Junctional Adhesion Molecule Research Articles

8038 Targeting Breast Cancer Stem Cells With Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

8038 Targeting Breast Cancer Stem Cells with Oncolytic Mammalian Orthoreovirus (MRV)

Abstract Disclosure: N.I. Roman Ortiz: None. J. Davydova: None. P. Danthi: None. J.H. Ostrander: None. Breast cancer is the most frequently diagnosed malignant cancer in women and is the second leading cause of cancer deaths among women in the United States. Patients with ER+ breast cancer can experience recurrence for more than 10 years after initial diagnosis. Breast cancer stem-like cells (BCSC) are slowly proliferative and exist as a minority sub-population (0-5%) within the tumor. Existing treatments target rapidly dividing cells, and BCSC are unresponsive to these therapies. Due to their self-renewal capabilities, BCSCs can fuel recurrence, metastasis, and resistance to therapies. Notably, no FDA-approved therapeutics selectively target BCSCs. Mammalian orthoreovirus (MRV) is an oncolytic virus tested in clinical trials for many cancer types including metastatic breast cancer. MRV was deemed safe but lacked efficacy as standalone treatment. We tested various laboratory MRV strains (T1L, R2) with T3D, a similar strain to the one evaluated in clinical trials. Our studies suggest that T3D is less effective in killing ER+ breast cancer cells and BCSCs compared to T1L and R2. To understand how T1L and R2 are more effective at killing ER+ breast cancer we aim to test the stages of MRV infection and how MRV induces cell death in ER+ therapy sensitive and resistant cells. MRV can enter cells by receptor mediated endocytosis through attachment with Junctional Adhesion Molecule A (JAM-A) and sialic acids as co-receptors. We have found that T1L attaches to cells in a JAM-A-dependent manner in ER+ sensitive cancer cells, while ER+ therapy resistant cells have increased resistance to JAM-A mediated endocytosis. Previous work has shown that apoptosis is involved in MRV-induced cell death. We found that apoptosis inhibitors in MRV-treated cells increase cell viability, suggesting that MRV-induces apoptotic cell death in ER+ breast cancer cells. Our current studies involve using RNA sequencing as a tool to assess gene expression of ER+ therapy sensitive and resistant cells infected with MRV at early and late stages of infection to determine key genes and pathways associated with cell death.We also aim to generate novel MRV strains with enhanced BCSC killing. We used a forward genetic approach and serially passaged MRV strains in BCSC enriched tumorsphere cultures of MCF-7 paclitaxel resistant (TaxR) cells to generate a selective oncolytic virus that can infect and kill BCSCs. Preliminary data has shown that serially passaged (SP) MRV strains (T1L SP, R2 SP) are more effective in decreasing cell viability and inhibiting tumorsphere formation compared to the parental strains (T1L and R2). Current studies are focused on sequencing SP clones to identify mutations that enhance BCSC death. We expect these studies to enhance MRV virotherapeutics for combined use with clinically relevant inhibitors (i.e. CDK4/6 inhibitors), anti-estrogens, or immunotherapy to prevent and treat metastatic ER+ breast cancer. Presentation: 6/2/2024

Epithelial neutrophil localization and tight junction Claudin-2 expression are innovative outcome predictors in inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) in clinical and endoscopic remission may still experience disease relapse. Therefore, there is a need to identify outcome predictors. Recently, the role of neutrophils in predicting outcomes in ulcerative colitis (UC) has been highlighted. Furthermore, the impairment of intestinal barrier plays a key role in forecasting disease outcomes in IBD. This observational study aimed to assess the predictive role of neutrophils according to tissue localization and intestinal barrier protein expression in IBD. IBD patients in clinical remission who underwent colonoscopy between January 2020 and June 2022 at two tertiary referral centres were enrolled. Patients with Mayo Endoscopic Score ≤1 (UC) and Simple Endoscopic Score ≤6 (Crohn's disease) were included. Histological activity was assessed using validated scores. Experienced pathologists evaluated neutrophil localization in the epithelium and lamina propria and immunohistochemical expression of Claudin-2 and junctional adhesion molecule A (JAM-A). Of 60 UC and 76 CD patients, 59.7% had histological activity. 25.8% of patients developed an adverse outcome within 12months. Neutrophils in the epithelium predicted adverse outcomes for UC (hazard ratio [HR] 5.198, p=0.01) and CD (HR 4.377, p=0.03) patients in endoscopic remission. Claudin-2 expression correlated with endoscopic and histological activity and predicted outcomes in UC. Similar results were found for JAM-A in CD despite this protein showing less specificity as a barrier predictor of outcome. This study highlights the potential role of epithelial neutrophil localization and Claudin-2 'leaky gut' expression as tools for predicting IBD outcomes and guiding further patient-tailored therapy.

