Expression of Adhesion Molecules in a Gastroduodenal Reflux Murine Model.

BACKGROUND: Various adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), have been shown to play a role in inflammation as well as contribute to tumor progression and prognosis. We hypothesized that gastroduodenal reflux upregulates ICAM-1 and VCAM-1 expression in the distal esophagus, serving as possible early markers of pathologic esophageal disease. METHODS: Normal human esophageal epithelial cells (HET1A), Barrett cells (CPB), and esophageal adenocarcinoma cells (FLO1 and OE33) were treated with deoxycholic acid at increasing concentrations for 24 hours. Adhesion molecule expression was assessed using immunoblotting. A surgical mouse reflux model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and the first portion of the duodenum (duodenum-gastroesophageal anastomosis). Esophageal sections were evaluated using hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence. RESULTS: Deoxycholic acid induced a significant increase in ICAM-1 and VCAM-1 expression in HET1A, CPB, FLO1, and OE33 cells. Animals undergoing duodenum-gastroesophageal anastomosis demonstrated a significant increase in mucosal hyperplasia (P < .0001) and cellular proliferation (P < .0001) compared with control animals. Immunofluorescence and Western blot analysis of the lower esophagus demonstrated significant upregulation of ICAM-1 (P = .005), with no change in VCAM-1 expression (P = .82). CONCLUSIONS: Our results reveal that ICAM-1 and VCAM-1 are upregulated in response to in vitro reflux treatment of normal esophageal epithelial cells. However, our investigation using a mouse reflux model found ICAM-1 is noticeably upregulated without a concomitant increase in VCAM-1. These findings identify ICAM-1, but not VCAM-1, as a potential player in early esophageal disease developing from chronic reflux exposure.

Toll-like Receptor 4 Mediates Reflux-Induced Inflammation in a Murine Reflux Model.

Dysregulation of toll-like receptor (TLR) signaling within the gastrointestinal epithelium has been associated with uncontrolled inflammation and tumorigenesis. We sought to evaluate the role of TLR4 in the development of gastroesophageal reflux-mediated inflammation and mucosal changes of the distal esophagus. Verified human esophageal Barrett's cells with high grade dysplasia (CPB) and esophageal adenocarcinoma cells (OE33) were treated with deoxycholic acid for 24 hours. Cells were pretreated with a TLR4-specific inhibitor peptide 2 hours prior to deoxycholic acid treatment. Inflammatory markers were evaluated using immunoblotting and enzyme-linked immunosorbent assay. A surgical reflux mouse model was generated by performing a side-to-side anastomosis between the second portion of the duodenum and the gastroesophageal junction. Control animals underwent laparotomy with incision and closure of the esophagus superior to the gastroesophageal junction (sham procedure). Esophageal sections were evaluated using hematoxylin and eosin staining and immunohistochemistry. Deoxycholic acid increased expression of inflammatory markers including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin 8. Pretreatment with a TLR4 inhibitor significantly decreased deoxycholic acid-induced inflammatory marker expression. C3H/HeNCrl mice demonstrated a significant increase in mucosal hyperplasia and proliferation following DGEA compared to sham procedure. TLR4 mutant mice (C3H/HeJ) undergoing DGEA demonstrated an attenuated hyperplastic and proliferative response compared to C3H/HeNCrl mice. Inhibition of TLR4 signaling attenuates reflux-induced inflammation in vivo. These findings identify TLR4 inhibition as a potential therapeutic target to halt the progression of pathologic esophageal changes developing in the setting of chronic gastroesophageal reflux disease.

Single-cell RNA-seq reveals a critical role of novel pro-inflammatory EndMT in mediating adverse remodeling in coronary artery-on-a-chip.

