Endoscopic ultrasound-guided gallbladder drainage as a first approach for jaundice palliation in unresectable malignant distal biliary obstruction: Prospective study.

OBJECTIVES: Endoscopic retrograde cholangiopancreatography (ERCP) represents the gold standard for jaundice palliation in patients with distal malignant biliary obstruction (DMBO). Biliary drainage using electrocautery lumen apposing metal stent (EC-LAMS) is currently a well-established procedure when ERCP fails. In a palliative setting the endoscopic ultrasound-guided gallbladder drainage (EUS-GBD) could represent an easy and valid option. We performed a prospective study with a new EC-LAMS with the primary aim to assess the clinical success rate of EUS-GBD as a first-line approach to the palliation of DMBO. METHODS: In all, 37 consecutive patients undergoing EUS-GBD with a new EC-LAMS were prospectively enrolled. Clinical success was defined as bilirubin level decrease >15% within 24 h and >50% within 14 days after EC-LAMS placement. RESULTS: The mean age was 73.5 ± 10.8 years; there were 17 male patients (45.9%). EC-LAMS placement was technically feasible in all patients (100%) and the clinical success rate was 100%. Four patients (10.8%) experienced adverse events, one bleeding, one food impaction, and two cystic duct obstructions because of disease progression. No stent-related deaths were observed. The mean hospitalization was 7.7 ± 3.4 days. Median overall survival was 4 months (95% confidence interval 1-8). CONCLUSION: Endoscopic ultrasound-guided gallbladder drainage with the new EC-LAMS is a valid option in palliative endoscopic biliary drainage as a first-step approach in low survival patients with malignant jaundice unfit for surgery. A smaller diameter EC-LAMS should be preferred, particularly if the drainage is performed through the stomach, to avoid potential food impaction, which could result in stent dysfunction.

Considerations for the Bioengineering of Advanced Cardiac In Vitro Models of Myocardial Infarction.

Despite the latest advances in cardiovascular biology and medicine, myocardial infarction (MI) remains one of the major causes of deaths worldwide. While reperfusion of the myocardium is critical to limit the ischemic damage typical of a MI event, it causes detrimental morphological and functional changes known as "reperfusion injury." This complex scenario is poorly represented in currently available models of ischemia/reperfusion injury, leading to a poor translation of findings from the bench to the bedside. However, more recent bioengineered in vitro models of the human heart represent more clinically relevant tools to prevent and treat MI in patients. These include 3D cultures of cardiac cells, the use of patient-derived stem cells, and 3D bioprinting technology. This review aims at highlighting the major features typical of a heart attack while comparing current in vitro, ex vivo, and in vivo models. This information has the potential to further guide in developing novel advanced in vitro cardiac models of ischemia/reperfusion injury. It may pave the way for the generation of advanced pathophysiological cardiac models with the potential to develop personalized therapies.

Proinflammatory Interleukin-33 Induces Dichotomic Effects on Cell Proliferation in Normal Gastric Epithelium and Gastric Cancer.

Interleukin (IL)-33 is a member of the interleukin (IL)-1 family of cytokines linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has a direct effect on human gastric epithelial cells (GES-1), the human gastric adenocarcinoma cell line (AGS), and the gastric carcinoma cell line (NCI-N87) by assessing its role in the regulation of cell proliferation, migration, cell cycle, and apoptosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assays, migration by wound healing assay, and apoptosis by caspase 3/7 activity assay and annexin V assay. Cell cycle was analyzed by means of propidium iodine assay, and gene expression regulation was assessed by RT-PCR profiling. We found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell lines, and it can stimulate proliferation and reduce apoptosis in normal epithelial cell lines. These effects were also confirmed by the analysis of cell cycle gene expression, which showed a reduced expression of pro-proliferative genes in cancer cells, particularly in genes involved in G0/G1 and G2/M checkpoints. These results were confirmed by gene expression analysis on bioptic and surgical specimens. The aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell-type-dependent manner.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis.

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFß1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFß1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFß1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFß1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

Current Technologies Based on the Knowledge of the Stem Cells Microenvironments.

The stem cell microenvironment or niche plays a critical role in the regulation of survival, differentiation and behavior of stem cells and their progenies. Recapitulating each aspect of the stem cell niche is therefore essential for their optimal use in in vitro studies and in vivo as future therapeutics in humans. Engineering of optimal conditions for three-dimensional stem cell culture includes multiple transient and dynamic physiological stimuli, such as blood flow and tissue stiffness. Bioprinting and microfluidics technologies, including organs-on-a-chip, are among the most recent approaches utilized to replicate the three-dimensional stem cell niche for human tissue fabrication that allow the integration of multiple levels of tissue complexity, including blood flow. This chapter focuses on the physico-chemical and genetic cues utilized to engineer the stem cell niche and provides an overview on how both bioprinting and microfluidics technologies are improving our knowledge in this field for both disease modeling and tissue regeneration, including drug discovery and toxicity high-throughput assays and stem cell-based therapies in humans.

