Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes.

Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure.

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-gamma activation.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) have reduced expression of apolipoprotein E (apoE) and peroxisome proliferator-activated receptor-gamma in lung tissues, and deficiency of both has been linked to insulin resistance. ApoE deficiency leads to enhanced platelet-derived growth factor signaling, which is important in the pathobiology of PAH. We therefore hypothesized that insulin-resistant apoE-deficient (apoE-/-) mice would develop PAH that could be reversed by a peroxisome proliferator-activated receptor-gamma agonist (eg, rosiglitazone). METHODS AND RESULTS: We report that apoE-/- mice on a high-fat diet develop PAH as judged by elevated right ventricular systolic pressure. Compared with females, male apoE-/- were insulin resistant, had lower plasma adiponectin, and had higher right ventricular systolic pressure associated with right ventricular hypertrophy and increased peripheral pulmonary artery muscularization. Because male apoE-/- mice were insulin resistant and had more severe PAH than female apoE-/- mice, we treated them with rosiglitazone for 4 and 10 weeks. This treatment resulted in markedly higher plasma adiponectin, improved insulin sensitivity, and complete regression of PAH, right ventricular hypertrophy, and abnormal pulmonary artery muscularization in male apoE-/- mice. We further show that recombinant apoE and adiponectin suppress platelet-derived growth factor-BB-mediated proliferation of pulmonary artery smooth muscle cells harvested from apoE-/- or C57Bl/6 control mice. CONCLUSIONS: We have shown that insulin resistance, low plasma adiponectin levels, and deficiency of apoE may be risk factors for PAH and that peroxisome proliferator-activated receptor-gamma activation can reverse PAH in an animal model.

Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia.

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation.

Complex interplay between T helper (Th) cells and macrophages contributes to the formation and progression of atherosclerotic plaques. While Th1 cytokines promote inflammatory activation of lesion macrophages, Th2 cytokines attenuate macrophage-mediated inflammation and enhance their repair functions. In spite of its biologic importance, the biochemical and molecular basis of how Th2 cytokines promote maturation of anti-inflammatory macrophages is not understood. We show here that in response to interleukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT6) and PPARgamma-coactivator-1beta (PGC-1beta) induce macrophage programs for fatty acid oxidation and mitochondrial biogenesis. Transgenic expression of PGC-1beta primes macrophages for alternative activation and strongly inhibits proinflammatory cytokine production, whereas inhibition of oxidative metabolism or RNAi-mediated knockdown of PGC-1beta attenuates this immune response. These data elucidate a molecular pathway that directly links mitochondrial oxidative metabolism to the anti-inflammatory program of macrophage activation, suggesting a potential role for metabolic therapies in treating atherogenic inflammation.

Proteomic profiles of serum inflammatory markers accurately predict atherosclerosis in mice.

At a population level, inflammatory markers have been shown to predict outcome and response to therapy in patients with atherosclerotic cardiovascular disease. However, current markers are not sufficiently sensitive or specific to provide clinical utility for managing individual patients. We hypothesize that measurement of multiple circulating disease-related inflammatory factors will be more informative, allowing the early identification of vascular wall disease activity. We have investigated whether protein microarray-based abundance measurements of circulating proteins can predict the severity of atherosclerotic disease. Using a longitudinal experimental design with apolipoprotein E-deficient mice and control C57Bl/6J and C3H/HeJ wild-type mice, we measured the time-related serum protein expression of 30 inflammatory markers using a protein microarray. We were able to identify a subset of proteins that classify and predict the severity of atherosclerotic disease with a high level of accuracy. The time-specific vascular expression of these markers was verified by showing that their gene expression in the mouse aorta correlated closely to the temporal pattern of serum protein levels. In conclusion, these data suggest that quantification of multiple disease-related inflammatory proteins can provide a more sensitive and specific methodology for assessing atherosclerotic disease activity in humans, and identify candidate biomarkers for such studies.