Junctional adhesion molecular 3 (JAM3) is a novel tumor suppressor and improves the prognosis in breast cancer brain metastases via the TGF-β/Smad signal pathway.

Breast cancer brain metastasis (BCBM) is a deadly clinical problem, and the exact underlying mechanisms remain elusive. Junctional adhesion molecule (JAM), a tight junction protein, is a key negative regulator of cancer cell invasion and metastasis. Junction adhesion molecule 3 (JAM3) expression in breast cancer was analyzed using bioinformatics methods and confirmed by PCR, western blotting, and immunofluorescence (IF) in cell lines. The effects of exogenous expression of JAM3 using lentiviral vectors on invasion, adhesion, and apoptosis were verified using transwell assays and flow cytometry. Differentially expressed genes (DEGs) were detected by RNA sequencing and verified by q‒PCR and Western blotting. The effect of JAM3 silencing using siRNA was assessed by an adhesion assay. Kaplan‒Meier analysis was applied to calculate the impact of JAM3 expression and classic clinicopathologic characteristics on survival. Bioinformatics analysis revealed that JAM3 expression was reduced in BCBM. Exogenous expression of JAM3 minimizes the ability of breast cancer cells to invade and adhere and promotes their apoptosis. Silencing JAM3 results in morphology changes and the recovery of invasion and adhesion to ECMs, and the TGF-β/Smad signaling pathway may be involved. JAM3 predicts less metastasis and good survival in patients with BCBM. Statistical analysis of BCBM samples detected by immunohistochemistry (IHC) and the associated clinicopathological characteristics revealed that low levels of JAM3 expression and high levels of TNF-β1 are linked to the clinical progression of both primary and metastatic breast tumors. Kaplan-Meier analysis revealed that a high expression level of JAM3 was associated with longer survival. JAM3 can serve as a key negative regulator of breast cancer cell invasion, apoptosis, and brain metastasis, possibly through the TGF/Smad signaling pathway. JAM3 is anticipated to be a promising biomarker for the diagnosis and prognosis of breast cancer.

Phytase, 25-hydroxyvitamin D3 and cocci vaccination to broilers fed a calcium and phosphorus-reduced diet under Eimeria spp. challenge: effects on growth performance and intestinal health

