Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Autophagy contributes to homeostasis in esophageal epithelium where high autophagic vesicle content marks basal cells with limited proliferation and enhanced self-renewal potential.

Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .

Eosinophilic esophagitis-associated epithelial remodeling may limit esophageal carcinogenesis.

Introduction: Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. Methods: In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. Results: In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Discussion: Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium.

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

A role for age-associated alterations in esophageal epithelium in eosinophilic esophagitis-associated fibrosis.

Subepithelial fibrosis occurs in a subset of eosinophilic esophagitis (EoE) patients and is associated with esophageal stricture. While mechanisms driving EoE fibrosis remain incompletely understood, findings from experimental systems support roles for epithelial-fibroblast crosstalk in this type of tissue remodeling. The current paradigm presents EoE as a progressive fibrostenotic disease in which aged patients develop fibrosis as a function of disease chronicity. In the current study we provide evidence that altered epithelial biology in the aging esophagus may also contribute to EoE-associated fibrosis. We find that induction of EoE inflammation in young and aged mice using the MC903/Ovalbumin protocol for the same time period results in increased lamina propria thickness uniquely in aged animals. Additionally, epithelial cells from aged mice less efficiently limit fibroblast contractility in collagen plug contraction assays compared to those from their young counterparts. Finally, to identify potential mechanisms through which aged esophageal epithelial cells may stimulate fibrotic remodeling, we perform cytokine array experiments in young and aged mice. These studies are significant as identification of age-associated factors that contribute to fibrotic remodeling may aid in the design of strategies toward early detection, prevention, and therapy of fibrostenotic EoE.

Increased Afterload Augments Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model.

Sunitinib, a multitargeted oral tyrosine kinase inhibitor, used widely to treat solid tumors, results in hypertension in up to 47% and left ventricular dysfunction in up to 19% of treated individuals. The relative contribution of afterload toward inducing cardiac dysfunction with sunitinib treatment remains unknown. We created a preclinical model of sunitinib cardiotoxicity using engineered microtissues that exhibited cardiomyocyte death, decreases in force generation, and spontaneous beating at clinically relevant doses. Simulated increases in afterload augmented sunitinib cardiotoxicity in both rat and human microtissues, which suggest that antihypertensive therapy may be a strategy to prevent left ventricular dysfunction in patients treated with sunitinib.

Myocardial apoptosis and mesenchymal stem cells with acute exercise.

Aerobic exercise confers many health benefits. However, numerous reports have shown that acute aerobic exercise can injure the heart. We tested the general hypothesis that acute moderate-intensity exercise in rodents induces cardiomyocyte damage and stimulates mesenchymal stem cells (MSCs) to increase paracrine-mediated protective effects on cardiomyocytes. A single session of treadmill running (13 m/min, 0% grade, for 45 min) in untrained C57BL/6 male mice (n = 18) increased cleaved poly ADP-ribose polymerase (PARP), a marker of apoptosis, in the myocardium 24 h postexercise. Microarray analysis of mouse myocardium identified 11 relevant apoptotic genes and several shifts in matrix remodeling transcripts over the postexercise window. Postexercise cardiomyocyte death was recapitulated in neonatal rat cardiomyocytes (NRCMs) by culturing cells in 2% plasma harvested from exercised rats. The increased cell death observed in exercise-treated NRCMs was attenuated by ß-adrenergic blockade, but not antioxidant treatment. MSC survival, proliferation, and chemotaxis showed no significant differences between sedentary and exercise plasma conditions, despite increased IL-6, TNF-α, IL-1ß, and IFN-γ secretions from MSCs treated with exercise plasma. NRCM survival was increased nearly 500% when cocultured with MSCs, but this effect was not altered under exercise plasma culture conditions. Our results suggest acute moderate-intensity aerobic treadmill running in exercise-naïve rodents induces temporal cardiomyocyte death due to plasma-borne factors, namely, catecholaminergic stress. Even though exercise conditions prompt an inflammatory response in MSCs, the exercise milieu does not alter the MSC-protective phenotype on cardiomyocytes.

Acute aerobic exercise increases exogenously infused bone marrow cell retention in the heart.

Stem cell therapy for myocardial infarction (MI) has been shown to improve cardiac function and reduce infarct size. Exercise training, in the form of cardiac rehabilitation, is an essential part of patient care post-MI. Hence, we tested the effects of acute and chronic aerobic exercise on stem cell retention and cardiac remodeling post-MI. Small epicardial MI's were induced in 12-month-old C57BL/6 mice via cryoinjury. Two weeks post-MI, vehicle infusion (N = 4) or GFP(+) bone marrow-derived cells (BMC) were injected (tail vein I.V.) immediately after acute exercise (N = 14) or sedentary conditions (N = 14). A subset of mice continued a 5-week intervention of chronic treadmill exercise (10-13 m/min; 45 min/day; 4 days/week; N = 7) or remained sedentary (N = 6). Exercise tolerance was assessed using a graded exercise test, and cardiac function was assessed with echocardiography. Acute exercise increased GFP(+) BMC retention in the infarcted zone of the heart by 30% versus sedentary (P < 0.05). This was not associated with alterations in myocardial function or gene expression of key cell adhesion molecules. Animals treated with chronic exercise increased exercise capacity (P < 0.05) and cardiac mass (P < 0.05) without change in left ventricular ejection fraction (LVEF), infarct size, or regional wall thickness (P = NS) compared with sedentary. While BMC's alone did not affect exercise capacity, they increased LVEF (P < 0.05) and Ki67(+) nuclei number in the border zone of the heart (P < 0.05), which was potentiated with chronic exercise training (P < 0.05). We conclude that acute exercise increases BMC retention in infarcted hearts and chronic training increases exogenous BMC-mediated effects on stimulating the cardiomyocyte cell cycle. These preclinical results suggest that exercise may help to optimize stem cell therapeutics following MI.

Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection.

BACKGROUND: Cardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects. METHODS: A robust, double emulsion production process was developed to encapsulate drugs into nanoparticles. Briefly in this proof of concept study, aspirin and prednisolone anti-inflammatory drugs were encapsulated in various poly-lactic glycolic acid polymer (PLGA) formulations. The resultant particle systems were characterized, co-delivered with GFP plasmid, and evaluated in harvested myocytes in culture for uptake. RESULTS: High quality nanoparticles were harvested from multiple production runs, with an average 64 ± 10 mg yield. Four distinct particle drug system combinations were characterized and evaluated in vitro: PLGA(50:50) Aspirin, PLGA(65:35) Prednisolone, PLGA(65:35) Aspirin and PLGA(50:50) Prednisolone Particles consisted of spherical shape with a narrow size distribution 265 ± 104 nm as found in scanning electron microscopy imaging. Prednisolone particles regardless of PLGA type were found on average ≈ 100 nm smaller than the aspirin types. All four groups demonstrated high zeta potential stability and re-constitution testing prior to in vitro. In vitro results demonstrated co uptake of GFP plasmid (green) and drug loaded particles (red) in culture with no incidence of toxicity. CONCLUSIONS: Nano formulated anti-inflammatories in combination with standalone gene product therapy may offer a clinical solution to maximize cardiac gene therapy product effects while minimizing the risk of the host response in the inflammatory myocardial environment.

A bioengineered hydrogel system enables targeted and sustained intramyocardial delivery of neuregulin, activating the cardiomyocyte cell cycle and enhancing ventricular function in a murine model of ischemic cardiomyopathy.

BACKGROUND: Neuregulin-1ß (NRG) is a member of the epidermal growth factor family possessing a critical role in cardiomyocyte development and proliferation. Systemic administration of NRG demonstrated efficacy in cardiomyopathy animal models, leading to clinical trials using daily NRG infusions. This approach is hindered by requiring daily infusions and off-target exposure. Therefore, this study aimed to encapsulate NRG in a hydrogel to be directly delivered to the myocardium, accomplishing sustained localized NRG delivery. METHODS AND RESULTS: NRG was encapsulated in hydrogel, and release over 14 days was confirmed by ELISA in vitro. Sprague-Dawley rats were used for cardiomyocyte isolation. Cells were stimulated by PBS, NRG, hydrogel, or NRG-hydrogel (NRG-HG) and evaluated for proliferation. Cardiomyocytes demonstrated EdU (5-ethynyl-2'-deoxyuridine) and phosphorylated histone H3 positivity in the NRG-HG group only. For in vivo studies, 2-month-old mice (n=60) underwent left anterior descending coronary artery ligation and were randomized to the 4 treatment groups mentioned. Only NRG-HG-treated mice demonstrated phosphorylated histone H3 and Ki67 positivity along with decreased caspase-3 activity compared with all controls. NRG was detected in myocardium 6 days after injection without evidence of off-target exposure in NRG-HG animals. At 2 weeks, the NRG-HG group exhibited enhanced left ventricular ejection fraction, decreased left ventricular area, and augmented borderzone thickness. CONCLUSIONS: Targeted and sustained delivery of NRG directly to the myocardial borderzone augments cardiomyocyte mitotic activity, decreases apoptosis, and greatly enhances left ventricular function in a model of ischemic cardiomyopathy. This novel approach to NRG administration avoids off-target exposure and represents a clinically translatable strategy in myocardial regenerative therapeutics.

Synergistic effects of SDF-1α chemokine and hyaluronic acid release from degradable hydrogels on directing bone marrow derived cell homing to the myocardium.