Genome-wide expression dynamics during mouse embryonic development reveal similarities to Drosophila development.

Gene transcription mediates many vital aspects of mammalian embryonic development. A comprehensive characterization and analysis of the dynamics of gene transcription in the embryo is therefore likely to provide significant insights into the basic mechanisms of this process. We used microarrays to map transcription in the mouse embryo in the important period from embryonic day 8 (e8.0) to postnatal day 1 (p1) during which the bulk of the differentiation and development of organ systems takes place. Analysis of these expression profiles revealed distinct patterns of gene expression which correlate with the differentiation of organs including the nervous system, liver, skin, lungs, and digestive system, among others. Statistical analysis of the data based on Gene Ontology (GO) group annotation showed that specific temporal sequence patterns in gene class utilization across development are very similar to patterns seen during the embryonic development of Drosophila, suggesting conservation of the temporal progression of these processes across 550 million years of evolution. The temporal profiles of gene expression and activation of processes revealed here provide intriguing insights into the mechanisms of mammalian development, embryogenesis, and organogenesis, as well as into the evolution of developmental processes.

Increased fibulin-5 and elastin in S100A4/Mts1 mice with pulmonary hypertension.

Transgenic mice overexpressing the calcium binding protein, S100A4/Mts1, occasionally develop severe pulmonary vascular obstructive disease. To understand what underlies this propensity, we compared the pulmonary vascular hemodynamic and structural features of S100A4/Mts1 with control C57Bl/6 mice at baseline, following a 2-week exposure to chronic hypoxia, and after 1 and 3 months "recovery" in room air. S100A4/Mts1 mice had greater right ventricular systolic pressure and right ventricular hypertrophy at baseline, which increased further with chronic hypoxia and was sustained after 3 months "recovery" in room air. These findings correlated with a heightened response to acute hypoxia and failure to vasodilate with nitric oxide or oxygen. S100A4/Mts1 mice, when compared with C57Bl/6 mice, also had impaired cardiac function judged by reduced ventricular elastance and decreased cardiac output. Despite higher right ventricular systolic pressures with chronic hypoxia, S100A4/Mts1 mice did not develop more severe PVD, but in contrast to C57Bl/6 mice, these features did not regress on return to room air. Microarray analysis of lung tissue identified a number of genes differentially upregulated in S100A4/Mts1 versus control mice. One of these, fibulin-5, is a matrix component necessary for normal elastin fiber assembly. Fibulin-5 was localized to pulmonary arteries and associated with thickened elastic laminae. This feature could underlie attenuation of pulmonary vascular changes in response to elevated pressure, as well as impaired reversibility.

Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease.

The propensity for developing atherosclerosis is dependent on underlying genetic risk and varies as a function of age and exposure to environmental risk factors. Employing three mouse models with different disease susceptibility, two diets, and a longitudinal experimental design, it was possible to manipulate each of these factors to focus analysis on genes most likely to have a specific disease-related function. To identify differences in longitudinal gene expression patterns of atherosclerosis, we have developed and employed a statistical algorithm that relies on generalized regression and permutation analysis. Comprehensive annotation of the array with ontology and pathway terms has allowed rigorous identification of molecular and biological processes that underlie disease pathophysiology. The repertoire of atherosclerosis-related immunomodulatory genes has been extended, and additional fundamental pathways have been identified. This highly disease-specific group of mouse genes was combined with an extensive human coronary artery data set to identify a shared group of genes differentially regulated among atherosclerotic tissues from different species and different vascular beds. A small core subset of these differentially regulated genes was sufficient to accurately classify various stages of the disease in mouse. The same gene subset was also found to accurately classify human coronary lesion severity. In addition, this classifier gene set was able to distinguish with high accuracy atherectomy specimens from native coronary artery disease vs. those collected from in-stent restenosis lesions, thus identifying molecular differences between these two processes. These studies significantly focus efforts aimed at identifying central gene regulatory pathways that mediate atherosclerotic disease, and the identification of classification gene sets offers unique insights into potential diagnostic and therapeutic strategies in atherosclerotic disease.

