Search

Rationale: Cilp1 (cartilage intermediate layer protein 1) is a secreted extracellular matrix protein normally associated with bone and cartilage development. Its function and mechanism of action in adult heart disease remain elusive. Objective: To establish the function and mechanism of action of Cilp1 in postmyocardial infarction (MI) cardiac remodeling. Methods and Results: We investigated the expression of Cilp1 in mouse models of pathological cardiac remodeling and human heart failure patients. Cilp1 was expressed predominantly in cardiac fibroblasts and upregulated in response to cardiac injury and in the heart and blood of patients with heart failure. We generated Cilp1 knock out (KO) and transgenic (Tg) mice with NCilp1 (N-terminal half of the protein) overexpressed in myofibroblasts. Cilp1 KO mice had better cardiac function, reduced number of immune cells and myofibroblasts, and enhanced microvascular survival after MI compared with wild-type littermates. Conversely, NCilp1-transgenic mice had augmented loss of cardiac function, increased number of myofibroblasts and infarct size after the MI injury. RNA-seq and gene ontology analysis indicated that cell proliferation and mTORC1 (mammalian Target of Rapamycin Complex 1) signaling were downregulated in KO hearts compared with wild-type hearts. In vivo BrdU labeling and immunofluorescence staining showed that myofibroblast proliferation in the Cilp1 KO heart was downregulated. Biaxial mechanical testing and extracellular matrix gene expression analysis indicated that while MI caused significant stiffness in wild-type hearts it had little effect on KO hearts. Upregulation of collagen expression after MI injury was attenuated in KO hearts. Recombinant CILP1 protein or NCilp1-conditioned medium promoted proliferation of neonatal rat ventricular cardiac fibroblasts via the mTORC1 signaling pathway. Conclusions: Our studies established a pathological role of Cilp1 in promoting post-MI remodeling, identified a novel function of Cilp1 in promoting myofibroblast proliferation, and suggested that Cilp1 may serve as a potential biomarker for pathological cardiac remodeling and target for fibrotic heart disease.

Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of patients with DMD, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional, and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD induced pluripotent stem cell lines, wherein the DMD open reading frame was restored via reframing or exon skipping. While DMD cardiomyocytes demonstrated morphological, structural, and functional deficits compared with control cardiomyocytes, cardiomyocytes from both corrected DMD lines were similar to control cardiomyocytes. Bulk RNA-sequencing of DMD cardiomyocytes showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in corrected DMD cardiomyocytes. We then corrected dysfunctional DMD cardiomyocytes by adenoviral delivery of Cas9/gRNA and showed that correction of DMD cardiomyocytes postdifferentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in cardiomyocytes and fibroblasts, which in corrected mice was reduced to similar levels as wild-type mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional, and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in induced pluripotent stem cell-derived cardiomyocytes and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA. Implications: These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the US, has a high metastatic rate and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, has been identified as a driver of metastasis and immune suppression in multiple cancer types. Here we use single cell RNA sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of mesenchymal tumor cells. We demonstrate that AXL-deficiency extends survival, reduces primary and metastatic burden and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression and a more active immune microenvironment compared to PDA in wild-type mice. Finally, we demonstrate that AXL-positive mesenchymal tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target for PDA patients.

Acute myocardial infarction (AMI), as the first manifestation of ischemic heart disease, is the most common cause of death in developed countries. A recent study showed that metastasis associated lung adenocarcinoma transcript 1 (MALAT1), a prognostic marker for lung cancer metastasis, could promote myocardial ischemia-reperfusion injury by regulating the levels of microRNA (miR)-145. In order to elucidate the biological function of MALAT1 in the pathogenesis of AMI and to explore the mechanisms underlying its action, an AMI rat model was established by ligation of the left anterior descending coronary artery. Downregulation of MALAT1 by siRNA transfection attenuated heart damage in an AMI model rat. The mouse cardiomyocyte cell line HL-1 was used to show that downregulation of nucleotide binding and oligomerization domain-like receptor C5 (NLRC5) and upregulation of miR-125b-5p were the results of MALAT1 silencing. TargetScan and a dual-luciferase reporter assay indicated that NLRC5 is a direct target of miR-125b-5p. Overexpression of miR-125b-5p significantly reduced hypoxia/reperfusion-induced apoptosis of HL-1 cells, an effect that could be blocked by NLCR5 overexpression. Taken together, these results suggest that MALAT1 reduced the protective effect of miR-125b-5p on injured cells through upregulation of NLCR5. This study highlights the role of MALAT1 in the pathogenesis of AMI and may guide future genetic therapeutic strategies for AMI treatment.