A study evaluated the effects of phytase, 25-hydroxyvitamin D3 (25OHD), and cocci vaccination on broilers fed a diet reduced in calcium (Ca) and available phosphorus (avP) under Eimeria challenge. A total of 840 one-day-old male broilers were assigned to a 2 × 5 factorial arrangement based on cocci vaccination and dietary treatments. Half of the birds were vaccinated against coccidia on d 1, and all birds were orally challenged with Eimeria spp. (sporulated oocysts: 12,500 of E. maxima, 12,500 of E. tenella, and 62,500 of E. acervulina) on d 14. Dietary treatments included: 1) a nutrient adequate diet (PC); 2) a diet reduced by 0.2% in Ca and avP (NC); 3) NC plus 1,500 FTU/kg of phytase (NC+PHY); 4) NC plus 3,000 IU/kg of 25OHD (NC+25OHD); 5) NC with both PHY and 25OHD (NC+PHY+25OHD). SAS was used for data analysis, with significance set at P ≤ 0.05. Pre-infection growth performance was comparable across the treatments. However, vaccinated birds exhibited higher body weight (BW) and body weight gain (BWG) from 0 to 6 d postinoculation (DPI; P < 0.05). The NC diet reduced BWG from 6 to 12 DPI and increased the feed conversion ratio (FCR) during 6 to 12 DPI and the overall period (0-26 d) compared to the PC birds. In contrast, the supplementation with phytase, 25OHD, or both, returned BWG and FCR to levels seen with the PC diet (P < 0.01). Vaccinated birds also had reduced gut permeability at 5 DPI, increased intestinal villus height, and lower expression levels of the tight junction proteins junctional adhesion molecule 2 (JAM2) and occludin (OCLN) at 6 DPI (P < 0.05). Interestingly, the cocci vaccine resulted in lower E. acervulina but higher E. tenella oocyst shedding at 6 DPI (P < 0.01). Interaction effects were observed for duodenal lesion scores and ileal crypt depth at 6 DPI (P < 0.05). In conclusion, coccidial vaccination improved growth performance, decreased intestinal permeability, enhanced intestinal morphology, and modulated tight junction protein gene expression under Eimeria infection. Reducing dietary Ca and avP levels adversely affected growth performance and FI during the recovery phase, but these negative effects could be mitigated by supplementing with phytase or 25OHD.

Impact of Epithelial Claudin-4 and Leukotriene B4 Receptor 2 in Normoganglionic Hirschsprung Disease Colon on Post Pull-through Enterocolitis

PurposeTo investigate whether Leukotriene B4 receptor 2 (BLT-2), an upstream regulator of tight junction protein (TJP) Claudin-4, and TJPs could be etiologic factors in Hirschsprung-associated enterocolitis (HAEC) after pull-through (PT) for Hirschsprung disease (HD). MethodsNormoganglionic colon (HD-N) and aganglionic rectum (HD-A) specimens from rectal/rectosigmoid (R/RS) or descending/transverse (D/T) HD were assessed using quantitative polymerase chain reaction (qPCR) for Occludin, TJP-1, TJP-2, Junctional adhesion molecule (JAM)-1, JAM-2, Claudin-1, Claudin-3, Claudin-4, and BLT-2 and immunoblotting for Claudin-4 using fresh specimens obtained intraoperatively (2021–2024; n = 17; R/RS = 15 and D/T = 2). Claudin-4 immunohistochemistry was also evaluated quantitatively using preserved (n = 29; R/RS = 20 and D/T = 9; 2009–2021) and fresh HD specimens for comparison with anorectal malformation patients having colostomy closure as controls (n = 42) and between HD-A versus HD-N, R/RS versus D/T, and HAEC (+) versus HAEC (−). Technically inadequate or transitional zone PT were excluded. ResultsSubjects were 123 PT cases. Mean ages at PT/colostomy closure (years) were R/RS: 2.7 ± 2.9, D/T: 1.6 ± 2.2, and controls: 1.4 ± 0.7. Postoperative HAEC occurred 18 times in 14 PT cases (grade I = 5, grade II = 13). Post-PT HAEC was significantly more frequent in D/T (50.0% versus 6.4%; p < 0.001); Claudin-4 was significantly lower in HD-N from post-PT HAEC cases, especially D/T (p < 0.05) on immunohistochemistry. Claudin-4 was significantly lower in HD-N/HD-A compared with controls on immunoblotting (p < 0.05) and immunohistochemistry (p < 0.001). qPCR showed TJP-1, TJP-2, JAM-1, JAM-2, Claudin-4, and BLT-2 were significantly lower in HD-N/HD-A compared with controls. ConclusionsLower Claudin-4 and BLT2 in post-PT HAEC HD-N (especially D/T) suggests generalized epithelial barrier derangement with possible etiologic implications for HAEC. Level of EvidenceⅡ

Junctions at the crossroads: the impact of mechanical cues on endothelial cell-cell junction conformations and vascular permeability.

