Kathryn L. Howe

Epithelia Under Metabolic Stress Perceive Commensal Bacteria as a Threat

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohns disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

Transforming Growth Factor-β Regulation of Epithelial Tight Junction Proteins Enhances Barrier Function and Blocks Enterohemorrhagic Escherichia coli O157:H7-Induced Increased Permeability

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an enteric pathogen that causes potentially fatal symptoms after intimate adhesion, modulation of intestinal epithelial signal transduction, and alteration of epithelial function (eg, barrier disruption). Although the epithelial barrier is critical to gut homeostasis, only a few agents, such as transforming growth factor (TGF)-beta, can enhance or protect epithelial barrier function. Our aims were to delineate the mechanism(s) behind TGF-beta-induced barrier enhancement and to determine whether TGF-beta could prevent EHEC-induced barrier disruption. Using monolayers of the human T84 colonic epithelial cell line, we found that TGF-beta induced a significant increase in transepithelial electrical resistance (a measure of paracellular permeability) through activation of ERK MAPK and SMAD signaling pathways and up-regulation of the tight junction protein claudin-1. Additionally, TGF-beta pretreatment of epithelia blocked the decrease in transepithelial electrical resistance and the increase in transepithelial passage of [(3)H]-mannitol caused by EHEC infection. EHEC infection was associated with reduced expression of zonula occludens-1, occludin, and claudin-2 (but not claudin-1 or claudin-4); TGF-beta pretreatment prevented these changes. These studies provide insight into EHEC pathogenesis by illustrating the mechanisms underlying TGF-beta-induced epithelial barrier enhancement and identifying TGF-beta as an agent capable of blocking EHEC-induced increases in epithelial permeability via maintenance of claudin-2, occludin, and zonula occludens-1 levels.

Dextran Sulfate Sodium-Induced Colonic Histopathology, but not Altered Epithelial Ion Transport, Is Reduced by Inhibition of Phosphodiesterase Activity

Inhibition of phosphodiesterase (PDE) activity is beneficial in models of arthritis and airway inflammation. Here we assessed the ability of PDE inhibitors to modulate colitis by exposing mice to 4% (w/v) dextran sulfate sodium (DSS) drinking water for 5 days with or without rolipram, an inhibitor of PDE type 4, or the nonselective PDE inhibitor, pentoxifylline (both at 5 mg/kg, i.p., twice daily). Controls received saline, vehicle, or drug only. Colonic histology, myeloperoxidase (MPO) and tumor necrosis factor-alpha (TNF-alpha) levels, and epithelial ion transport (baseline and stimulated by electrical nerve stimulation, carbachol, and forskolin) were examined. DSS-treated mice displayed a variable diarrhea, significant histopathology in the mid-distal colon, elevated MPO activity, and reduced (>50%) responses to all three pro-secretory stimuli. Treatment with rolipram, and to a lesser extent pentoxifylline, significantly reduced the severity of the colonic histopathology and MPO levels. Neither PDE inhibitor had any affect on the diminished ion transport events caused by DSS-induced colitis. However, although stimulated ion transport events were still reduced 3 days after DSS treatment, colonic segments from DSS + rolipram-treated mice displayed enhanced recovery in their secretory responsiveness, particularly to carbachol. These findings indicate that specific PDE4 inhibition can significantly reduce the tissue damage that accompanies colitis and enhance recovery of normal colonic function.

Transforming growth factor-β1 protects against intestinal epithelial barrier dysfunction caused by hypoxia-reoxygenation.

ABSTRACT Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-&bgr;1 (TGF-&bgr;1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-&bgr;1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2bbe) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-&bgr;1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-&bgr;1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 – 3 h) and Caco-2bbe (1 h) monolayers. Transforming growth factor-&bgr;1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-&bgr;1–treated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P < 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P < 0.05 vs. normoxia-EHEC–infected cells), but this was not protected by TGF-&bgr;1 pretreatment. Transforming growth factor-&bgr;1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-&bgr; could lead to novel therapies in enteric diseases characterized by HR injury.