VEGF-mediated phosphorylation of eNOS regulates angioblast and embryonic endothelial cell proliferation.

To evaluate potential roles of nitric oxide (NO) in the regulation of the endothelial lineage and neovascular processes (vasculogenesis and angiogenesis) we evaluated endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) expression in 7.2-8.5 days post-coitum (dpc) mouse embryos. Analysis revealed that p-eNOS((S1177)) but not P-eNOS((S617)) or P-eNOS((T495)) was expressed in a subpopulation of angioblasts (TAL-1(+)/Flk-1(+)/CD31(-)/CD34(-)/VE-Cadherin(-)) at 7.2 dpc. A role of the VEGF/Akt1/eNOS signaling pathway in the regulation of the endothelial cell (EC) lineage was suggested by the strong correlation observed between cell division and p-eNOS((S1177)) expression in both angioblasts and embryonic endothelial cells (EECs, TAL-1(+)/Flk-1(+)/CD31(+)/CD34(+)/VE-Cadherin(+)). Our studies using Akt1 null mouse embryos show a reduction in p-eNOS((S1177)) expression in angioblast and EECs that is correlated with a decrease in endothelial cell proliferation and results in changes in VEGF-induced vascular patterning. Further, we show that VEGF-mediated cell proliferation in Flk-1(+) cells in allantoic cultures is decreased by pharmacological inhibitors of the VEGF/Akt1/eNOS signaling pathways. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation on Ser1177 regulates angioblast and EEC division, which underlies the formation of blood vessels and vascular networks.

Protective role of acetylcholine and the cholinergic system in the injured heart.

This review explores the roles of the cholinergic system in the heart, comprising the neuronal and non-neuronal cholinergic systems. Both systems are essential for maintaining cardiac homeostasis by regulating the release of acetylcholine (ACh). A reduction in ACh release is associated with the early onset of cardiovascular diseases (CVDs), and increasing evidence supports the protective roles of ACh against CVD. We address the challenges and limitations of current strategies to elevate ACh levels, including vagus nerve stimulation and pharmacological interventions such as cholinesterase inhibitors. Additionally, we introduce alternative strategies to increase ACh in the heart, such as stem cell therapy, gene therapy, microRNAs, and nanoparticle drug delivery methods. These findings offer new insights into advanced treatments for regenerating the injured human heart.

Evolving Concepts of the SCORE System: Subtracting Cholesterol from Risk Estimation: A Way for a Healthy Longevity?

The role of cholesterol, mainly low-density lipoproteins (LDL-C), as a causal risk factor for atherosclerotic cardiovascular disease (ASCVD) is now established and accepted by the international scientific community. Based on this evidence, the European and American guidelines recommend early risk stratification and "rapid" achievement of the suggested target according to the risk estimation to reduce the number of major cardiovascular events. Prolonged exposure over the years to high levels of LDL-C is one of the determining factors in the development and progression of atherosclerotic plaque, on which the action of conventional risk factors (cigarette smoking, excess weight, sedentary lifestyle, arterial hypertension, diabetes mellitus) as well as non-conventional risk factors (gut microbiota, hyperuricemia, inflammation), alone or in combination, favors the destabilization of the atherosclerotic lesion with rupture/fissuration/ulceration and consequent formation of intravascular thrombosis, which leads to the acute clinical manifestations of acute coronary syndromes. In the current clinical practice, there is a growing number of cases that, although extremely common, are emblematic of the concept of long-term exposure to the risk factor (LDL hypercholesterolemia), which, not adequately controlled and in combination with other risk factors, has favored the onset of major cardiovascular events. The triple concept of "go lower, start earlier and keep longer!" should be applied in current clinical practice at any level of prevention. In the present manuscript, we will review the current evidence and documents supporting the causal role of LDL-C in determining ASCVD and whether it is time to remove it from any score.

Chlorella-enriched hydrogels protect against myocardial damage and reactive oxygen species production in anin vitroischemia/reperfusion model using cardiac spheroids.