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature's specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

F11R RNA trinucleotide over-edited by ADAR in gastric and colorectal cancers: Cross-cohort validation, gene expression regulation, and diagnostic significance

The F11 receptor (F11R) gene encoding junctional adhesion molecule A has been associated with gastric cancer (GC) and colorectal cancer (CRC), in which its role and regulation remain to be further elucidated. Recently F11R was also identified as a potential target of adenosine-to-inosine (A-to-I) mediated by the adenosine deaminases acting on RNA (ADARs). Herein, using RNA-Seq and experimental validation, our current study revealed an F11R RNA trinucleotide over-edited by ADAR, with its regulation of gene expression and clinical significance in four GC and three CRC cohorts. Our results found an over-edited AAA trinucleotide in an AluSg located in the F11R 3′-untranslated region (3′-UTR), which showed editing levels correlated with elevated ADAR expression across all GC and CRC cohorts in our study. Overexpression and knockdown of ADAR in GC and CRC cells, followed by RNA-Seq and Sanger sequencing, confirmed the ADAR-mediated F11R 3′-UTR trinucleotide editing, which potentially disrupted an RBM45 binding site identified by crosslinking immunoprecipitation sequencing (CLIP-seq) and regulated F11R expression in luciferase reporter assays. Moreover, the F11R trinucleotide editing showed promising predictive performance for diagnosing GC and CRC across GC and CRC cohorts. Our findings thus highlight both the potential biological and clinical significance of an ADAR-edited F11R trinucleotide in GC and CRC, providing new insights into its application as a novel diagnostic biomarker for both cancers.

Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba – one of the four Pard3 proteins expressed in the zebrafish – is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba - one of the four Pard3 proteins expressed in the zebrafish - is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

Effects of dietary vitamin D3 and carbohydrate levels on growth performance, carbohydrate metabolism, and intestinal health of yellow catfish (Pelteobagrus fulvidraco)

The study was performed to determine effects of vitamin D3 (VD3) and carbohydrate levels in the diets on growth, carbohydrate absorption and metabolism, and intestinal health of yellow catfish (Pelteobagrus fulvidraco). Four groups of yellow catfish with an average weight of 4.12 ± 0.00 g were provided with diets containing varying proportions of VD3 and carbohydrate. These diets included two VD3 levels at 1000 IU/kg (basic VD3) and 16,100 IU/kg diet (high VD3), each containing carbohydrate levels of 25% and 35%, respectively. After analyzing dietary VD3 and carbohydrate contents, these groups were labeled as follows: the control (30.78% carbohydrates +1040 IU/kg), the VD3 group (30.67% carbohydrates +16,041 IU/kg), the high-carbohydrate group (HCD group, 39.65% carbohydrates +1017 IU/kg), and the HCD + VD3 group (39.67% carbohydrates +16,061 IU/kg). Each tank consisted of three replicates, with each replicate containing 30 fish. The experiment continued for 10 weeks. Compared with the control, HCD group reduced growth performance, the muscular layer thickness, the zonulae occludentes1 and 3 (zo1, zo3), junctional adhesion molecules 3a (jam3a), claudin1, occludin, phosphoglucomutase 1 (pgm1) mRNA expression, but increased intestinal villi height, glycogen content, glycogen synthase (gs), glycogen branching enzyme (gbe), sodium/glucose cotransporter 1 and 2 (sglt1, sglt2) and glucose transporter 2 (glut2) mRNA expression, solute carrier family 5 member 1 (SGLT1) protein expression, and dietary VD3 group alleviated HCD-induced variations of these indices mentioned above. Compared to the control, HCD group significantly suppressed pyruvate carboxylase b (pcxb), phosphoenolpyruvate carboxykinase (pepck), interleukin10 (il10), NFE2 like bZIP transcription factor 2 (nrf2), superoxide dismutase 2 (sod2) and catalase 1 (cat1) mRNA expression, and also inhibited the activities of glutathione peroxidase (GPX), catalase (CAT), total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC), but increased hexokinase 1 (hk1), glucokinase (gk), 1-phosphofructokinase (pfkb), interleukin 1β (il1β), interleukin 6 (il6), tumor necrosis factor a and β (tnfa, tnfβ) mRNA expression, and malondialdehyde (MDA) content; dietary VD3 group further suppressed gluconeogenic genes (pcxb, pepck) expression, but significantly enhanced the expression of glycolytic genes (hk1, gk, pfkp). Dietary VD3 also alleviated HCD-induced variations of these indices involved in antioxidant and inflammatory responses in yellow catfish intestine. Based on these results, our study indicated that dietary VD3 contributed to alleviate HCD-induced adverse effects on intestinal carbohydrate utilization and health. Our study also elucidated the innovative insights into the interaction of HCD and VD3 in fish, and provided the potential strategies for improving dietary carbohydrate utilization of fish via VD3 addition in aquafeeds. These findings can potentially contribute to cost savings for businesses and serve as a reference for optimizing feed formulations.

