HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5+)-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation.NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

Selepressin, a novel selective V1A receptor agonist: Effect on mesenteric flow and gastric mucosa perfusion in the endotoxemic rabbit.

Intestinal or mesenteric ischemia generally leads to inflammation and injury, potentially developing hypoxia, causing cell death and tissue necrosis. This in turn can lead to sepsis and shock. Conversely, following shock, the intestinal tract is a main organ to experience ischemic/reperfusion injury. Increased intestinal cell-membrane permeability through mesenteric ischemia provoking bacterial translocation and gut-barrier injury can lead to sepsis and multi-organ failure. Hypotension induced by systemic vasodilation and vascular leak in systemic inflammatory response syndrome and sepsis is countered by immediate fluid resuscitation and vasopressor administration, primarily norepinephrine (NE), with possible arginine vasopressin (AVP) supplementation, an agonist of vasopressin V1A and V2 receptors. Selepressin is a selective V1A-receptor agonist, avoiding potential V2 receptor-associated adverse effects. Selepressin, non-selective AVP, and NE effects on mesenteric blood flow (MBF) and gastric mucosa perfusion (GMP) were compared in control rabbits and a lipopolysaccharide-induced, fluid-resuscitated rabbit endotoxemia model. AVP induced a pronounced decrease in MBF and GMP in non-endotoxemic and endotoxemic rabbits, whereas the reduction after selepressin treatment was significantly less for both indicators in the endotoxemic animals. By contrast, NE increased the MBF and did not affect GMP in both groups. Selepressin and AVP induced a pronounced dose-dependent increase in mesenteric vascular resistance in non-endotoxemic and endotoxemic rabbits, tending to be less in endotoxemic animals, whereas a minor increase in both groups was observed with NE. Therefore, in this safety study, the risk for mesenteric ischemia on selepressin treatment was not inferior to AVP, being less in endotoxemic than in non-endotoxemic animals.

JAK2V617F mutation prevalence on Chilean adults suffering from primary mesenteric and portal venous thromboses.

INTRODUCTION: Mesenteric and portal venous thromboses are rare diseases with high mortality rates and are strongly associated with hepatic cirrhosis, and abdominal inflammatory or tumoral processes, but in some cases can be the first sign of myeloproliferative neoplasm (MPN) or hereditary thrombophilia. JAK2V617F mutation detection is an important diagnostic tool for MPN patients. The aim of this study was to describe the JAK2V617F mutation prevalence on Chilean patients suffering from a primary splanchnic venous thrombosis (SVT), in order to assess how it relates to primary MVT and PVT in our specific population. METHODS: A retrospective observational study was conducted in patients referred to the University of Chile Clinical Hospital with mesenteric and/or portal venous thrombosis diagnosis over a 7-year period. Patients with primary thrombosis underwent hereditary thrombophilia study and JAK2V617F mutation screening. RESULTS: A total of 123 patients had splanchnic venous thrombosis (mesenteric and/or portal) as their main discharge diagnosis. Sixty patients (49%) had primary mesenteric or portal venous thrombosis (no attributable secondary cause). Hereditary thrombophilia and MPN were diagnosed in 21.6% and 43.3% of SVT patients, respectively. Twenty SVT patients remained without an etiologic diagnosis. In MPN patients, almost all had the JAK2V617F mutation (92.3%). About 16% of patients who had positive JAK2V617F mutation did not meet diagnostic criteria for MPN. CONCLUSIONS: In this Chilean cohort, half of mesenteric or portal venous thrombosis showed no secondary cause. In this group, the main causes were MPN and hereditary thrombophilia. Nearly, all MPN patients had JAK2V617F mutation, but there was a group of patients having JAK2V617F mutation but did not meet MPN criteria.

Prothrombin Arg541Trp Mutation Leads to Defective PC (Protein C) Pathway Activation and Constitutes a Novel Genetic Risk Factor for Venous Thrombosis.

OBJECTIVE: Defective PC (protein C) pathway predisposes patients to venous thromboembolism (VTE) and is mostly, but not exclusively, attributed to hereditary PC or PS (protein S) deficiencies and activated PC resistance caused by factor V Leiden mutation. Approach and Results: In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the 3 pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. CONCLUSIONS: In summary, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function.

