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Background Esophageal pathology such as perforation or malignancy often necessitates esophageal reconstruction. However, the optimal method of esophageal primary repair and anastomosis are unclear. We aim to determine stress to failure of esophageal anastomosis for full thickness and mucosal‐submucosal anastomotic techniques. Methods Six esophagi from freshly sacrificed 60 kilogram swine were transected at the mid thoracic portion of the esophagus. Esophageal anastomoses were created and subsequently primarily anastomosed using 2‐0 running continuous polyglactin (Vicryl, Ethicon, Somerville, NJ) suture either as full thickness or partial thickness (mucosa‐submucosa alone). The sutured specimens were then mounted on cannulae. Using a syringe pump system, the esophagi were infused with colored saline while monitoring pressure continuously with optical tracking for detection of fluid leak. Data acquisition software was used to collect stress‐to‐failure data. Synchronized video and pressure data were analyzed post hoc for visualization of the leak and concurrent pressure to determine failure pressure. Results The full‐thickness anastomosis failed at a mean ± (SEM) pressure of 15.33 ± (1.45) mmHg while the mucosal‐submucosal anastomosis failed at a mean ± (SEM) pressure of 36.33 ± (3.76) mmHg, ( P = 0.02, unpaired t ‐test with Welch's correction). MATLAB (MathWorks, Natick, MA) was used calculate the statistical results. Conclusion While the optimal in vivo method for esophageal anastomosis is unclear, we present evidence that an anastomosis incorporating only the mucosa and submucosa may be superior to full thickness. Additional research is needed to further clarify optimal technique and post‐operative outcomes. Support or Funding Information Dr. Aho reports funding from a Grant from the National Heart, Lung, and Blood Institute T32 HL105355, a component of the National Institutes of Health (NIH).

Tissue fibrosis represents an increasing cause of morbidity and mortality worldwide, with fibroblast‐derived myofibroblasts playing an essential pathogenic role. Myofibroblasts are characterized by their increased expression of smooth muscle actin (α‐SMA), and contribute to tissue fibrosis through their increased abundance, matrix deposition and tissue contraction. Unlike in physiological wound repair, where fibroblast activation is spontaneously reversible, the fibroblast activation associated with pathological fibrosis is perpetuated, and fibroblasts maintain their activated phenotype even after the initial injury has been resolved. Previous studies have shown that aberrant epigenetic signaling may drive sustained fibroblast activation during fibrosis and several small‐molecule epigenetic modifiers have been shown to be effective in protecting against fibrosis in various animal models, however no systematic effort has been reported to identify epigenetic small molecule regulators of myofibroblast activation. Here we screened a library of 98 well‐annotated epigenetic modifying compounds from Mayo Clinic's collaboration with Nanosyn (Santa Clara, CA) for their ability to inhibit fibroblast to myofibroblast activation. To analyze compound effects, we designed a high‐content image‐based screening method to quantify α‐SMA expression, organization, and area coverage. For each metric we calculated an independent Z‐score, and to identify which compounds most effectively blocked myofibroblast activation we created a robust, multiparametric “Deep Z Analysis” which stacked together individual Z‐scores. In the initial screen each compound was run in triplicate using three concentrations (0.5, 2.5, and 10 μM). Screen hits were defined by any compound which displayed an estimated IC50 < 2.5μM, resulting in a hit rate of 12.2%. These 12 hits were then validated for their efficacy in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. In support of our overall screening approach, among the primary screen hits were compounds previously shown to be antifibrotic, including: bromodomain inhibitors JQ1 and CPI‐0610, DNA methyl transferase inhibitor azacitidine, and Poly (ADP‐ribose) Polymerase (PARP) inhibitor ABT‐888. While several epigenetic therapies are already approved by the FDA for cancer treatment, no epigenetic drugs for the treatment of fibrotic diseases have reached the clinic. Our approach will help identify epigenetic modifiers attractive for further detailed research, with a long term goal of defining new targets to move forward towards eventual therapies for lung fibrosis.

