Higher Levels Of Fibrosis Research Articles

In Vivo Evaluation of Tissue Biocompatibility of Calcium Silicate-based and Epoxy Resin-based Sealers.

Calcium silicate-based sealers are an alternative to be used into root canal, mainly to their biological properties. However, some biological parameters need to be determined in an in vivo animal research model. So, the aim of the present study was to evaluate in vivo the tissue biocompatibility of a calcium silicate-based sealer (EndoSequence BC Sealer) and an epoxy resin-based sealer (AH-Plus). Polyethylene tubes were filled with freshly mixed sealers and implanted in connective subcutaneous tissue of 25 rats (5/euthanasia day) (Rattus norvegicus albinus). Empty tubes were used as controls and no tubes as sham. Histopathological (hematoxylin eosin) and histochemical (Picrosirius red) examinations were conducted at 3, 7, 15, 30 and 60 days (five rats/day) after the implantation procedure (n=5/group). The type/intensity of inflammation and collagenesis was analyzed statistically with Friedman or Kruskal-Wallis/Dunn tests (P<0.05). The profile of inflammation induced by AH-Plus (Median=2, Range=2-3) was significantly greater than that by Endosequence BC Sealer (Median=1, Range=1-1) during the 15-day experimentation period (P=0.018). After 30 days, both materials produced similar tissue reaction (P>0.05). AH-Plus and Endosequence BC Sealer (Median=2, Range=1-2) induced a high level of fibrosis after 60-day than control (Median=1, Range=1-1) and sham (Median=0, Range=0-0) groups (P<0.001) of fibrosis based in type I collagen increase (P=0.025 and P=0.021, respectively). Tissue necrosis was not observed and the bioceramic sealer showed significant signs of endocytosed (Median=1, Range=1-1) material after 7 days than other groups (Median=0, Range=0-0) (P<0.05). The calcium silicate-based sealer induced tissue repair faster than the epoxy resin-based sealer tested. However, both materials showed adequate biocompatibility and tolerance by subcutaneous tissues, with few differences in inflammatory profiles, formation of granulation tissue, and collagenesis. It may be concluded that calcium silicate-based sealer (EndoSequence BC Sealer) and an epoxy resin-based sealer (AH-Plus) presented suitable biocompatibility.

Overview of the safety, efficiency, and potential mechanisms of finerenone for diabetic kidney diseases.

Diabetic kidney disease (DKD) is a common disorder with numerous severe clinical implications. Due to a high level of fibrosis and inflammation that contributes to renal and cardiovascular disease (CVD), existing treatments have not effectively mitigated residual risk for patients with DKD. Excess activation of mineralocorticoid receptors (MRs) plays a significant role in the progression of renal and CVD, mostly by stimulating fibrosis and inflammation. However, the application of traditional steroidal MR antagonists (MRAs) to DKD has been limited by adverse events. Finerenone (FIN), a third-generation non-steroidal selective MRA, has revealed anti-fibrotic and anti-inflammatory effects in pre-clinical studies. Current clinical trials, such as FIDELIO-DKD and FIGARO-DKD and their combined analysis FIDELITY, have elucidated that FIN reduces the kidney and CV composite outcomes and risk of hyperkalemia compared to traditional steroidal MRAs in patients with DKD. As a result, FIN should be regarded as one of the mainstays of treatment for patients with DKD. In this review, the safety, efficiency, and potential mechanisms of FIN treatment on the renal system in patients with DKD is reviewed.

Multiscale computational modeling of aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common cardiovascular disease that affects millions of people worldwide. The disease is characterized by the formation of calcium nodules on the aortic valve leaflets, which can lead to stenosis and heart failure if left untreated. The pathogenesis of CAVD is still not well understood, but involves several signaling pathways, including the transforming growth factor beta (TGF ) pathway. In this study, we developed a multiscale computational model for TGF -stimulated CAVD. The model framework comprises cellular behavior dynamics, subcellular signaling pathways, and tissue-level diffusion fields of pertinent chemical species, where information is shared among different scales. Processes such as endothelial to mesenchymal transition (EndMT), fibrosis, and calcification are incorporated. The results indicate that the majority of myofibroblasts and osteoblast-like cells ultimately die due to lack of nutrients as they become trapped in areas with higher levels of fibrosis or calcification, and they subsequently act as sources for calcium nodules, which contribute to a polydispersed nodule size distribution. Additionally, fibrosis and calcification processes occur more frequently in regions closer to the endothelial layer where the cell activity is higher. Our results provide insights into the mechanisms of CAVD and TGF signaling and could aid in the development of novel therapeutic approaches for CAVD and other related diseases such as cancer. More broadly, this type of modeling framework can pave the way for unraveling the complexity of biological systems by incorporating several signaling pathways in subcellular models to simulate tissue remodeling in diseases involving cellular mechanobiology.