JAM3 promotes cervical cancer metastasis by activating the HIF-1α/VEGFA pathway.

Cervical cancer is the fourth most common cancer and the leading cause of mortality among women worldwide. Tumor metastasis is an important cause of poor prognosis. Determining the exact mechanisms of metastasis and potential targeted therapies is urgently needed. Junctional adhesion molecule 3 (JAM3) is an important member of the TJ tight junction (TJ) family, and its biological function in cervical cancer needs to be further clarified. We found that JAM3 was highly expressed in cervical cancer patients with lymph node metastasis and that high expression of JAM3 promoted cervical cancer cell metastasis both in vitro and in vivo. In addition, overexpression of JAM3 induces epithelial-mesenchymal transition (EMT). Moreover, silencing JAM3 suppressed cervical cancer cell migration and invasion in vitro. Finally, JAM3 overexpression activated the HIF-1α/VEGFA pathway. In conclusion, our results suggested that JAM3 promotes cervical cancer cell migration and invasion by activating the HIF-1α/VEGFA pathway. JAM3 may be a promising biomarker and effective therapeutic target for cervical cancer.

Expression of genes related to ileal barrier function in heritage and modern broiler chickens

ABSTRACT 1. An experiment was conducted to determine differences in the expression of genes encoding intestinal barrier proteins between fast, medium and slow-growing chickens. Chicken breeds Athens Canadian Random Bred (ACRB), Longenecker’s Heritage (LHR), RedBro, Hubbard H1 (HH1), Cobb500 and Ross708 were raised from hatch for 35 d. 2. Ileal samples were collected at embryonic day E19 (−2 days post-hatch), hatch and d 7, 14, 21, 28 and 35 post-hatch to assess the expression of genes encoding tight junction proteins (claudins, CLDN; occludin, OCLN; zonula occludens, ZO; and junctional adhesion molecules, JAM), mucin (Muc2), immunoglobulin A (IgA), polymeric immunoglobulin receptor (pIgR) and fatty acid binding protein (FABP2). 3. Expression of CLDN-1 was increased (p < 0.0001) in LHR compared to Cobb500 while CLDN-5 was increased (p < 0.0001) in ACRB, HH1, RedBro and Ross708 compared to LHR as well as in ACRB compared to Cob500. Occludin was upregulated (p = 0.01) in ACRB and LHR compared to Ross708 at d 14 post-hatch. Expression of ZO-1 was upregulated (p = 0.001) in LHR compared to Ross708, HH1 and Cobb500. Tight junction genes, except CLDN-4, JAM-2 and JAM-3 were downregulated (p < 0.0001) at hatch and d 7 post-hatch. Expression of Muc2 was increased (p < 0.0001) in LHR compared to RedBro and from −2 d to d 7 post-hatch. 4. Immunoglobulin A was increased (p = 0.001) in LHR compared to Ross708 and HH1 at −2 d post-hatch and in LHR compared to ACRB, Cobb500 and Ross708 at hatch. In addition, IgA expression was increased in all breeds at d 14 post-hatch while pIgR was upregulated (p = 0.02) in Cobb500 and Ross708 compared to ACRB, HH1, LHR and RedBro at hatch. 5. The gene expression patterns suggest that selection for growth may have not induced changes in junctional complexes and immune defence genes. However, the results confirmed that the expression of these genes is age dependent.