Microalgae have emerged as promising photosynthetic microorganisms for biofabricating advanced tissue constructs, with improved oxygenation and reduced reactive oxygen species (ROS) production. However, their use in the engineering of human tissues has been limited due to their intrinsic growth requirements, which are not compatible with human cells. In this study, we first formulated alginate-gelatin (AlgGel) hydrogels with increasing densities ofChlorella vulgaris. Then, we characterised their mechanical properties and pore size. Finally, we evaluated their effects on cardiac spheroid (CS) pathophysiological response under control and ischemia/reperfusion (I/R) conditions. Our results showed that the addition ofChlorelladid not affect AlgGel mechanical properties, while the mean pore size significantly decreased by 35% in the presence of the 107cells ml-1microalgae density. Under normoxic conditions, the addition of 107Chlorellacells ml-1significantly reduced CS viability starting from 14 d in. No changes in pore size nor CS viability were measured for hydrogels containing 105and 106Chlorellacells ml-1. In our I/R model, allChlorella-enriched hydrogels reduced cardiac cell sensitivity to hypoxic conditions with a corresponding reduction in ROS production, as well as protected against I/R-induced reduction in cell viability. Altogether, our results support a promising use ofChlorella-enriched Alg-Gel hydrogels for cardiovascular tissue engineering.

Correction: Outcomes of lumen apposing metal stent placement in patients with surgically altered anatomy: Multicenter international experience.

[This corrects the article DOI: 10.1055/a-2411-1814.].

Outcomes of lumen apposing metal stent placement in patients with surgically altered anatomy: Multicenter international experience.

Background and study aims Although outcomes of lumen-apposing metal stents (LAMS) placement in native anatomy have been reported, data on LAMS placement in surgically altered anatomy (SAA) are sparse. We aimed to assess outcomes of LAMS placement in patients with SAA for different indications. Patients and methods This was an international, multicenter, retrospective, observational study at 25 tertiary care centers through November 2023. Consecutive patients with SAA who underwent LAMS placement were included. The primary outcome was technical success defined as correct placement of LAMS. Secondary outcomes were clinical success and safety. Results Two hundred and seventy patients (125 males; average age 61 ± 15 years) underwent LAMS placement with SAA. Procedures included EUS-directed transgastric ERCP (EDGE) and EUS-directed transenteric ERCP (EDEE) (n = 82), EUS-guided entero-enterostomy (n = 81), EUS-guided biliary drainage (n = 57), EUS-guided drainage of peri-pancreatic fluid collections (n = 48), and EUS-guided pancreaticogastrostomy (n = 2). Most cases utilized AXIOS stents (n = 255) compared with SPAXUS stents (n = 15). Overall, technical success was 98%, clinical success was 97%, and the adverse event (AE) rate was 12%. Using AGREE classification, five events were rated as Grade II, 21 events as Grade IIIa, and six events as IIIb. No difference in AEs were noted among stent types ( P = 0.52). Conclusions This study shows that placement of LAMS is associated with high technical and clinical success rates in patients with SAA. However, the rate of AEs is noteworthy, and thus, these procedures should be performed by expert endoscopists at tertiary centers.

Photosynthetic microorganisms for the oxygenation of advanced 3D bioprinted tissues.

3D bioprinting technology has emerged as a tool that promises to revolutionize the biomedical field, including tissue engineering and regeneration. Despite major technological advancements, several challenges remain to be solved before 3D bioprinted tissues could be fully translated from the bench to the bedside. As oxygen plays a key role in aerobic metabolism, which allows energy production in the mitochondria; as a consequence, the lack of tissue oxygenation is one of the main limitations of current bioprinted tissues and organs. In order to improve tissue oxygenation, recent approaches have been established for a broad range of clinical applications, with some already applied using 3D bioprinting technologies. Among them, the incorporation of photosynthetic microorganisms, such as microalgae and cyanobacteria, is a promising approach that has been recently explored to generate chimerical plant-animal tissues where, upon light exposure, oxygen can be produced and released in a localized and controlled manner. This review will briefly summarize the state-of-the-art approaches to improve tissue oxygenation, as well as studies describing the use of photosynthetic microorganisms in 3D bioprinting technologies. STATEMENT OF SIGNIFICANCE: 3D bioprinting technology has emerged as a tool for the generation of viable and functional tissues for direct in vitro and in vivo applications, including disease modeling, drug discovery and regenerative medicine. Despite the latest advancements in this field, suboptimal oxygen delivery to cells before, during and after the bioprinting process limits their viability within 3D bioprinted tissues. This review article first highlights state-of-the-art approaches used to improve oxygen delivery in bioengineered tissues to overcome this challenge. Then, it focuses on the emerging roles played by photosynthetic organisms as novel biomaterials for bioink generation. Finally, it provides considerations around current challenges and novel potential opportunities for their use in bioinks, by comparing latest published studies using algae for 3D bioprinting.