Bone marrow fibrosis is associated with non-response to CD19 CAR T-cell therapy in B-acute lymphoblastic leukemia.

CD19 directed CAR T-cell therapy is used to treat relapsed/refractory B-cell acute lymphoblastic leukemia. The role of the pre-CAR bone marrow (BM) stromal microenvironment in determining response to CAR T-cell therapy has been understudied. We performed whole transcriptome analysis, reticulin fibrosis assessment and CD3 T-cell infiltration on BM core biopsies from pre- and post-CAR timepoints for 61 patients, as well as on a cohort of 54 primary B-ALL samples. Pathways of fibrosis, extracellular matrix development, and associated transcription factors AP1 and TGF-β3, were enriched and upregulated in nonresponders (NR) even prior to CAR T cell therapy. NR showed significantly higher levels of BM fibrosis compared to complete responders by both clinical reticulin assessment and AI-assisted digital image scoring. CD3+ T cells showed a trend toward lower infiltration in NR. NR had significantly higher levels of pre-CAR fibrosis compared to primary B-ALL. High levels of fibrosis were associated with lower overall survival after CAR T-cell therapy. In conclusion, BM fibrosis is a novel mechanism mediating nonresponse to CD19-directed CAR T-cell therapy in B-ALL. A widely used clinically assay for quantitating myelofibrosis can be repurposed to determine patients at high risk of non-response. Genes and pathways associated with BM fibrosis are a potential target to improve response.

Abstract P222: Effect Of Western Diet On Pathophysiology And Cardiac Gene Expression Profile In Human AT1 Receptor Over-Expressing Hypertensive Transgenic Mice

Human angiotensin II type 1 receptor (hAT1R) gene overexpression may lead to pathophysiological outcomes due to an overdrive of the renin angiotensin system. Our transgenic (TG) mice containing Hap-I (hypertensive genotype) of human AT1R gene are more prone to develop metabolic syndrome (MetS) as compared to TG mice with Hap-II (normotensive genotype). The increased risk of MetS, especially in hypertension, is compounded by the effects of aging and Western diet (WD), which may lead to cardiac complications. However, the underlying mechanisms are not well examined. In this regard, we studied the pathophysiological changes and alterations in transcription profile in the heart of aged Hap-I and Hap-II TG mice following exposure to WD. Aged mice (20-24 months) were maintained on a regular diet or high fat diet with 2% NaCl (WD) for 16 weeks. Following administration of WD, blood pressure increased significantly in Hap-I animals (~15 mmHg), in comparison to Hap-II (~7 mmHg). With respect to Hap-II, aged Hap-I mice show larger heart weight-to-body weight ratio and higher levels of fibrosis on treatment with WD. The TF Activation Profiling (Signosis) after WD treatment shows an increased activation of transcription factors including USF1, CEBP, STAT1, HNF1 in the nuclear extracts from heart of Hap-I TG mice as compared to the Hap-II animals. Competition assay (Promoter-binding TF profiling plate arrays, Signosis) shows stronger binding to the promoter for these TFs binding to the promoter of hAT1R, leading to overexpression of hAT1R in HAP-I TG animals. Transcriptomic analysis from RNA sequencing revealed that WD significantly altered the expression of &gt;500 genes from cardiac tissue of WD treated Hap-I aged mice (compared to control diet treated age-matched mice). Bioinformatics analysis, using Qiagen IPA software, identified major alterations in main canonical pathways involved in cardiac function, inflammation, and oxidative damage. Overall, these results indicate that Western diet promotes hypertension, hypertrophy, and fibrosis in the heart of aged mice. Results from these studies will assist in the identification of novel molecules and mechanisms involved in hypertension and associated cardiac pathophysiology.

FIB‑4 index and NAFLD fibrosis score are useful indicators for screening high‑risk groups of non‑viral hepatocellular carcinoma.

Non-viral hepatocellular carcinoma (HCC) tends to appear in non-cirrhotic livers, rendering it difficult to screen for a high-risk group. The present study aimed to identify the most suitable indicator for screening high-risk groups of non-viral HCC. A total of 190 patients with non-viral HCC, including 126 with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH), were enrolled in the present study. A total of two cut-off values, for low and high levels of fibrosis, were set for each of the indicators, including the Child-Pugh score (CPS; 6 and 7), platelet counts (15.8 and 10x104/µl), albumin-bilirubin (ALBI) score (-2.60 and -2.27), fibrosis 4 index (FIB-4 index; 1.30 and 2.67) and NAFLD fibrosis score (NFS; -1.455 and 0.675). The ratio of the number of patients who fell outside the cut-off value for all patients was defined as the overlooking rate. The overlooking rates of CPS, platelet counts, ALBI score, FIB-4 index and NFS for the low fibrosis cut-off value were 41.0, 48.9, 35.8, 4.2 and 5.8%, respectively. When performing analysis limited to the NAFLD cases, those of the FIB-4 index and NFS were 4.8 and 6.3%, respectively. Those for the high fibrosis cut-off value were 79.5, 73.2, 62.6, 30.0 and 37.4%, respectively. On the whole, the present study demonstrates that the cut-off values of ≥1.30 for the FIB-4 index or ≥-1.455 for the NFS may be used to screen high-risk groups of HCC among patients with non-viral hepatitis.