A three-dimensional microengineered human coronary artery-on-a-chip was developed for investigation of the mechanism by which low and oscillatory shear stress (OSS) induces pro-atherogenic changes. Single-cell RNA sequencing revealed that OSS induced distinct changes in endothelial cells (ECs) including pro-inflammatory endothelial-to-mesenchymal transition (EndMT). OSS promoted pro-inflammatory EndMT through the Notch1/p38 MAPK-NF-κB signaling axis. Moreover, OSS-induced EC phenotypic changes resulted in proliferation and extracellular matrix (ECM) protein up-regulation in smooth muscle cells (SMCs) through the RANTES-mediated paracrine mechanism. IL-37 suppressed OSS-induced pro-inflammatory EndMT and thereby abrogated SMC proliferation and ECM protein remodeling. Overall, this study provides insights into endothelial heterogeneity under atheroprone shear stress and identifies the mechanistic role of a novel EC subtype in promoting adverse vascular remodeling. Further, this study demonstrates that anti-inflammatory approach is capable of mitigating vascular pathobiology evoked by atheroprone shear stress.

Simvastatin down-regulates osteogenic response in cultured human aortic valve interstitial cells.

BACKGROUND: Aortic valve interstitial cells have been implicated in the pathogenesis of aortic stenosis. In response to proinflammatory stimuli, aortic valve interstitial cells undergo an osteogenic phenotypic change. The purpose of this study was to determine whether the anti-inflammatory effects of statins prevent osteogenic activity in cultured aortic valve interstitial cells. METHODS: Human aortic valve interstitial cells were isolated from hearts explanted for cardiac transplantation. To test whether simvastatin down-regulates TLR4-induced osteogenic response, aortic valve interstitial cells were treated with simvastatin with and without TLR4 agonist lipopolysaccharide (LPS), and osteogenic markers were measured. Simvastatin's influence on in vitro calcium deposition was assessed by alizarin red staining. Knockdown of postreceptor signaling proteins (MyD88 and TRIF) was performed to determine which of 2 TLR4-associated pathways mediates the osteogenic response. Expression levels of TLR4-induced nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and TLR4 expression were assessed after treatment with simvastatin. Statistical testing was done by analysis of variance (P < .05). RESULTS: Simvastatin decreased LPS-induced ALP and Runx2 expression and inhibited in vitro calcium deposition in aortic valve interstitial cells. Knockdown of MyD88 and TRIF attenuated the osteogenic response. Simvastatin attenuated TLR4-dependent NF-κB signaling and down-regulated TLR4 levels. CONCLUSIONS: Simvastatin prevented TLR4-induced osteogenic phenotypic changes in isolated aortic valve interstitial cells via down-regulation of TLR4 and inhibition of NF-κB signaling. These data offer mechanistic insight into a possible therapeutic role for simvastatin in the prevention of aortic stenosis.

Secretory Phospholipase A2 Inhibition Attenuates Adhesive Properties of Esophageal Barrett's Cells.

BACKGROUND: Gastroesophageal reflux and Barrett's esophagus are significant risk factors for the development of esophageal adenocarcinoma. Group IIa secretory phospholipase A2 (sPLA2) catalyzes the production of various proinflammatory metabolites and plays a critical role in promoting reflux-induced inflammatory changes within the distal esophagus. We hypothesized that inhibition of sPLA2 in human Barrett's cells would attenuate adhesion molecule expression via decreased activation of nuclear factor kappa B (NF-κB) and decrease cell proliferation, possibly mitigating the invasive potential of Barrett's esophagus. MATERIALS AND METHODS: Normal human esophageal epithelial cells (HET1A) and Barrett's cells (CPB) were assayed for baseline sPLA2 expression. CPB cells were treated with a specific inhibitor of sPLA2 followed by tumor necrosis factor-α. Protein expression was evaluated using immunoblotting. Cell proliferation was assessed using an MTS cell proliferation assay kit. Statistical analysis was performed using the Student's t-test or analysis of variance, where appropriate. RESULTS: CPB cells demonstrated higher baseline sPLA2 expression than HET1A cells (P = 0.0005). Treatment with 30 µM sPLA2 inhibitor significantly attenuated intercellular adhesion molecule-1 (P = 0.004) and vascular cell adhesion molecule-1 (P < 0.0001) expression as well as decreased NF-κB activation (P = 0.002). sPLA2 inhibition decreased cell proliferation in a dose-dependent manner (P < 0.001 for 15, 20, and 30 µM doses). CONCLUSIONS: sPLA2 inhibition in human Barrett's cells decreases cellular adhesive properties and NF-κB activation as well as decreases cell proliferation, signifying downregulation of the inflammatory response and possible attenuation of cellular malignant potential. These findings identify sPLA2 inhibition as a potential chemopreventive target for premalignant lesions of the esophagus.

