harness and decipher the inherent complexity of high‐volume data. Large datasets are associated with a variable degree of stochastic noise that contributes to the balance of an ordered, multistable state with the capacity to evolve in response to stimulus, thus exhibiting a hallmark feature of biological criticality. In this context, the network theory has become an invaluable tool to map relationships that integrate discrete elements that collectively direct global function within a particular –omic category, and indeed, the prioritized focus on the functional whole of the genomic, transcriptomic, or proteomic strata over single molecules is a primary tenet of systems biology analyses. This new biology perspective allows inspection and prediction of disease conditions, not limited to a monogenic challenge, but as a combination of individualized molecular permutations acting in concert to effect a phenotypic outcome. Bioinformatic integration of multidimensional data within and between biological layers thus harbors the potential to identify unique biological signatures, providing an enabling platform for advances in clinical and translational science.