Poor cell engraftment in the myocardium is a limiting factor towards the use of bone marrow derived cells (BMCs) to treat myocardial infarction (MI). In order to enhance the engraftment of circulating BMCs in the myocardium following MI, we have developed in situ forming hyaluronic acid (HA) hydrogels with degradable crosslinks to sustain the release of recombinant stromal cell-derived factor-1 alpha (rSDF-1α) and HA to the injured myocardium. Both rSDF-1α and the crosslinkable HA macromer stimulate BMC chemotaxis up to 4-fold in vitro through CXCR4 and CD44 receptor signaling, respectively. Moreover, the HA macromer binds rSDF-1α with a dissociation constant of 36 ± 5 µM through electrostatic interaction. When formed into hydrogels via photoinitiated crosslinking, release of encapsulated rSDF-1α and crosslinked HA was sustained for over 7 days, and these molecules significantly increased BMC chemotaxis in vitro. When applied to the heart following experimental MI in mice, the HA gel containing rSDF-1α significantly increased the number of systemically infused BMCs in the heart by ~8.5 fold after 7 days, likely through both systemic and local effects of released molecules. We conclude that sustained release of rSDF-1α and HA from our engineered HA hydrogels enhances BMC homing to the remodeling myocardium better than delivery of rSDF-1α alone.

A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues.

Engineered myocardial tissues can be used to elucidate fundamental features of myocardial biology, develop organotypic in vitro model systems, and as engineered tissue constructs for replacing damaged heart tissue in vivo. However, a key limitation is an inability to test the wide range of parameters (cell source, mechanical, soluble and electrical stimuli) that might impact the engineered tissue in a high-throughput manner and in an environment that mimics native heart tissue. Here we used microelectromechanical systems technology to generate arrays of cardiac microtissues (CMTs) embedded within three-dimensional micropatterned matrices. Microcantilevers simultaneously constrain CMT contraction and report forces generated by the CMTs in real time. We demonstrate the ability to routinely produce ~200 CMTs per million cardiac cells (<1 neonatal rat heart) whose spontaneous contraction frequency, duration, and forces can be tracked. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that both the dynamic force of cardiac contraction as well as the basal static tension within the CMT increased with boundary or matrix rigidity. Cell alignment is, however, reduced within a stiff collagen matrix; therefore, despite producing higher force, CMTs constructed from higher density collagen have a lower cross-sectional stress than those constructed from lower density collagen. We also study the effect of electrical stimulation on cell alignment and force generation within CMTs and we show that the combination of electrical stimulation and auxotonic load strongly improves both the structure and the function of the CMTs. Finally, we demonstrate the suitability of our technique for high-throughput monitoring of drug-induced changes in spontaneous frequency or contractility in CMTs as well as high-speed imaging of calcium dynamics using fluorescent dyes. Together, these results highlight the potential for this approach to quantitatively demonstrate the impact of physical parameters on the maturation, structure, and function of cardiac tissue and open the possibility to use high-throughput, low volume screening for studies on engineered myocardium.

Periostin and periostin-like factor in the human heart: possible therapeutic targets.

BACKGROUND: Although numerous signaling pathways have been identified in adult heart disease, our ability to diagnose and treat human cardiomyopathies remains limited. A family of proteins, which includes periostin and periostin-like factor (PLF), has been identified during heart development and disease. Based on recent findings, these proteins are candidate therapeutic agents for heart disease. METHODS: Affymetrix GeneChip Expression Analysis as well as northern and western blot analyses were used to determine periostin and PLF expression in humans. Periostin-like factor levels were determined, by western blot analysis, in the rat animal model used to study myocardial loading and unloading. In vivo and in vitro effects of overexpressing PLF by infection with adenovirus were assessed by calculating cardiac myocyte cross-sectional area and determining the level of protein synthesis, respectively. RESULTS AND CONCLUSIONS: Our findings on PLF suggest that this periostin isoform plays a crucial role in adult cardiac myocyte growth following mechanical overload, thus, implicating its potential as a therapeutic target. In addition, we believe that the differences between the periostin and PLF are of functional significance.

Unloading-induced remodeling in the normal and hypertrophic left ventricle.

To date, no study has assessed the degree of similarity between left ventricular (LV) reverse remodeling and atrophic remodeling. Stable LV hypertrophy was induced by creation of an arteriovenous fistula (AVF) in Lewis rats (32 days). LV unloading was induced by heterotopic transplantation of normal (NL-HT) and/or hypertrophic (AVF-HT) hearts (7 days). We compared indexes of remodeling in AVF, NL-HT, and AVF-HT groups with those of normal controls. LV unloading induced decreases in cardiomyocyte size in NL-HT and AVF-HT hearts. NL-HT and AVF-HT LV were both characterized by relative increases in collagen concentration that were largely a reflection of decreases in myocyte volume. NL-HT and AVF-HT LV were associated with similar increases in matrix metalloproteinase (MMP-2 and -9) zymographic activity, without change in the abundance of the tissue inhibitors of the MMPs. In contrast, AVF-HT, but not NL-HT, was associated with a dramatic increase in collagen cross-linking. Our findings suggest an overall similarity in the response of the normal and hypertrophic LV to surgical unloading. However, the dramatic increase in collagen cross-linking after just 1 wk of unloading suggests a potential difference in the dynamics of collagen metabolism between the two models. Further studies will be required to determine the precise molecular mechanisms responsible for these differences in extracellular matrix regulation. However, with respect to these and related issues, heterotopic transplantation of hypertrophied hearts will be a useful small animal model for defining mechanisms of myocyte-matrix interactions during decreased loading conditions.