Factors Associated with Advanced Fibrosis in Patients with Nonalcoholic Fatty Liver Disease

Introduction. Nonalcoholic fatty liver disease includes conditions such as nonalcoholic steatosis and nonalcoholic steatohepatitis, with varying degrees of fibrosis that can progress to cirrhosis and hepatocellular carcinoma. Although the gold standard for its diagnosis remains liver biopsy, many non-invasive methods have been proposed to aid in both the diagnosis of the disease and the evaluation of the presence of liver fibrosis, which is a strong and independent factor for liver related mortality. Objectives. The objectives of this study were: 1) to identify the clinical and laboratory features associated with the presence of advanced fibrosis in individuals with biopsy-confirmed nonalcoholic fatty liver disease; 2) to evaluate the performance of non-invasive markers in identifying patients with advanced fibrosis. Methods. A cross-sectional-analytic study that evaluated patients with nonalcoholic fatty liver disease treated in the outpatient clinic of a university hospital of reference in hepatology, between January 2013 and December 2016. Results. 81 patients aged 53.3 ± 9.8 years were included in this study; 39.5% were men and 70.1% were obese. When comparing patients with advanced fibrosis to those without advanced fibrosis, patients with advanced fibrosis had a lower proportion of males than females (17.6 vs. 45.4%, p = 0.038), a higher proportion of hypothyroidism (29.4 vs. 6.3%, p = 0.017) and a higher median AST (52 vs. 31 U/L, p = 0.005). In logistic regression analysis, only hypothyroidism was independently associated with advanced fibrosis (OR = 4.975; CI 95% 1.050 - 23.574; p = 0.043). Spearman correlation analysis showed that higher levels of fibrosis on liver biopsy, were associated with higher levels of TSH (r = 0.304; p = 0.036), AST (r = 0.277; p = 0.019), GGT (r = 0.284; p = 0.017) and LDL (r = 0.258; p = 0.037). Regarding the performance of the non-invasive markers, the area under the ROC curve of Fibrosis 4 score was 0.723 (p = 0.008), that of Nonalcoholic fatty liver disease fibrosis score was 0.713 (p = 0.022), that of gamma-glutamyl transferase platelet ratio was 0.697 (p = 0.019) and that of aspartate-to-alanine aminotransferase ratio was 0.689 (p = 0.031). Conclusions. Hypothyroidism is a factor independently associated with advanced fibrosis. In the outpatient setting, non-invasive markers may be useful in identifying patients with advanced fibrosis.

Abstract P4-09-02: A molecular classification system for basal-like breast cancer based on the tumor microenvironment is prognostic for survival