Physically-based simulation of elastic-plastic fusion of 3D bioprinted spheroids.

Spheroids are microtissues containing cells organized in a spherical shape whose diameter is usually less than a millimetre. Depending on the properties of the environment they are placed in, some nearby spheroids spontaneously fuse and generate a tissue. Given their potential to mimic features typical of body parts and their ability to assemble by fusing in permissive hydrogels, they have been used as building blocks to 3D bioprint human tissue parts. Parameters controlling the shape and size of a bioprinted tissue using fusing spheroid cultures include cell composition, hydrogel properties, and their relative initial position. Hence, simulating, anticipating, and then controlling the spheroid fusion process is essential to control the shape and size of the bioprinted tissue. This study presents the first physically-based framework to simulate the fusion process of bioprinted spheroids. The simulation is based on elastic-plastic solid and fluid continuum mechanics models. Both models use the 'smoothed particle hydrodynamics' method, which is based on discretizing the continuous medium into a finite number of particles and solving the differential equations related to the physical properties (e.g. Navier-Stokes equation) using a smoothing kernel function. To further investigate the effects of such parameters on spheroid shape and geometry, we performed sensitivity and morphological analysis to validate our simulations within-vitrospheroids. Through ourin-silicosimulations by changing the aforementioned parameters, we show that the proposed models appropriately simulate the range of the elastic-plastic behaviours ofin-vitrofusing spheroids to generate tissues of desired shapes and sizes. Altogether, this study presented a physically-based simulation that can provide a framework for monitoring and controlling the geometrical shape of spheroids, directly impacting future research using spheroids for tissue bioprinting.

Long-term endoscopic surveillance in HBV compensated cirrhotic patients treated with Tenofovir or Entecavir for 11 years.

BACKGROUND: Long-term administration of TDF/ETV in patients with HBV-related compensated cirrhosis reduces HCC and decompensation events but the effect of this regimen on development/regression of oesophageal varices (EV) is currently unknown. AIM: To assess the risk of EV development/progression in this population. METHODS: A total of 186 Caucasian HBV-monoinfected compensated cirrhotics were enrolled in a long-term cohort study from TDF/ETV introduction. Upper GI endoscopies were performed according to Baveno recommendations. Primary endpoint was development/progression of oesophageal/gastric varices over time. RESULTS: At TDF/ETV start, median age was 61 years, 80% males, 60% HBV-DNA undetectable, 63% NUCs previously exposed, 73% normal ALT, 40% platelets <150,000/mmc and 25 (13%) with low-risk varices (LRV). During 11 years of antiviral therapy and 666 endoscopies performed, 9 patients either developed or had a progression of oesophageal or gastric varices with an 11-year cumulative probability of 5.1% (95% CI 3-10%); no patient bled. Out of 161 patients without EV at baseline, the 11-year probably was 4.5% with all varices developing within the first six years of treatment. In 25 patients with LRV at baseline, the 11-year probability of progression or regression was 9.3% and 58%, respectively. Only baseline platelet count (HR 0.96, p = 0.028) was associated with LRV development at multivariate analysis: platelet ≤90,000/mmc (AUROC 0.70) had 98.1% specificity, 42.9% sensitivity, 50% PPV for LRV onset. CONCLUSIONS: In compensated cirrhotic patients under long-term effective TDF/ETV treatment, the 11-year risk of developing/progressing EV is negligible, thus challenging the current endoscopic surveillance recommendations in patients without EV at baseline.

New Perspectives in Endoscopic Treatment of Gastroesophageal Reflux Disease.

Gastroesophageal reflux disease has a high incidence and prevalence in the general population. Clinical manifestations are heterogenous, and so is the response to medical treatment. Proton pump inhibitors are still the most common agents used to control reflux symptoms and for healing esophagitis, but they are not a one-size-fits-all solution for the disease. Patients with persistent troublesome symptoms despite medical therapy, those experiencing some adverse drug reaction, or those unwilling to take lifelong medications deserve valid alternatives. Anti-reflux Nissen fundoplication is an effective option, but the risk of adverse events has limited its spread. In recent years, advancements in therapeutic endoscopy have been made, and three major endoluminal alternatives are now available, including (1) the delivery of radiofrequency energy to the esophago-gastric junction, (2) transoral incisionless fundoplication (TIF), and (3) anti-reflux mucosal interventions (ARMI) based on mucosal resection (ARMS) and mucosal ablation (ARMA) techniques to remodel the cardia. Endoscopic techniques have shown interesting results, but their diffusion is still limited to expert endoscopists in tertiary centers. This review discusses the state of the art in the endoscopic approach to gastroesophageal reflux disease.