Wnt Signaling Mediates Pro-Fibrogenic Activity in Human Aortic Valve Interstitial Cells.

BACKGROUND: Proinflammatory activation of toll-like receptor-4 (TLR4) drives phenotypic changes in aortic valve interstitial cells (AVICs) and produces a fibrogenic phenotype that mediates valvular fibrosis and contributes to aortic stenosis. Prior work identified upregulated Wnt signaling in AVICs taken from valves affected by aortic stenosis. Our purpose was to determine the contribution of Wnt signaling to TLR4-dependent fibrogenic activity in isolated human AVICs. METHODS: Human AVICs were isolated from hearts explanted for cardiac transplantation (N = 4). To test whether Wnt signaling contributed to TLR4-dependent fibrogenic activity, AVICs were treated with Wnt inhibitor (Dkk1) prior to TLR4 activation (LPS) and fibrogenic markers assessed. To determine the mediator of TLR4-to-Wnt signaling, expression of the key Wnt ligand, Wnt3a, was assessed after TLR4 activation and neutralizing antibodies confirmed the identity of the mediator. Fibrogenic activity was assessed after AVICs were treated with recombinant Wnt3a. Statistics were by analysis of variance (P < .05). RESULTS: TLR4 activation upregulated in vitro collagen deposition, type IV collagen and MMP2 expression, and Dkk1 inhibited these responses (P < .05). Expression of Wnt3a was upregulated after TLR4 activation (P < .05). Anti-Wnt3a neutralizing antibodies abrogated TLR4-dependent type IV collagen and MMP2 expression (P < .05). Wnt3a upregulated type IV collagen and MMP2 expression independent of TLR4 activation (P < .05). CONCLUSIONS: This study found that TLR4-dependent fibrogenic activity was mediated through Wnt signaling. The mediator of profibrogenic TLR4-to-Wnt signaling was a key Wnt ligand, Wnt3a. The abrogation of TLR4-induced fibrogenic activity in human AVICs by Wnt blockade illustrates a potential therapeutic role for Wnt inhibition in treatment and/or prevention of aortic stenosis.

Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level.

Background and Objectives: Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication. Methods and Results: AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression. Conclusions: This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.

Overexpression of connective tissue growth factor is associated with tumor progression and unfavorable prognosis in endometrial cancer.

OBJECTIVES: This study was to explore the prognostic value of connective tissue growth factor (CTGF) expression in endometrial cancer (EC). METHODS: We compared CTGF expression in 198 samples from patients with endometrial cancer and 50 samples from patients with healthy endometrial tissues as determined by immunohistochemistry. RESULTS: Expression of CTGF was significantly higher in endometrial cancers as compared to normal endometrial tissues. Positive CTGF expression displayed a strong association with CA125 level, histological grade, depth of myometrial invasion and the International Federation of Gynecology and Obstetrics (FIGO) stage. Our findings revealed histological grade, depth of myometrial invasion, FIGO stage, vascular/lymphatic invasion, and the CTGF expression are related to 5-year survival in patients with endometrial cancer. Positive CTGF expression, lymph node status, as well as vascular/lymphatic invasion, were identified as independent prognostic factors in endometrial cancer. CONCLUTIONS: Over-expression of CTGF is an independent prognostic factor that will allow the successful differentiation of high-risk population from the group of patients with stage III-IV endometrial cancer. The up-regulation of CTGF may contribute to the progression of endometrial cancer and serve as a new prognostic biomarker in patients with endometrial cancer survival.