Abstract Recent advancements in molecular profiling have revealed distinct breast cancer subtypes, but many clinical NGS assays rely on gene panels, such as PAM50, limiting their clinical utility. Basal-like breast cancer (BLBC), one of the most aggressive subtypes, has highly variable molecular and clinical characteristics. Now, the tumor microenvironment (TME) is recognized as a vital participant in tumor progression and therapeutic response. The development of more refined classifications based on the TME, capable of accounting for tissue heterogeneity, may improve NGS clinical utility for BLBC. Here, we apply our transcriptomic-based approach, recently described by Bagaev et. al., to classify the BLBC TME into discrete immune portraits, to potentially improve clinical outcomes and facilitate therapeutic decisions. We collected a cohort of 1,708 BLBC samples based on the expression levels of 50 genes (PAM50) from 10 publicly available datasets, with clinical outcomes available (n = 819). Using methodology described by Bagaev et. al., 31 functional gene expression signatures (Fges) were selected, and unsupervised dense Louvain clustering was performed to identify TME subtypes. A novel RNA-seq deconvolution algorithm was used to determine the cell types within the TME. Validation of the histological features, including stroma, tumor infiltrating lymphocytes (TILs), and tertiary lymphoid structures (TLS), relative to gene expression patterns in the TME subtypes was performed by automated and manual annotation of BLBC H&amp;E slides (n = 146) from an independent TCGA cohort. Overall survival (OS) analysis was performed using Kaplan-Meier and Cox regression methods. We revealed 5 BLBC subtypes with distinct expression patterns: immune-enriched, non-fibrotic (IE, 19%), B-cell–enriched, TLS-like (TLS, 25.5%), granulocyte-enriched (G, 12.8%), fibrotic (F, 28%), and immune desert (D, 17.7%) (Table). IE tumors featured an active immune TME, with high immune checkpoint expression and T cell activity. The TLS subtype also had an immune-rich TME, presenting high levels of B cells, T helper cells, and TLS (p &amp;lt; 0.001). The TLS subtype exhibited the highest number of stromal TILs on TCGA H&amp;E slides. The G subtype was characterized by high expression of granulocytes and granulocyte traffic molecules. In accordance with our findings, deconvolution predicted the highest percent of neutrophils in the G subtype (p &amp;lt; 0.001). The F subtype demonstrated the highest levels of angiogenesis, stromal Fges, and VEGFR1-3, FGFR1, and EGFR expression. By histological evaluation, 84% of F subtype samples demonstrated a medium or high level of fibrosis. The D subtype showed a high proliferation rate and low stromal and immune Fges. Indicative of proliferation rate, CCNB1 and cyclin B1 were highest in G, D, and IE subtypes. OS analysis revealed a significant association between TME subtypes (TLS = baseline; log HR G = 0.87, p &amp;lt; 0.05; IE = 0.39, p = 0.18; F = 0.99, p &amp;lt; 0.05; D = 1.21, p &amp;lt; 0.05), and survival outcomes. The immune-enriched subtypes, IE and TLS, demonstrated good prognosis and higher expression of immune checkpoint genes, while immune desert D and granulocyte-enriched G subtypes exhibited the worst OS. Using our transcriptomic-based approach, BLBC was classified into 5 distinct subtypes, each with unique therapeutic vulnerabilities. Further investigation of these TME subtypes may lead to potential clinical utility as a prognostic tool to improve clinical decision making. Table. Characteristics of Basal-like breast cancer (BLBC) tumor microenvironment (TME) subtypes. TLS - Tertiary lymphoid structures; CAF - cancer-associated fibroblasts Citation Format: Svetlana Khorkova, Diana Shamsutdinova, Vladimir Kushnarev, Lev Popyvanov, Daniil Dymov, Anastasia Zotova, Ivan Valiev, Zoya Antysheva, Anna Love, Jessica H. Brown, Alexander Bagaev, Nikita Kotlov, Nathan Fowler. A molecular classification system for basal-like breast cancer based on the tumor microenvironment is prognostic for survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-02.

PECULIARITIES OF THE FUNCTIONAL STATE OF THE LIVER IN PATIENTS WITH CHRONIC HEPATITIS C IN THE PRESENCE OF CHRONIC PANCREATITIS.

The aim: To study the features of the functional state of the liver in patients with chronic hepatitis C (CHC) in the presence of CP, depending on the enzymatic activity of the pancreas. Materials and methods: 72 patients were under observation: 52 with CHC and CP with exocrine secretory insufficiency (EI) of the pancreas and 20 - with CHC and CP without EI. In all patients, the degree of liver fibrosis, levels of aminotransferases, total bilirubin, gamma-glutamyltransferase, albumin, stool coproscopy and pancreatic fecal elastase-1 (FE-1) were determined. Results: It was revealed that in patients with CHC combined with CP+EI of the pancreas, higher activity of the necroinflammatory process and deeper stages of liver fibrosis is more often noted than in patients with preserved exocrine function of the pancreas. A statistically significant association was established between the degree of liver fibrosis and the presence of EI of the pancreas (p=0.03), namely, in patients with CHC and CP with EI of the pancreas, the degree of fibrosis F2-4 was 2.8 times more frequent. Also, higher levels of aminotransferases and lower levels of albumin were noted in this group of patients than in patients with CHC and CP with preserved exocrine function of the pancreatic gland. Conclusions: In patients with CHC combined with CP+EI of the pancreas, higher levels of fibrosis and necroinflammatory activity of the liver are more often detected, as well as a tendency to lower albumin levels, than in patients with CHC and CP without EI.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

Prognostic implications of fibrosis in low risk aortic stenosis patients

Introduction and objectivesAmong patients with aortic stenosis (AS), interstitial fibrosis has been associated with progression to heart failure and is a marker of poorer prognosis. We aimed to assess the impact of myocardial fibrosis on clinical events after aortic valve replacement (AVR) in low risk, severe AS. MethodsWe prospectively followed 56 severe AS patients with ejection fraction >40%, who underwent AVR with simultaneous myocardial biopsies and collagen volume fraction (CVF) determination. Baseline and follow-up echocardiographic parameters were assessed. Outcomes were all-cause death and the combined endpoint of all-cause death or non-fatal cardiovascular hospitalization. ResultsPatients were predominantly women (67.9%) and mean age was 66±12 years. At follow-up, there was a significant decrease in transaortic gradients and wall stress, as well as regression in indexed LV mass. Patients who suffered a fatal event or the combined endpoint had a higher degree of fibrosis (27.1±20.7% vs. 15.4±11.8%, p=0.035; 24.0±18.2% vs. 15.3±12.0%, p=0.038, respectively). Patients with CVF≥15.4% had higher rates of all-cause death (37.5% vs. 97.0%, p=0.001) and lower survival free of the combined endpoint of all-cause death or non-fatal cardiovascular hospitalization (0% vs. 91.2%, p<0.001). CVF was the only independent predictor of all-cause death (hazard ratio (HR) 1.88; 95% confidence interval (CI): 1.08-3.29 for each 10% increase; p=0.026) and all-cause death or cardiovascular hospitalization (HR 1.73; 95% CI: 1.03-2.911 for each 10% increase; p=0.038). ConclusionsIn low risk AS patients, higher levels of fibrosis are independent predictors of all-cause death and the composite of all-cause death or non-fatal cardiovascular hospitalization. Further advances in anti-fibrotic therapies in AS are needed.