The Use of a New Dedicated Electrocautery Lumen-Apposing Metal Stent for Gallbladder Drainage in Patients with Acute Cholecystitis.

Aims: Lumen-apposing metal stents (LAMSs) in ultrasonography-guided gallbladder drainage (EUS-GBD) have become increasingly important for high-risk surgical patients. Our study aims to evaluate the technical and clinical success, safety, and feasibility of endoscopic ultrasonography-guided gallbladder drainage using a new dedicated LAMS. Methods: This is a retrospective multicenter study that included all consecutive patients not suitable for surgery who were referred to a tertiary center for EUS-GBD using a new dedicated electrocautery LAMS for acute cholecystitis at eight different centers. Results: Our study included 54 patients with a mean age of 76.48 years (standard deviation: 12.6 years). Out of the 54 endoscopic gallbladder drainages performed, 24 (44.4%) were cholecysto-gastrostomy, and 30 (55.4%) were cholecysto-duodenostomy. The technical success of LAMS placement was 100%, and clinical success was achieved in 23 out of 30 patients (76.67%). Adverse events were observed in two patients (5.6%). Patients were discharged after a median of 5 days post-stenting. Conclusions: EUS-GBD represents a valuable option for high-surgical-risk patients with acute cholecystitis. This new dedicated LAMS has demonstrated a high rate of technical and clinical success, along with a high level of safety.

In vitro modeling of myocardial ischemia/reperfusion injury with murine or human 3D cardiac spheroids.

Myocardial infarction (MI) is the primary cause of death worldwide, but there are no clinically relevant models to study MI. Here, we describe an ischemia/reperfusion (I/R) injury model typical of MI using mouse or human 3D in vitro cardiac spheroids (CSs). First, we demonstrated the culture and maintenance of CSs. Then, we detailed how to expose CSs to pathophysiological oxygen concentrations to induce I/R injury. The protocol can be used in combination with viability, contractility, and mRNA expression level measurements. For complete details on the use and execution of this protocol, please refer to Sharma et al. (2022).

Taking It Personally: 3D Bioprinting a Patient-Specific Cardiac Patch for the Treatment of Heart Failure.

Despite a massive global preventative effort, heart failure remains the major cause of death globally. The number of patients requiring a heart transplant, the eventual last treatment option, far outnumbers the available donor hearts, leaving many to deteriorate or die on the transplant waiting list. Treating heart failure by transplanting a 3D bioprinted patient-specific cardiac patch to the infarcted region on the myocardium has been investigated as a potential future treatment. To date, several studies have created cardiac patches using 3D bioprinting; however, testing the concept is still at a pre-clinical stage. A handful of clinical studies have been conducted. However, moving from animal studies to human trials will require an increase in research in this area. This review covers key elements to the design of a patient-specific cardiac patch, divided into general areas of biological design and 3D modelling. It will make recommendations on incorporating anatomical considerations and high-definition motion data into the process of 3D-bioprinting a patient-specific cardiac patch.

Fibulin-3 Deficiency Protects Against Myocardial Injury Following Ischaemia/ Reperfusion in in vitro Cardiac Spheroids.

Myocardial infarction (MI, or heart attack) is a leading cause of death worldwide. Myocardial ischaemia reperfusion (I/R) injury typical of MI events is also associated with the development of cardiac fibrosis and heart failure in patients. Fibulin-3 is an extracellular matrix component that plays a role in regulating MI response in the heart. In this study, we generated and compared in vitro cardiac spheroids (CSs) from wild type (WT) and fibulin-3 knockout (Fib-3 KO) mice. These were then exposed to pathophysiological changes in oxygen (O2) concentrations to mimic an MI event. We finally measured changes in contractile function, cell death, and mRNA expression levels of cardiovascular disease genes between WT and Fib-3 KO CSs. Our results demonstrated that there are significant differences in growth kinetics and endothelial network formation between WT and Fib-3 KO CSs, however, they respond similarly to changes in O2 concentrations. Fib-3 deficiency resulted in an increase in viability of cells and improvement in contraction frequency and fractional shortening compared to WT I/R CSs. Gene expression analyses demonstrated that Fib-3 deficiency inhibits I/R injury and cardiac fibrosis and promotes angiogenesis in CSs. Altogether, our findings suggest that Fib-3 deficiency makes CSs resistant to I/R injury and associated cardiac fibrosis and helps to improve the vascular network in CSs.