Predictors of Acute Kidney Injury Following Aortic Arch Surgery.

BACKGROUND: Acute kidney injury (AKI) following open aortic arch surgery is a frequent complication associated with increased morbidity and mortality. The primary purpose of this study was to evaluate risk factors for postoperative AKI in patients who underwent open aortic arch surgery utilizing hypothermic circulatory arrest (HCA). MATERIALS AND METHODS: Included were 295 patients undergoing surgery between January 2011 and March 2018. AKI was defined according to Kidney Disease: Improving Global Outcomes guidelines. Preoperative and intraoperative variables were stratified by no AKI versus any AKI, and bivariate analysis was performed. Multivariable logistic regression analysis used statistically and clinically significant characteristics from the bivariate analysis. RESULTS: Of the 295 patients, 93 (32%) developed AKI. In the bivariate analysis, significant predictors of AKI included the following: history of hypertension (P < 0.001), diabetes (P = 0.03), operative urgency (P = 0.009), cardiopulmonary bypass (CPB) time (P < 0.0001), HCA time (0.02), total intraoperative transfusions (P = 0.002), and concomitant procedures (coronary artery bypass grafting, or mitral/tricuspid interventions, P = 0.0009). In the multivariable analysis, significant predictors of AKI were history of hypertension (P = 0.03) and CPB time (P = 0.02). Age, operative urgency, circulatory arrest time, and any intraoperative transfusion were not significant in the multivariable analysis. CONCLUSION: In conclusion, given that CPB time is the only modifiable risk factor identified in the analysis, approaches to reducing bypass time should continue to be the focus of decreasing risk for postoperative AKI in HCA cases.

Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection.

Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff. Methods. Escherichia coli was incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015. In vitro techniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff. Results. Mean bacterial colony formation in PBS was greater for E. coli incubated in the presence of GTM (1.48 × 10(7) CFU/mL) versus without (9.95 × 10(5) CFU/mL) following 20-hour incubation (p = 0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) with Enterococcus faecalis. Conclusion. In vitro experiments support a facilitating role of GTM on colony formation of E. coli in PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence of E. faecalis, but results were inconclusive. Further studies are recommended.

Comparative Surgical Outcomes for Endometrial Cancer Patients 65 Years Old or Older Staged With Robotics or Laparotomy.

BACKGROUND: This study aimed to assess the safety of robotic surgery for older women undergoing surgery for endometrial cancer. METHODS: A retrospective chart review of women undergoing surgery for endometrial cancer between October 2010 and December 2012 was conducted at the authors' institution. This cohort was divided by age (≥65 vs <65 years) and surgical approach (laparotomy vs robotic surgery). Postoperative morbidity and mortality were compared using standard statistical analysis. RESULTS: Of 228 patients identified, 73 (32 %) were 65 years old or older, and 98 (43 %) had undergone robotic surgery. Among the robotic surgery patients, women 65 years old or older had a higher Charlson comorbidity score (7.6 vs 4.9; p < 0.01) and were more likely to undergo pelvic lymphadenectomy (73 vs 39 %; p < 0.01). The complication rates did not differ between the groups except for increased urinary retention in the older group (15 % vs 3 %; p = 0.04). Older patients had a longer hospital stay (2.2 vs 1.3 days; p < 0.01) and a similar rate of discharge home (100 vs 96 %; p = 0.09). For the patients 65 years old or older, robotic surgery was associated with less blood loss (131 vs 235 ml; p = 0.03), a lower rate of ileus (0 vs 15 %; p = 0.04), a lower perioperative surgical complication rate (4 vs 30 %; p = 0.01), a shorter hospital stay (2.2 vs 4.4 days; p < 0.01), and a similar rate of discharge home (96 vs 91 %; p = 0.45) compared with laparotomy. CONCLUSION: Robotic surgery appears to be associated with less postoperative morbidity than laparotomy for endometrial cancer staging in women 65 years old or older. The complication rates after robotic surgery were similar between the two age groups.