Thrombin and factor Xa promote premature senescence and remodeling in areas at risk of the left appendage and in atrial endothelial cells: potential modulation by SGLT1 and/or SGLT2 inhibition

Abstract Introduction Atrial fibrillation (AF), the most common cardiac arrhythmia, promotes prothrombotic responses particularly in the distal part of the left atrial appendage (LAA) characterized by reduced flow and low shear stress. AF occurrence and duration are closely associated with endothelial dysfunction and left atrium remodeling. The contribution of coagulation factors thrombin and factor Xa (FXa) to LA remodeling remains poorly studied. This study investigated whether thrombin and FXa promote endothelial dysfunction and profibrotic and prothrombotic responses in the distal and proximal parts of the LAA. Since SGLT2 inhibitors have shown pronounced cardiac protection, the contribution of SGLT2 was examined. Methods LAAs were harvested from porcine hearts. Proximal (low stasis, high shear) and distal (high stasis, low shear) parts were cut and incubated with a coagulation factor (thrombin or FXa). Left atrial endothelial cells (AECs) were collected enzymatically, cultured and used at passage 1. The level of oxidative stress was assessed using the redox-sensitive probe dihydroethidium (DHE), fibrosis by Sirius red histological staining, senescence-associated β-galactosidase (SA-β-gal) activity using X-gal staining in tissues and the fluorogenic substrate C12FDG and flow cytometry in cells. The expression of target proteins was detected by immunofluorescence staining and Western blot analysis. Results The distal part of LAA displayed higher levels of oxidative stress in the endothelium and the atrial wall than the proximal part. SA-β-gal activity, ICAM-1, VCAM-1, and MMP-9 staining was more pronounced in the endothelium of the distal part compared to the proximal part whereas eNOS staining was decreased. The distal LAA wall showed higher levels of fibrosis. Western blot analysis showed higher expression levels of tissue factor (TF), VCAM-1, MCP-1, MMP-9, TGF-β and SGLT-2 in the distal part compared to the proximal one. Both thrombin and FXa increased these signals to a greater extent in the distal part of the LAA whereas the eNOS expression level was decreased. Exposure of AECs to either thrombin or FXa induced a pro-oxidant response and increased the expression level of ICAM-1, TGF-β, MMP-2, MMP-9, SGLT1 and SGLT-2. Thrombin promoted SA-β-gal activity in AECs. The selective SGLT-2 inhibitor empagliflozin and the dual SGLT1/2 inhibitor sotagliflozin prevented the pro-oxidant response and SA-β-gal activity in response to thrombin. Conclusion Thus, the distal part of the LAA is prematurely affected by endothelial dysfunction and senescence associated with a pro-oxidant, pro-remodeling and pro-inflammatory response that is further increased by thrombin and FXa. Hence, the coagulation cascade appears to prematurely promote atrial endothelium and wall dysfunction predominantly in areas at risk that may pave the way to thrombus formation. The findings also suggest a potential protective effect of SGLT1 and/or SGLT2 inhibition on atrial dysfunction. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG, Biberach, Germany

Abstract P479: Pathogenesis And Altered Cardiac Transcriptomic Landscape In Western Diet Treated Aged Mice Overexpressing Human Angiotensin Type 1 Receptor