Role of endothelial cell-selective adhesion molecule in the regulation of pulmonary vascular resistance

Endothelial cell-selective adhesion molecule (ESAM) is a member of the endothelial cell junction adhesion molecules family, which plays an important role in the maintenance of endothelial barrier function in the pulmonary circulation. The role of ESAM in affecting pulmonary vascular resistance remains unknown. To examine the role of ESAM in pulmonary vascular resistance, whole lungs were isolated from 16-18 weeks old ESAM knock out (KO) mice and wild type (WT) littermates, ventilated and perfused. We found that pulmonary vascular resistance is significantly increased in the lung of ESAM KO mice compared to WT mice. Bradykinin-induced, endothelium-dependent reduction in the pulmonary vascular resistance was impaired in ESAM KO mice, whereas the direct NO donor, sodium nitroprusside-induced endothelium independent responses remained similar in WT and ESAM KO mice. U46619 mediated increases in pulmonary vascular resistance was significantly higher in ESAM KO mice. Inhibition of the NO synthesis with the L-NAME (0.2 mM for 10 minutes) augmented the U46619-mediated increase in pulmonary vascular resistance in WT mice, but it did not affect the U46619 response in the ESAM KO mice. The mRNA expression of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor, was increased in the lungs of ESAM KO mice, whereas expression of collagen isoforms (type I and type III) and fibronectin were similar in WT and ESAM KO mice. Collectively, we propose that ESAM plays an important role in the maintenance of pulmonary vascular resistance, likely via preserving endothelial nitric oxide production in pulmonary arteries. AHA predoctoral award ID 917057. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Da-Cheng-Qi decoction improves severe acute pancreatitis capillary leakage syndrome by regulating tight junction-associated proteins.

To investigate mechanisms underlying the effects of Da-Cheng-Qi decoction (DCQD) on severe acute pancreatitis (SAP) capillary leakage syndrome. In this study, a SAP rat model was established using retrograde perfusion of 5% sodium taurocholate into the biliopancreatic duct. The study included three randomized groups: control, SAP (modeling), and DCQD (via gavage at 2h pre-modeling and 2 and 4h post-modeling). HPLC was used to analyzed major components of DCQD. Pathological changes and capillary permeability in the rat pancreatic tissues were examined. mRNA levels of claudin 5, occludin, zonula occludin-1 (ZO-1), and junctional adhesion molecules (JAM-C) were assessed using qRT-PCR. Tight junction-associated protein expression was evaluated using immunofluorescence and Western blot analyses. Human umbilical vein endothelial cells (HUVECs) were used to investigate the mechanism m of DCQD. Serum levels of amylase, TNF-α, IL-1β, IL-2, and IL-6 were higher in the SAP group compared to the DCQD group (p < 0.05). DCQD treatment significantly attenuated rat pancreas damage (p < 0.05) and reduced tissue capillary permeability compared to the SAP group (p < 0.05). Claudin 5, occludin, and ZO-1 expression in the rat tissues was upregulated, but JAM-C was downregulated by DCQD treatment (p < 0.05). HUVEC permeability was improved by DCQD in a dose-time-dependent manner compared to the SAP group (p < 0.05). DCQD also upregulated claudin 5, occludin, and ZO-1 expression in vitro (p < 0.05). DCQD can improve capillary permeability in both in vivo and in vitro models of SAP by upregulating expression of claudin 5, occludin, and ZO-1, but not JAM-C.