Mouse strain-specific differences in vascular wall gene expression and their relationship to vascular disease.

OBJECTIVE: Different strains of inbred mice exhibit different susceptibility to the development of atherosclerosis. The C3H/HeJ and C57Bl/6 mice have been used in several studies aimed at understanding the genetic basis of atherosclerosis. Under controlled environmental conditions, variations in susceptibility to atherosclerosis reflect differences in genetic makeup, and these differences must be reflected in gene expression patterns that are temporally related to the development of disease. In this study, we sought to identify the genetic pathways that are differentially activated in the aortas of these mice. METHODS AND RESULTS: We performed genome-wide transcriptional profiling of aortas from C3H/HeJ and C57Bl/6 mice. Differences in gene expression were identified at baseline as well as during normal aging and longitudinal exposure to high-fat diet. The significance of these genes to the development of atherosclerosis was evaluated by observing their temporal pattern of expression in the well-studied apolipoprotein E model of atherosclerosis. CONCLUSIONS: Gene expression differences between the 2 strains suggest that aortas of C57Bl/6 mice have a higher genetic propensity to develop inflammation in response to appropriate atherogenic stimuli. This study expands the repertoire of factors in known disease-related signaling pathways and identifies novel candidate genes for future study. To gain insights into the molecular pathways that are differentially activated in strains of mice with varied susceptibility to atherosclerosis, we performed comprehensive transcriptional profiling of their vascular wall. Genes identified through these studies expand the repertoire of factors in disease-related signaling pathways and identify novel candidate genes in atherosclerosis.

Genome-wide expression profiling of a cardiac pressure overload model identifies major metabolic and signaling pathway responses.

Cardiac hypertrophy is a predictor of cardiovascular morbidity and mortality independent of other risk factors. Pressure overload induces the development of left ventricular hypertrophy (LVH) and left atrial enlargement (LAE) in the mammalian heart. To systematically investigate the transcriptional changes, which mediate these processes, we have performed a genome-wide transcriptional profiling of each of the four heart chambers from mice subjected to transverse aortic constriction (TAC). A major new finding of this analysis is that during enlargement the left atrium undergoes radical changes in gene transcription that may play a significant role in pathophysiology. Structural changes in the LA and LV are correlated with significant changes in the transcriptional profile of these chambers, with thousands of differentially expressed known and novel factors. Statistical analysis of the results identified Gene Ontology biological process groups with significant group-wide changes, including angiogenesis, energy pathways, fatty acid oxidation, oxidative phosphorylation, cytoskeletal and matrix reorganization, and G-protein coupled receptor (GPCR) signaling. To facilitate future research, a searchable annotated Internet database has been constructed that allows access to the expression data presented here. Further study of these genes and processes will lead to better understanding of pathways involved in the pathophysiology of the cardiac response to pressure overload.

Transcriptional profiling of the heart reveals chamber-specific gene expression patterns.

Cardiac chamber-specific gene expression is critical for the normal development and function of the heart. To investigate the genetic basis of cardiac anatomical specialization, we have undertaken a nearly genome-wide transcriptional profiling of the four heart chambers and the interventricular septum. Rigorous statistical analysis has allowed the identification of known and novel members of gene families that are felt to be important in cardiac development and function, including LIM proteins, homeobox proteins, wnt and T-box pathway proteins, as well as structural proteins like actins and myosins. In addition, these studies have allowed the identification of thousands of additional differentially expressed genes, for which there is little structural or functional information. Clustering of genes with known and unknown functions provides insights into signaling pathways that are essential for development and maintenance of chamber-specific features. To facilitate future research in this area, a searchable internet database has been constructed that allows study of the chamber-specific expression of any gene represented on this comprehensive microarray. It is anticipated that further study of genes identified through this effort will provide insights into the specialization of heart chamber tissues, and their specific roles in cardiac development, aging, and disease.