Overexpression of human angiotensin II type 1 receptor (hAT1R) may lead to pathophysiological outcomes due to overactivation of the renin angiotensin system. We have shown that transgenic (TG) mice containing Hap-I (hypertensive genotype) of human AT1R gene are more prone to develop metabolic syndrome (MetS) as compared to TG mice with Hap-II (normotensive genotype). The increased risk of MetS, especially in hypertension, compounded by the effects of aging and Western diet (WD), which may lead to cardiac complications. However, the underlying mechanisms are not well examined. For this purpose, we studied the pathophysiological changes and gene expression profile alterations in the heart of aged Hap-I and Hap-II TG mice following exposure to WD. Aged mice (20-24 months of age) were maintained on a regular diet or high fat diet with 2% NaCl (WD) for 16 weeks. On regular diet, aged Hap-I mice presented higher (~9 mmHg) systolic blood pressure with respect to age-matched Hap-II animals. Following administration of WD, blood pressure increased in both groups of mice, but to a larger extent in Hap-I animals (~15 mmHg), in comparison to Hap-II (~7 mmHg). With respect to Hap-II, aged Hap-I mice on regular diet tended to have larger heart weight-to-body weight ratio and higher levels of fibrosis. Western Diet treatment exacerbated these differences. RNA sequencing data from cardiac tissue of WD treated Hap-I aged mice (compared to control diet treated age-matched mice) revealed that WD significantly altered the expression of &gt;500 genes (p-adj. &lt;0.05). Bioinformatics analysis, using Qiagen IPA software, identified major alterations in main canonical pathways involved in cardiac function, inflammation, and oxidative damage. Top hits in the disease and biological function category included arrhythmia, chamber enlargement, and cell death. Importantly, IRF3, IRF7, IFNG and STAT1 were among the top upstream regulators significantly affected by WD. Overall, these results indicate that Western diet promotes hypertension, hypertrophy, and fibrosis in the heart of aged mice. Results from these studies will assist in the identification of novel molecules and mechanisms involved in hypertension and associated cardiac pathophysiology.

Reverse translation approach generates a signature of penetrating fibrosis in Crohn’s disease that is associated with anti-TNF response

ObjectiveFibrosis is a common feature of Crohn’s disease (CD) which can involve the mesenteric fat. However, the molecular signature of this process remains unclear. Our goal was to define the...

The combination of carboxy-terminal propeptide of procollagen type I blood levels and late gadolinium enhancement at cardiac magnetic resonance provides additional prognostic information in idiopathic dilated cardiomyopathy-A multilevel assessment of myocardial fibrosis in dilated cardiomyopathy.

AimsTo determine the prognostic value of multilevel assessment of fibrosis in dilated cardiomyopathy (DCM) patients.Methods and resultsWe quantified fibrosis in 209 DCM patients at three levels: (i) non‐invasive late gadolinium enhancement (LGE) at cardiac magnetic resonance (CMR); (ii) blood biomarkers [amino‐terminal propeptide of procollagen type III (PIIINP) and carboxy‐terminal propeptide of procollagen type I (PICP)], (iii) invasive endomyocardial biopsy (EMB) (collagen volume fraction, CVF). Both LGE and elevated blood PICP levels, but neither PIIINP nor CVF predicted a worse outcome defined as death, heart transplantation, heart failure hospitalization, or life‐threatening arrhythmias, after adjusting for known clinical predictors [adjusted hazard ratios: LGE 3.54, 95% confidence interval (CI) 1.90–6.60; P < 0.001 and PICP 1.02, 95% CI 1.01–1.03; P = 0.001]. The combination of LGE and PICP provided the highest prognostic benefit in prediction (likelihood ratio test P = 0.007) and reclassification (net reclassification index: 0.28, P = 0.02; and integrated discrimination improvement index: 0.139, P = 0.01) when added to the clinical prediction model. Moreover, patients with a combination of LGE and elevated PICP (LGE+/PICP+) had the worst prognosis (log‐rank P < 0.001). RNA‐sequencing and gene enrichment analysis of EMB showed an increased expression of pro‐fibrotic and pro‐inflammatory pathways in patients with high levels of fibrosis (LGE+/PICP+) compared to patients with low levels of fibrosis (LGE‐/PICP‐). This would suggest the validity of myocardial fibrosis detection by LGE and PICP, as the subsequent generated fibrotic risk profiles are associated with distinct cardiac transcriptomic profiles.ConclusionThe combination of myocardial fibrosis at CMR and circulating PICP levels provides additive prognostic value accompanied by a pro‐fibrotic and pro‐inflammatory transcriptomic profile in DCM patients with LGE and elevated PICP.