The role of ischemic preconditioning in the expression of apoptosis-related genes in a rat model of intestinal ischemia-reperfusion injury.

PURPOSE: To analyze the effects of ischemic preconditioning (IPC) in the expression of apoptosis-related genes in rat small intestine subjected to ischemia and reperfusion. METHODS: Thirty anesthetized rats underwent laparotomy and were drive into five groups: control (CG); ischemia (IG); ischemia and reperfusion (IRG); IPC and ischemia (IG+IPC); IPC and ischemia and reperfusion (I/RG+IPC). Intestinal ischemia was performed by clamping the superior mesenteric artery for 60 minutes, whereas reperfusion lasted for 120 minutes. IPC was carried out by one cycle of 5 minutes of ischemia followed by 10 minutes of reperfusion prior to the prolonged 60-minutes-ischemia and 120-minutes-reperfusion. Thereafter, the rats were euthanized and samples of small intestine were processed for histology and gene expression. RESULTS: Histology of myenteric plexus showed a higher presence of neurons presenting pyknotic nuclei and condensed chromatin in the IG and IRG. IG+IPC and I/RG+IPC groups exhibited neurons with preserved volume and nuclei, along with significant up-regulation of the anti-apoptotic protein Bcl2l1 and down-regulation of pro-apoptotic genes. Moreover, Bax/Bcl2 ratio was lower in the groups subjected to IPC, indicating a protective effect of IPC against apoptosis. CONCLUSION: Ischemic preconditioning protect rat small intestine against ischemia/reperfusion injury, reducing morphologic lesions and apoptosis.

The role of ischemic preconditioning in the expression of apoptosis-related genes in a rat model of intestinal ischemia-reperfusion injury

Abstract Purpose: To analyze the effects of ischemic preconditioning (IPC) in the expression of apoptosis-related genes in rat small intestine subjected to ischemia and reperfusion. Methods: Thirty anesthetized rats underwent laparotomy and were drive into five groups: control (CG); ischemia (IG); ischemia and reperfusion (IRG); IPC and ischemia (IG+IPC); IPC and ischemia and reperfusion (I/RG+IPC). Intestinal ischemia was performed by clamping the superior mesenteric artery for 60 minutes, whereas reperfusion lasted for 120 minutes. IPC was carried out by one cycle of 5 minutes of ischemia followed by 10 minutes of reperfusion prior to the prolonged 60-minutes-ischemia and 120-minutes-reperfusion. Thereafter, the rats were euthanized and samples of small intestine were processed for histology and gene expression. Results: Histology of myenteric plexus showed a higher presence of neurons presenting pyknotic nuclei and condensed chromatin in the IG and IRG. IG+IPC and I/RG+IPC groups exhibited neurons with preserved volume and nuclei, along with significant up-regulation of the anti-apoptotic protein Bcl2l1 and down-regulation of pro-apoptotic genes. Moreover, Bax/Bcl2 ratio was lower in the groups subjected to IPC, indicating a protective effect of IPC against apoptosis. Conclusion: Ischemic preconditioning protect rat small intestine against ischemia/reperfusion injury, reducing morphologic lesions and apoptosis.

The role of ischemic preconditioning in gene expression related to inflammation in a rat model of intestinal ischemia-reperfusion injury

Abstract Purpose: To investigate the gene expression related to inflammation on mice subjected to intestinal ischemia and reperfusion (I/R) and treated with ischemic preconditioning (IPC). Methods: Thirty rats (EPM-Wistar), distributed in five groups of six animals each, were underwent anesthesia and laparotomy. The ischemia time was standardized in 60 minutes and the reperfusion time 120 minutes. IPC was standardized in 5 minutes of ischemia followed by 10 minutes of reperfusion accomplished before I/R. The control group was submitted only to anesthesia and laparotomy. The other groups were submitted to ischemia, I/R, ischemia + IPC and I/R + IPC. It was collected a small intestine sample to analyses by Quantitative Polymerase Chain Reaction in real Time (RT-qPCR) and histological analyses. It was studied 27 genes. Results: The groups that received IPC presented downregulation of genes, observed in of genes in IPC+ischemia group and IPC+I/R group. Data analysis by clusters showed upregulation in I/R group, however in IPC groups occurred downregulation of genes related to inflammation. Conclusion: The ischemia/reperfusion promoted upregulation of genes related to inflammation, while ischemic preconditioning promoted downregulation of these genes.