Electroacupuncture can Modify Stress, Low-Grade Inflammation in the Duodenum, and Damage to the Intestinal Barrier in Rats with Functional Dyspepsia through the CRF Signaling Pathway

In the domain of functional gastrointestinal disorders, Functional Dyspepsia (FD) stands out due to its widespread occurrence internationally. Historically, electroacupuncture (EA) has been employed as a therapeutic modality for FD, demonstrating notable clinical efficacy. This research aimed to delve into the impact of EA on stress responses, minor duodenal inflammatory processes, and the integrity of the intestinal barrier within FD-affected rodent models while also elucidating the underlying mechanisms. Thirty-six male Wistar rats were evenly distributed into three cohorts: a normal, a modeled FD, and an EA treatment group. The FD condition in the rats, barring those in the normal, was induced through a series of multifactorial procedures. For the EA cohort, the rats received electroacupuncture at the acupoints RN12 (Zhongwan) and ST36 (Zusanli) for 20 minutes daily over a span of one week. The gastric residue rate (GRR), intestinal propulsion rate (IPR), and changes in emotional state were measured in each group of rats. Additionally, serum levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) were detected, and the duodenal inflammatory condition and intestinal mucosal barrier status were observed through staining and fluorescence. The expression levels of Claudin-1, Junctional Adhesion Molecule 1 (JAM-1), Corticotropin-Releasing Factor (CRF), and Corticotropin-Releasing Factor Receptor 1 (CRF-R1) were also detected. The study demonstrated that EA had a positive effect on body weight and food intake, GRR, and IPR in FD rats. Additionally, the EA group showed a decrease in serum levels of CRH, ACTH, and CORT, as well as a decrease in the number of duodenal mast cells and tryptase content. Furthermore, the expression of tight junction proteins Claudin-1 and JAM-1 was increased in the EA group compared to the model group. EA also reduced the levels of CRF and CRF-R1 in the hypothalamus and duodenum. EA has been shown to improve the stress state of FD rats, inhibit the activation of mast cells in the duodenum, and reduce low-grade inflammatory response and damage to the intestinal mucosal barrier. It is believed that EA achieves these effects by modulating the expression of CRF and its receptors in the brain-gut interaction pathway through the CRF signaling pathway. This provides a new approach to treating FD.

Cellular adhesion molecules in drug-naïve and previously medicated patients with schizophrenia-spectrum disorders

BackgroundEndothelial inflammation may be involved in the pathogenesis of schizophrenia, and cellular adhesion molecules (CAMs) on endothelial cells may facilitate leukocyte binding and transendothelial migration of cells and inflammatory factors. The aim of the present study was to assess levels of soluble cellular adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule (MADCAM), junctional adhesion molecule (JAM-A) and neural cadherin (N-CAD) in patients with schizophrenia compared to healthy controls. MethodsThe study population consists of 138 patients with schizophrenia-spectrum disorder, of whom 54 were drug-naïve, compared to 317 general population controls. The potential confounders age, gender, smoking and body mass index (BMI) were adjusted for in linear regression models. ResultsThe total patient group showed significantly higher levels of ICAM-1 (p < 0.001) and VCAM-1 (p < 0.001) compared to controls. Previously medicated patients showed higher ICAM-1 levels compared to drug-naïve patients (p = 0.042) and controls (p < 0.001), and elevated VCAM-1 levels compared to controls (p < 0.001). Drug-naive patients had elevated levels of VCAM-1 (p = 0.031) compared to controls. ConclusionsIn our study, patients with schizophrenia – including the drug-naïve – have higher levels of soluble CAMs compared to healthy controls. These findings suggest activation of the endothelial system as in inflammation.