Introduction Cardiogenic transcription factors (TFs), Gata4, Mef2c, and Tbx5, can directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the Hand2 TF and the Akt1 kinase. Although these cardiac reprogramming factors are key regulators of heart development, their expression and biological functions are not limited to the heart, and the mechanism by which these cardiac factors orchestrate reprogramming remains unclear. Purpose We sought to study the molecular mechanisms by which cardiac reprogramming factors contribute to cell fate conversion using a genome-wide approach. Methods Here, we used chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to explore the genomic binding sites of reprogramming TFs and the landscape of active enhancers, annotated by H3K27ac histone modification, during cardiac reprogramming. Results We found that reprogramming TFs rapidly silence fibroblast enhancers and synergistically activate cardiac enhancers which were predominantly enriched with Mef2 motifs. Addition of Hand2 and Akt1 to GMT expands TF co-occupancy and thereby activates additional cardiac enhancers, which further augments cardiac gene expression. Moreover, additional cardiac enhancers were sequentially activated during the reprogramming process, in accordance with the temporal acquisition of functional phenotypes in induced cardiac-like myocytes. We discovered that subsets of reprogramming enhancers are conserved among other cardiogenic processes, and a collection of reprogramming enhancers displayed unique spatial expression patterns in the developing heart in transgenic mouse embryos. Conclusion Our study describes the epigenomic dynamics that underlie cardiac reprogramming which is cooperatively orchestrated by reprogramming factors to convert fibroblasts toward a cardiac lineage. Acknowledgement/Funding NIH (AR-067294, HL-130253, HL-138426, HD-087351), Fondation Leducq Transatlantic Networks of Excellence in Cardiovascular Research

Background & AimsPancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression. The progression of PDA is a highly complex and dynamic process featuring changes in cancer cells and stromal cells; however, a comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression.MethodsWe employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models.ResultsOur data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression. We also found distinct populations of macrophages with increasing inflammatory features during PDA progression. In addition, we noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts are dynamically regulated by epigenetic mechanisms.ConclusionThis study systematically outlines the landscape of cellular heterogeneity during the progression of PDA. It strongly improves our understanding of the PDA biology and has the potential to aid in the development of therapeutic strategies against specific cell populations of the disease.