Despite recent therapeutic developments, pulmonary fibrosis remains a debilitating disease with poor prognosis. In order to create effective treatments, promising molecular targets need to be identified and validated. Across multiple organs, mesenchymal derived myofibroblasts play an essential role in matrix deposition and tissue contraction. We previously carried out a high‐content, RNAi screen to characterize myofibroblast cytoskeletal phenotypes (α‐SMA and organized F‐actin) in cultured fibroblasts. Hits from this screen, including STAT3 (Signal transducer and activator of transcription 3), were validated in primary lung fibroblasts from patients with IPF (idiopathic pulmonary fibrosis) on compliant hydrogels. To further evaluate STAT3 as a therapeutic target, we have focused on LLL12, a small molecule inhibitor of STAT3. LLL12 dose‐dependently (0.3, 1.0μM) reduced RNA expression of profibrotic genes ( ACTA2 , CTGF , COL1A1 , and FN1 ) as well as protein deposition of collagen III and fibronectin, in TGFβ stimulated cultured fibroblasts. LLL12 also reduced contractile force, as measured by traction force microscopy, to levels comparable to that seen with inhibition of Rho kinase (Y27632, 20μM). In an effort to support the role of STAT3 as a multi‐organ regulator of tissue fibrosis we confirmed similar effects of LLL12 in primary human hepatic stellate cells and cardiac fibroblasts. Using the bleomycin model of pulmonary fibrosis we assessed the in vivo efficacy of STAT3 inhibition. The lungs of the mice treated therapeutically (day 14–28) with LLL12 (5 mg/kg i.p. daily) showed reduced collagen and fibrotic matrix content measured by hydroxyproline and trichrome histology. RNA expression of COL1A1 , FN1 , CTGF , and ACTA2 also decreased (~50%) in the mice treated with LLL12. Importantly, in the mice which did not receive bleomycin, LLL12 had no effect on RNA expression of these genes. These data not only validate the RNAi screening approach, but also point towards STAT3 as an important molecular target for regulation of myofibroblast activation in pulmonary fibrosis and other fibrotic diseases. Support or Funding Information Supported by NIH RO1 HL092961 and HL113796, and Boehringer Ingelheim.

In pathologies of the heart, blood vessels, and airways, and in cancer as well, the ultimate therapeutic target is often modulation of cellular contractile force. For discovering new therapies, drug screening technologies typically use biochemical or structural surrogates for contractile force rather than contractile force itself. Because such approaches are indirect, some hits are false while other potential hits could be missed. To fill this gap, we developed a new high‐throughput method that utilizes cellular contractile force directly as the reporter ‐‐called contractile force screening (CFS)‐‐ and established feasibility of CFS in the context of drug discovery in asthma. During an acute asthma attack, contractile forces generated by airway smooth muscle cells act to constrict the airway and thus obstruct airflow. To dilate constricted airways, asthma patients use bronchodilators to reduce these contractile forces and thus allow the airway to open more fully, but currently available bronchodilator medications often fail to relax that muscle sufficiently, especially in severe asthma. To address this unmet need for more effective bronchodilator medications, we implemented CFS to evaluate 10,000 small molecule compounds selected randomly from the Chembridge DIVERset® library. Acrylamide gels were made in 96‐well plates and fluorescent markers and collagen were coated onto gel surfaces. Upon each gel we cultured primary human airway smooth muscle (HASM) cells to near confluence. Drug effects were quantified as the ‘force response ratio’, namely, the ratio of the contractile forces before and after drug addition. By screening mixtures of 8 compounds simultaneously, we were able to evaluate 2000 compounds per week in quadruplicate. After retesting individual compounds from initially positives, we identified 2 individual compounds that relaxed HASM cells in a dose‐dependent and a non‐toxic manner. The mechanism of action is still under investigation but we found that these compounds work independent of cAMP. CFS provides a convenient, rapid, and inexpensive platform for drug discovery based upon a physiological endpoint, namely, cell contractile force. Furthermore, CFS technology has the potential to become a new high‐content assay for drug discovery in any disease process in which cellular contractile force plays a central role. Support or Funding Information NIH P50HL107171 (CADET phase I), R01EY019696 (Eye BRP), NRF GRN (NRF‐2013S1A2A2035518)

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5′-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P =7.12×10 −7 ). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.

Abstract The neutral amino acid glutamine plays a central role in TGF-β (transforming growth factor-β)–induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5 (solute carrier transporter 1A5). In the present work, we demonstrated that profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and that targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat patients with fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from idiopathic pulmonary fibrosis lungs. The expression of profibrotic targets, cell migration, and anchorage-independent growth by TGF-β required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy; suppressed mTOR, HIF (hypoxia-inducible factor), and Myc signaling; and impaired mitochondrial function, ATP production, and glycolysis. Pharmacological inhibition of SLC1A5 by the small-molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving and attenuated fibrosis in a bleomycin-treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, providing a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.