Metabolic Environment Alters the Transcriptomic Landscape of the Aged Heart

Eating disorders at older age are coupled with a number of unfavorable health outcomes, including cardiovascular disease; but mechanisms underlying the process remain to be clarified. For this purpose, we studied alterations in gene expression profile of hearts from aged mice following exposure to Western diet. Specifically, we used transgenic mice expressing the haplotype-I (Hap-I: hypertensive genotype) of the human angiotensin receptor type 1 (AT1R) gene because these animals are more prone to develop metabolic syndrome-related disorders. For comparison, mice containing haplotype-II of human AT1R (Hap-II: normotensive genotype) were used. Aged mice (20-24 months of age) were maintained on a regular diet or high fat diet with 2% NaCl (Western diet) for 16 weeks. Blood pressure was monitored by telemetry, whereas cardiac anatomy and gene expression profile were established on heart specimens, using histological and RNA-seq analysis. On regular diet, old Hap-I mice presented higher (~9 mmHg) systolic blood pressure, with respect to aged-matched Hap-II animals. Following administration of Wester diet, blood pressure increased in both groups of mice, but to a larger extent in Hap-I animals (~15 mmHg), in comparison to Hap-II (~7 mmHg). With respect to Hap-II, aged Hap-I mice on regular diet tended to have larger heart weight-to-body weight ratio, increased cell size, and higher levels of fibrosis. Western Diet treatment exacerbated these differences. RNA sequencing data from cardiac tissue of Hap-I aged mice revealed that Western diet significantly altered the expression of >500 genes (p-adj. <0.05). Bioinformatics analysis, using Qiagen IPA software, identified major alterations in main canonical pathways involved in cardiac function, inflammation, and oxidative damage. Top hits in the disease and biological function category included arrhythmia, chamber enlargement, and cell death. Importantly, IRF3, IRF7, IFNG and STAT1 were among the top upstream regulators significantly affected by Western diet. Overall, these results indicate that Western diet promotes hypertension, hypertrophy, and fibrosis in the heart of aged mice. These alterations are paralleled by perturbation of cardiac transcriptional profile, including upstream gene regulators.

Diabetic phenotype and prognosis of patients with heart failure and preserved ejection fraction in a real life cohort

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome, with several underlying etiologic and pathophysiologic factors. The presence of diabetes might identify an important phenotype, with implications for therapeutic strategies. While diabetes is associated with worse prognosis in HFpEF, the prognostic impact of glycemic control is yet unknown. Hence, we investigated phenotypic differences between diabetic and non-diabetic HFpEF patients (pts), and the prognostic impact of glycated hemoglobin (HbA1C).MethodsWe prospectively enrolled 183 pts with HFpEF (78 ± 9 years, 38% men), including 70 (38%) diabetics (type 2 diabetes only). They underwent 2D echocardiography (n = 183), cardiac magnetic resonance (CMR) (n = 150), and were followed for a combined outcome of all-cause mortality and first HF hospitalization. The prognostic impact of diabetes and glycemic control were determined with Cox proportional hazard models, and illustrated by adjusted Kaplan Meier curves.ResultsDiabetic HFpEF pts were younger (76 ± 9 vs 80 ± 8 years, p = 0.002), more obese (BMI 31 ± 6 vs 27 ± 6 kg/m2, p = 0.001) and suffered more frequently from sleep apnea (18% vs 7%, p = 0.032). Atrial fibrillation, however, was more frequent in non-diabetic pts (69% vs 53%, p = 0.028). Although no echocardiographic difference could be detected, CMR analysis revealed a trend towards higher LV mass (66 ± 18 vs 71 ± 14 g/m2, p = 0.07) and higher levels of fibrosis (53% vs 36% of patients had ECV by T1 mapping > 33%, p = 0.05) in diabetic patients.Over 25 ± 12 months, 111 HFpEF pts (63%) reached the combined outcome (24 deaths and 87 HF hospitalizations). Diabetes was a significant predictor of mortality and hospitalization for heart failure (HR: 1.72 [1.1–2.6], p = 0.011, adjusted for age, BMI, NYHA class and renal function). In diabetic patients, lower levels of glycated hemoglobin (HbA1C < 7%) were associated with worse prognosis (HR: 2.07 [1.1–4.0], p = 0.028 adjusted for age, BMI, hemoglobin and NT-proBNP levels).ConclusionOur study highlights phenotypic features characterizing diabetic patients with HFpEF. Notably, they are younger and more obese than their non-diabetic counterpart, but suffer less from atrial fibrillation. Although diabetes is a predictor of poor outcome in HFpEF, intensive glycemic control (HbA1C < 7%) in diabetic patients is associated with worse prognosis.

A difficult-to-diagnose fibromatosis-like metaplastic carcinoma of the breast: a case report

BackgroundFibromatosis-like metaplastic carcinoma (FLMCa), classified as a metaplastic carcinoma of the breast, is a very rare type of metaplastic carcinoma. We report a case of FLMCa that was difficult to diagnose.Case presentationThe patient was a 56-year-old postmenopausal woman who presented with a left-sided breast mass. A 1.3-cm irregular mass was found in the lower outer quadrant of the left breast on breast ultrasonography. She underwent core needle biopsy and vacuum-assisted biopsy, but the pathological findings only revealed inflammatory cell infiltration and a high level of fibrosis, with no malignant findings. At 3 months follow-up, she underwent a repeat breast ultrasonography, which revealed an increase in the size of the mass to 1.8 cm, and a repeat core needle biopsy, which showed a few spindle cells and squamous cells positive for cytokeratin (CK)5/6 and AE1/AE3, leading to the suspicion of FLMCa. Since the amount of tissue was insufficient to establish a definitive diagnosis, she underwent a lumpectomy. We found low-grade and slightly atypical spindle cells and partly atypical spindle cell carcinoma and squamous cell carcinoma. CK5/6 and α-SMA were positive, thus confirming FLMCa. Because the margins on the edge of the nipple side and anterior side were “ink on tumor”, she underwent a mastectomy and sentinel lymph node biopsy. After the surgery, she received adjuvant chemotherapy. At 3 years and 8 months of follow-up, no recurrent or metastatic lesions were identified in her body.ConclusionsFLMCa should be considered in the differential diagnosis when collagenous fibers are proliferating and malignancy is clinically suspected. Immunohistochemical analysis may be helpful in confirming this diagnosis.