The role of ischemic preconditioning in gene expression related to inflammation in a rat model of intestinal ischemia-reperfusion injury.

PURPOSE: To investigate the gene expression related to inflammation on mice subjected to intestinal ischemia and reperfusion (I/R) and treated with ischemic preconditioning (IPC). METHODS: Thirty rats (EPM-Wistar), distributed in five groups of six animals each, were underwent anesthesia and laparotomy. The ischemia time was standardized in 60 minutes and the reperfusion time 120 minutes. IPC was standardized in 5 minutes of ischemia followed by 10 minutes of reperfusion accomplished before I/R. The control group was submitted only to anesthesia and laparotomy. The other groups were submitted to ischemia, I/R, ischemia + IPC and I/R + IPC. It was collected a small intestine sample to analyses by Quantitative Polymerase Chain Reaction in real Time (RT-qPCR) and histological analyses. It was studied 27 genes. RESULTS: The groups that received IPC presented downregulation of genes, observed in of genes in IPC+ischemia group and IPC+I/R group. Data analysis by clusters showed upregulation in I/R group, however in IPC groups occurred downregulation of genes related to inflammation. CONCLUSION: The ischemia/reperfusion promoted upregulation of genes related to inflammation, while ischemic preconditioning promoted downregulation of these genes.

MiR-146a protects small intestine against ischemia/reperfusion injury by down-regulating TLR4/TRAF6/NF-κB pathway.

Previous studies reported that miR-146a was involved in small intestine ischemia-reperfusion (I/R) injury, but the mechanism is largely vague. Here, we aimed to identify the change of miR-146a in patients with mesenteric ischemia and explore the potential regulatory mechanism of miR-146a in intestine epithelial cells survival under ischemia and I/R injury. The plasma of 20 patients with mesenteric ischemia and 25 controls was collected to examine the miR-146a expression by qPCR. Rat intestinal epithelial cells (IEC-6) and 24 male Sprague-Dawley rats were included to build ischemia and I/R model in vitro and in vivo. The qPCR results showed that miR-146a decreased both in the plasma of patients with mesenteric ischemia and in IEC-6 cells and rat small intestine tissues in ischemia and I/R model compared to controls. Both the in vitro and in vivo results showed that I/R resulted in more severe apoptotic injury than ischemia. Cleaved-caspase 3, TLR4, TRAF6, and nuclear NF-κB p65 were up-regulated accompanying reduced XIAP and SOCS3 expression in intestinal ischemia and I/R injury. After up-regulation of miR-146a in IEC-6 cells, increased cell survival and decreased cell apoptosis were observed, concomitant with decreased cleaved-caspase 3 and down-regulated TLR4/TRAF6/NF-κB pathway. What is more, this protective effect was blocked by TRAF6 overexpression and increased nuclear NF-κB p65 nuclear. Taken together, this study revealed that miR-146a expression was decreased in small intestine ischemia and I/R injury. And miR-146a improves intestine epithelial cells survival under ischemia and I/R injury through inhibition TLR4, TRAF6, and p-IκBα, subsequently leading to decreased NF-κB p65 nuclear translocation.

Another gastroenteritis?

Portal vein thrombosis (PVT) is an important, but often delayed or missed differential diagnosis in patients presenting with abdominal pain. In this case report we present a previously healthy 42-year-old patient with persistent upper abdominal pain for five days. Being a common complication in patients suffering from liver cirrhosis, PVT is an unusual finding in healthy individuals. However, gene mutation leading to a hypercoagulable state can be associated with thrombotic events in the portal venous system. Investigation for underlying disorders such as myeloproliferative neoplasm (MPN), paroxysmal nocturnal hemoglobinuria (PNH), antiphospholipid antibody syndrome are crucial.

MicroRNA profiling of the intestine during hypothermic circulatory arrest in swine.