The adult mammalian heart has a limited capacity to regenerate upon injury, such as myocardial infarction (MI). MI in adult mouse hearts results in a reduced cardiac function, due to extensive loss of cardiomyocytes and formation of scar tissue by activated fibroblasts. In contrast, the newborn postnatal day 1 (P1) mouse heart is able to regenerate and restore its cardiac function following injury. However, this regenerative ability dramatically declines after P7. The molecular properties and mechanisms that facilitate neonatal heart regeneration remain unknown. To gain further insight into the realm of cardiac regeneration, our lab performed transcriptome analysis by bulk and single cell RNA-seq on P1 and P8 hearts at various time points following MI surgery. We also performed enhancer profiling by H3K27ac ChIP-seq. Coupling of both these approaches has uncovered two different gene sets that that may promote the regenerative ability of neonatal hearts: developmental genes and injury-induced genes. Amongst the neonatal-specific developmental genes, Igf2bp3, a RNA-binding protein, and Hic2, a transcriptional regulator, have been shown to promote neonatal rat ventricular cardiomyocyte (NRVM) proliferation and are currently being characterized for their involvement in neonatal heart regeneration by using gain-of-function and loss-of-function in vivo approaches. Amongst the injury-induced genes, we identified mitogens secreted from macrophages that promote NRVM proliferation. This is in line with the differential immune response elicited by P1 hearts upon MI-injury. We have also shown that an epicardial secreted factor in P1 hearts can enhance human umbilical vein endothelial cell tube formation, indicative of its role in angiogenesis. In summary, by using a multi-layered genomic analysis, we identified various genes that are involved in different cellular responses during neonatal heart regeneration. This comprehensive analysis of the neonatal regenerative response provides insights into the molecular basis of neonatal heart regeneration, and can potentially identify genes that can be modulated to facilitate adult heart regeneration.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes is an attractive therapeutic strategy to restore cardiac function following injury. The cardiac transcription factors Gata4, Mef2c, and Tbx5 are sufficient to directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the addition of Hand2 and Akt1. However, the mechanisms by which these transcription factors orchestrate this cell fate conversion remains elusive. To understand the mechanistic basis of cardiac reprogramming, we performed a genome-wide analysis of cardiogenic transcription factor binding sites and enhancer activation throughout cardiac reprogramming. We found that cardiogenic transcription factors act cooperatively by co-occupying regulatory elements enriched with Mef2 binding sites during cardiac reprogramming. Importantly, these transcription factors when overexpressed in isolation are incapable of activating reprogramming enhancer elements. Further, we discovered that the early reprogramming enhancer landscape most closely resembles that of the neonatal heart. Acquisition of enhancers associated with cardiac maturation occurred at later stages in reprogramming, with addition of Hand2 and Akt1 augmenting activation of this more mature enhancer landscape. Additionally, we constructed a cardiac reprogramming gene regulatory network which demonstrated downregulation of the EGF receptor signaling pathway. We found that inhibition of EGF receptor signaling augments reprogramming toward the cardiac phenotype. Our findings demonstrate that cardiac reprogramming is coordinated by synergistic transcriptional activation across a broad landscape of cardiac enhancers and define the epigenetic landscape of the cardiac phenotype.

Cancer can be conceptualized as arising from somatic mutations resulting in a single renegade cell escaping from the constraints of multicellularity. Thus, the era of precision medicine has led to intense focus on the cancer cell to target these mutations that result in oncogenic signaling and sustain malignancy. However, in pancreatic ductal adenocarcinoma (PDAC) there are only four abundantly common driver mutations (KRAS, CDKN2A, TP53, and SMAD4), which are not currently actionable. Thus, precision therapy for PDAC must look beyond the cancer cell. In fact, PDAC is more than a collection of renegade cells, instead representing an extensive, supportive ecosystem, having developed over several years, and consisting of numerous interactions between the cancer cells, normal mesenchymal cells, immune cells, and the dense extracellular matrix. In this issue, Huang and colleagues demonstrate how elucidation of these complex relationships within the tumor microenvironment (TME) can be exploited for therapeutic intervention in PDAC. They identify in a subset of PDAC with mutations in TGFβ signaling, that a paracrine signaling axis can be abrogated to modulate the TME and improve outcomes.

Customizable mobile services are usually expressed with complex services composed of different atomic services. Fine-grained atomic mobile services are not so convenient for end users to reuse. Considering that in identical or similar service domains, a great deal of the business logics and functions are reusable within the scope. So we present a template-based framework to allow reuse of services and to achieve rapid mobile application development. The reusable fine-grained service logics and functions are encapsulated into comparatively coarse-grained templates, from which the designers can create the personalized composite services and edit the templates efficiently.

Many countries regulate the quality of food and drugs, yet it remains unclear whether markets can be relied upon to deliver high quality in the absence of regulation, notably where companies can advertise the superior quality of their products. We present evidence from two field experiments in China's infant formula industry, which has seen a trust crisis after several safety scandals. We show that disclosure of information about product quality can have a negative impact on consumers' purchase decisions and self-reported trust in the industry, as information reminds consumers of past scandals and draws their attention to safety risks.

Pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death in the US, has a high metastatic rate that is associated with persistent immune suppression. Axl, a member of the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinase family, has been identified as a critical factor that drives metastasis and immune suppression in many cancer types. We have shown that pharmacological inhibition of Axl significantly reduces tumor progression and metastasis and improves response to chemotherapy in multiple preclinical PDAC models. Here we demonstrate that Axl-deficient PDAC bearing mice have longer median survival, smaller tumors, fewer metastases and higher sensitivity to chemotherapy (gemcitabine) compared to Axl wild-type (WT) PDAC mice. Furthermore, Axl-deficient PDAC displays a more differentiated histology and has a more inflammatory and active immune microenvironment. Single cell RNA sequencing of PDAC genetically engineered mouse models reveals that Axl is expressed highly in tumor cells that have a mesenchymal-like phenotype and Axl expression correlates with classic markers of mesenchymal tumor cells at the RNA and protein level. To investigate the importance of Axl on tumor cells, CRISPR-Cas9 was used to knockout Axl in PDAC cells. Human and murine Axl-deficient PDAC cells displayed a more differentiated (epithelial) phenotype in vitro including reduced migratory capacity compared to Axl WT parental cells. Orthotopic injection of Axl-deficient murine PDAC cells into Axl WT mice resulted in longer median survival and fewer metastases compared to WT mice injected with WT PDAC cells. However, Axl-deficient mice injected with WT PDAC cell injection showed similar median survival and metastases as WT mice injected with WT PDAC cells. These in vivo results indicate that Axl expression on tumor cells is critical for PDAC progression and metastasis. In vitro experiments using WT and Axl-deficient human PDAC cells show that Axl is required for TGFβ-mediated epithelial to mesenchymal transition, probably through regulating the expression of Smad2 and Smad4. Interesting, Axl depletion has no effect on the mRNA level of Smad2 and Smad4, but does decrease the protein level, indicating that Axl might be involved in post-translational regulation (e.g., protein stability) of canonical TGFβ signaling intermediates. In summary, our study demonstrates that Axl expressed by pancreatic cancer cells is critical to tumor cell plasticity, tumor progression and metastasis. Citation Format: Wenting Du, Huocong Huang, Jason Toombs, Zhaoning Wang, Abdel Hosein, Natalie Burton, Rolf A. Brekken. Axl is critical for pancreatic cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1037.

In this paper, we show an end-user oriented tool suite for mobile application development. The advantages of this tool suite are that the graphical user interface (GUI), as well as the application logic can both be developed in a rapid and simple way, and web-based services on the Internet can be integrated into our platform by end-users. This tool suite involves three sub-systems, namely ServiceAccess, EasyApp and LSCE. ServiceAccess takes charge of the registration and management of heterogeneous services, and can export different form of services according to the requirements of the other sub-systems. EasyApp is responsible for developing GUI in the form of mobile app. LSCE takes charge of creating the application logic that can be invoked by mobile app directly. Finally, a development case is presented to illustrate the development process using this tool suite. The URL of demo video: https://youtu.be/mM2WkU1_k-w.

In this paper, we present an ecosystem for mobile application development by end-users. An advantage of this ecosystem is that the graphical user interface (GUI), as well as the application logic, can both be developed in a rapid and simple way. This ecosystem is mainly implemented through development and integration of two sub-systems, namely EasyApp and LSCE. EasyApp is responsible for developing the mobile app with the compatibility of multiple mobile platforms, while LSCE is in charge of creating the service process that can be invoked by mobile app directly. A case study is presented to illustrate the development process using this ecosystem.

According to several model test results of Francis turbines, complete model efficiency hill charts were constructed. The formation and inevitability of diversified hydraulic phenomena on model efficiency hill chart for typical head range were analyzed and the difference is compared, as well as characteristics and commonness toward the curves are discussed and summarized. Furthermore, hydraulic performance and geometric features are presented by particularly analyzing the efficiency hill charts. The inherent characteristics of Francis turbine is expressed by all kinds of curves on the model efficiency hill charts, and these curves can be adjusted and moved in a small range but cannot be removed out. Due to wide range of unit speed in terms of medium-low-head hydraulic turbines, incipient cavitation curve on suction side can be observed and it is positioned close to the operation zone; however, it fails to be visualized for the high-head turbine. The blade channel vortex curves are in the vicinity of optimum region for low-head hydraulic turbines, while high-head shows reverse trend. The interaction between zero incidence angle and zero circulation curve has a significant influence on isoefficiency circles. All comparisons and analyses could provide hydraulic design basis and retrofit references.