Urinary vitronectin identifies patients with high levels of fibrosis in kidney grafts

BackgroundIn kidney transplantation, fibrosis represents the final and irreversible consequence of the pathogenic mechanisms that lead to graft failure, and in the late stages it irremediably precedes the loss of renal function. The invasiveness of kidney biopsy prevents this condition from being frequently monitored, while clinical data are rather unspecific. The objective of this study was to find noninvasive biomarkers of kidney rejection.MethodsWe carried out proteomic analysis of the urinary Extracellular Vesicles (uEVs) from a cohort of kidney transplant recipients (n = 23) classified according to their biopsy-based diagnosis and clinical parameters as interstitial fibrosis and tubular atrophy (IFTA), acute cellular rejection (ACR), calcineurin inhibitors toxicity (CNIT) and normal kidney function (NKF).ResultsShotgun mass spectrometry of uEV-proteins identified differential expression of several proteins among these different groups. Up to 23 of these proteins were re-evaluated using targeted proteomics in a new independent cohort of patients (n = 41) classified in the same diagnostic groups. Among other results, we found a differential expression of vitronectin (VTN) in patients displaying chronic interstitial and tubular lesions (ci and ct mean > 2 according to Banff criteria). These results were further confirmed by a pilot study using enzyme-linked immunosorbent assay (ELISA).ConclusionUrinary vitronectin levels are a potential stand-alone biomarker to monitor fibrotic changes in kidney transplant recipients in a non-invasive fashion.

Abstract 2768: Development of a bioluminescent pancreatic orthotopic KPC model for immuno-oncology drug discovery

Abstract Introduction: The tumor microenvironment (TME), composed of immune cells, angiogenic vascular cells, lymphatic endothelial cells, and cancer-associated fibroblasts, is an essential part of cancer malignancy, which cannot manifest without an important interplay between cancer cells and their local environment. Therapies targeting various components of the TME are now being used in the clinic and, accordingly, highly relevant preclinical models mimicking the patient TME are urgently needed. Orthotopic tumor models, engrafted into relevant organs of tumor origin, provide a valuable tool allowing researchers to examine cancer progression more accurately and interrogate drugs more faithfully. Methods: We have previously established a Kras (G12D)/Trp53 null/Pdx1-cre (KPC) mouse allograft tumor model, mPA6115, to study innovative treatments of pancreatic ductal adenocarcinoma (PDAC) which ranks as the 10th most common cancer and the 4th leading cause of cancer-related death in the United States, and which has limited standard of care options. The mPA6115 KPC MuPrime™ model recapitulates human PDAC tumors in many aspects including: morphological small ductal tumors with enriched stromal contents including high levels of fibrosis and M2 macrophages; key features of a highly hostile immune microenvironment; and most importantly, the model is non-responsive to a series of immune checkpoint inhibitors. These features make mPA6115 an ideal model to test novel therapies for PDAC. In order to enable real-time monitoring of orthotopically engrafted tumor growth using optical imaging, we generated and characterized a firefly luciferase (Luc) expressing model via lentivirus transfection of the syngeneic cell line derived from parental KPC MuPrime tumors. Results: We successfully established the bioluminescent mPA6115-luc line in vivo via orthotopic inoculation into the pancreas of C57BL/6 mice. Tumor progression was monitored using IVIS® bioluminescent imaging with additional survival readouts. Benchmark efficacy studies showed that mPA6115-luc was resistant to treatment with an anti-PD-1 immune checkpoint inhibitor. However, gemcitabine produced a better response in mPA6115-luc than the parental model. Baseline immune profiling and IHC staining showed enrichment of myeloid-derived suppressor cells and macrophages in the TME, obvious fibrosis, and angiogenesis in line with parental KPC MuPrime model data. Conclusions: We have established and characterized a bioluminescent orthotopic KPC mouse model mPA6115-luc, compatible with optical imaging to facilitate preclinical evaluation of innovative treatments for PDAC. Citation Format: Yanrui Song, Phillip Shuzong Wang, Yinfei Yin, Henry Q. X. Li, Davy Xuesong Ouyang. Development of a bioluminescent pancreatic orthotopic KPC model for immuno-oncology drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2768.