AIM: To perform a profiling analysis of changes in intestinal microRNA (miRNA) expression during hypothermic circulatory arrest (HCA). METHODS: A total of eight piglets were randomly divided into HCA and sham operation (SO) groups. Under general anesthesia, swine in the HCA group were subjected to hypothermic cardiopulmonary bypass at 24 °C followed by 80 min of circulatory arrest, and the reperfusion lasted for 180 min after cross-clamp removal. The counterparts in the SO group were only subjected to median sternotomy. Histopathological analysis was used to detect mucosal injury, and Pick-and-Mix custom miRNA real-time polymerase chain reaction (PCR) panels containing 306 unique primer sets were utilized to assay unpooled intestinal samples harvested from the two groups. RESULTS: The intestinal mucosa of the animals that were subjected to 24 °C HCA exhibited representative ischemic reperfusion injury of grade 2 or 3 according to the Chiu score. Such intestinal mucosal injuries, with the subepithelial space and epithelial layer lifting away from the lamina propria, were accompanied by shortened and irregular villi. On the contrary, the intestinal mucosa remained normal in the sham-operated animals. In total, twenty-five miRNAs were differentially expressed between the two groups (15 upregulated and 10 downregulated in the HCA group). Among these, eight miRNAs (miR-122, miR-221-5p, miR-31, miR-421-5p, miR-4333, miR-499-3p, miR-542 and let-7d-3p) were significantly dysregulated (four higher and four lower). The expression of miR-122 was significantly (5.37-fold) increased in the HCA group vs the SO group, indicating that it may play a key role in HCA-induced mucosal injury. CONCLUSION: Exposure to HCA caused intestinal miRNA dysregulation and barrier dysfunction in swine. These altered miRNAs might be related to the protection or destruction of the intestinal barrier.

Intestinal ischemia-reperfusion of macaques triggers a strong innate immune response.

AIM: To investigate inflammatory injury in the intestinal mucosa after intestinal ischemia-reperfusion (IIR) with Toll-like receptor (TLR)-mediated innate immunity. METHODS: Ten macaques were randomized into control and IIR groups. The distribution and expression level of TLR2, TLR4, MD2, nuclear factor (NF)-κB p65 and interferon (IFN)-γ were measured by immunohistochemical stain and western blotting. The mRNA expression of TLR4, TLR2, MD2, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured by reverse transcriptase-polymerase chain reaction. The cytokine levels in blood and intestinal tissues were measured by ELISA. RESULTS: Obvious hemorrhage and erosion of mucosae were seen in the IIR group. Expression of TLR2, TLR4, MD2, NF-κB p65 and IFN-γ was significantly higher in the IIR group than in the control group (0.13 ± 0.04, 0.22 ± 0.04, 0.16 ± 0.06, 0.65 ± 0.12, 0.38 ± 0.10 vs 0.07 ± 0.04, 0.08 ± 0.03, 0.04 ± 0.02, 0.19 ± 0.06, 0.14 ± 0.05, P < 0.05). In addition, the expression of TLR2, TLR4, MD2, IL-1ß and TNF-α mRNA in the IIR group were significantly higher than those of control group(1.52 ± 0.15, 1.39 ± 0.06, 1.94 ± 0.12, 1.48 ± 0.15, 0.66 ± 0.08 vs 0.31 ± 0.05, 0.5 ± 0.04, 0.77 ± 0.05, 0.35 ± 0.08, 0.18 ± 0.04, P < 0.05). Furthermore, IL-1ß, IL-6 and TNF-α levels in the macaques ileum and plasma were significantly higher than in the control group (plasma: 86.3 ± 15.2, 1129 ± 248.3, 77.8 ± 16.2 vs 29.5 ± 7.3, 19.8 ± 8.2, 5.6 ± 1.7; ileum: 273.4. ± 44.7, 1636 ± 168.0, 205.5 ± 30.7 vs 76.8 ± 20.5, 663.4 ± 186.9, 49.0 ± 9.4; P < 0.05). CONCLUSION: After IIR, general inflammatory injury in the intestinal mucosa is correlated with a strong innate immune response, mediated by activation of the TLR-NF-κB-cytokine pathway.