Abstract 2025: Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A

Abstract Pancreatic tumors display an abundance of cancer-associated fibroblasts (CAFs), which negatively affect prognosis and therapy response. Oncolytic virotherapy exploits viruses that preferentially lyse epithelial cancer cells as opposed to normal cells. Interestingly, we have observed that oncolytic reoviruses are able to infect and lyse CAFs, in addition to epithelial cancer cells. Targeting CAFs, in addition to cancer cells, could be advantageous to increase therapy effectiveness. It could serve as a conduit for viral spread and simultaneously disrupt the desmoplastic barrier around tumors, thereby also accelerating the influx of other therapeutics and immune cells. We previously found that the proneness of CAFs to lysis by oncolytic reovirus correlates with the cell surface expression levels of the reovirus entry receptor junction adhesion molecule A (JAM-A). However, most pancreatic CAFs do not express JAM-A. Therefore, a genome-wide CRISPR/Cas9 screen was employed to identify the genes regulating JAM-A expression on fibroblasts, which can subsequently be targeted to sensitize CAFs to reovirus. Pancreatic stellate cells with a moderate JAM-A expression level were transduced with a gRNA library making a knockout of one gene per cell. The highest and lowest JAM-A expressing cells were sorted and sequenced to identify the gRNAs that regulate JAM-A expression. Clonal CRISPR/Cas9-generated knockouts of a top negative regulator were generated and infected with reovirus, followed by cell viability assays to quantify their susceptibilities to reovirus-induced cell death. F11R, the gene encoding JAM-A, was identified as the top positive regulator of JAM-A expression in the CRISPR/Cas9 screen, verifying the validity of the screen. The top negative regulators identified were Fibroblast Growth Factor Receptor 1 (FGFR1) and Zinc finger E-box Binding homeobox 1 (Zeb1), thereby serving as potential therapeutic targets to sensitize CAFs to reovirus treatment. Using clonal Zeb1 knock-outs, Zeb1 was confirmed as a strong regulator of JAM-A expression. Zeb1 knockout in JAM-A negative pancreatic fibroblasts caused a robust upregulation of JAM-A and sensitized these inherently resistant fibroblasts to reovirus-directed cytolysis. Additionally, the clinically approved drug Mocetinostat, previously described to inhibit Zeb1, upregulated JAM-A expression on CAFs and increased cell lysis by reovirus. Altogether, our data show that Zeb1 is a strong negative regulator of JAM-A expression on fibroblasts and that Zeb1 inhibition can sensitize CAFs to reovirus-induced cell death. This research provides a rationale for combining Zeb1 inhibitory drugs with oncolytic reovirus treatment to improve killing of CAFs, which in turn could boost overall tumor eradication. Citation Format: Nicole Dam, Tom J. Harryvan, Bernhard Schmierer, Lukas J.A.C. Hawinkels, Vera Kemp. Zeb1 downregulation sensitizes pancreatic cancer-associated fibroblasts to killing by oncolytic reovirus through upregulation of the reovirus receptor junction adhesion molecule A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2025.

JAM3: A prognostic biomarker for bladder cancer via epithelial-mesenchymal transition regulation.

Understanding the intricate relationship between prognosis, immune function, and molecular markers in bladder cancer (BC) demands sophisticated analytical methods. To identify novel biomarkers for predicting prognosis and immune function in BC patients, we combined weighted gene co-expression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression analysis. This was conducted using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Ultimately, we screened the junctional adhesion molecule 3 (JAM3) as an independent risk factor in BC. High levels of JAM3 were linked to adverse clinical parameters, such as higher T and N stages. Additionally, a JAM3-based nomogram model accurately predicted 1-, 3- and 5-year survival rates of BC patients, indicating potential clinical utility. Functional enrichment analysis revealed that high JAM3 expression activated the calcium signaling pathway, the extracellular matrix (ECM)-receptor interaction, and the PI3K-Akt signaling pathway, and was positively correlated with genes associated with epithelial-mesenchymal transition (EMT). Subsequently, we found that overexpression of JAM3 promoted the migration and invasion abilities in BC cells, regulating the expression levels of N-cadherin, matrix metallopeptidase 2 (MMP2), and Claudin-1 thereby promoting EMT levels. Additionally, we showed that JAM3 was negatively correlated with anti-tumor immune cells such as CD8+ T cells, while positively correlated with pro-tumor immune cells such as M2 macrophages, suggesting its involvement in immune cell infiltration. The immune checkpoint CD200 also showed a positive correlation with JAM3. Our findings revealed that elevated JAM3 levels are predictive of poor prognosis and immune cell infiltration in BC patients by regulating the EMT process.