Management of Lower Extremity Ischaemia During Type A Dissection Repair

Introduction Type A aortic dissection can present with malperfusion syndrome. Lower limb ischaemia may resolve with repair of the dissection; however, the long duration of repair and/or persistent ischaemia may leave tissue at risk of necrosis with potential for substantial morbidity. Report Here, the case of a 67 year old man who presented with a Type A aortic dissection with malperfusion of the lower extremities is described. The Vascular Surgery and Cardiac Surgery divisions were consulted simultaneously. A decision was made to perform bilateral superficial femoral artery cannulation with perfusion initially via the right axillary artery prior to sternotomy, then via the cardiopulmonary bypass circuit. At completion of the arch replacement, lower extremity inflow was reassessed with formal repair of the femoral arteries. Post-operatively, the patient had complete return of motor-sensory function, despite densely ischaemic symptoms at presentation several hours previously. Discussion Cannulation of the superficial femoral arteries serves as a means to limit limb ischaemia intra-operatively and potentially improve outcomes in the setting of Type A aortic dissection with lower extremity malperfusion.

Blunt innominate artery trauma requiring repair and carotid ligation

Traumatic dissection of the innominate artery is a rare clinical entity. Management of a patient with motorsensory compromise and dissection extending to the subclavian and right common carotid arteries is quite rare and can be quite involved. Here we present such a case and discuss the unique peri-operative decision-making in the context of what is reported in the literature. Restoration of motorsensory function is critical and in this case, requiring a multi-disciplinary team.

IP185. Institutional Retrospective Review of Ultrasound-Accelerated Arterial and Venous Thrombolysis

Objectives: We sought to review all thrombolysis cases and determine whether ultrasound-accelerated thrombolysis (EKOS): (1) reduces the amount of tissue plasminogen activator (tPA) used, (2) decreases the number of adjunctive procedures, and/or (3) impacts technical success, compared to standard catheter-directed thrombolysis (CDT). Methods: Adult patients treated with EKOS or CDT for arterial or venous occlusion between January 2013 and September 2016 had their index hospitalization retrospectively reviewed. Treatment of arteriovenous fistula and grafts, pulmonary arteries, and femoral-popliteal deep veins was excluded. EKOS working catheter length, tPA dosing, and adjunctive procedures were decided by the treating surgeon. Our primary outcome variable was technical success, defined as free flow through treated vessels without residual thrombus. Data extracted from medical records included patient age, catheter length, total tPA, adjunctive procedures, technical success, and complications (death, hemorrhagic events). Results: Therewere51EKOSand61 standardCDTcasesbetweenJanuary 2013 and September 2014 (Table). Average age was higher for EKOS, with more men and significantly more arterial cases, as compared to CDT. Casemix (EKOS:CDT) included upper extremity arterial (0:2), iliac/lower extremity arterial (38:13), upper extremity venous (7:5), iliocaval/lower extremity venous (4:39), andmesenteric arterial (0:4). Lengthof stay, total tPAdose, number of adjunctive procedures, and catheter length (a surrogate for treatment length) were not significantly different. Technical success was greater in EKOS-treated patients compared to CDT, with arterial and venous success rates better in EKOS (arterial: 77.5% vs 64.7%, venous: 63.6% vs 54.4%), although this was not statistically significant (P 1⁄4 .9). In CDT, there was one amputation, but no strokes or deaths vs one hemorrhagic stroke causing death that occurred with EKOS. Conclusions: At our institution, EKOS is used in greater proportion on slightly older, predominantlymale patients with iliac/lower extremity arterial lesions. EKOS does not appear to reduce LOS, total tPA dose, or number of adjunctive procedures. The trend toward higher technical success in EKOS cases likely reflects differences in the pathophysiology of treating arterial vs venous occlusions andmay be an important consideration in case selection, which must ultimately be supported by reduced re-intervention rates.

PC224 Thrombin-Mediated Human Aortic Endothelial Barrier Dysfunction Alters MicroRNA Pathways Involved in Cell Survival, Injury, and Cancer

Objectives: Endothelial cells (ECs) must maintain an effective physiologic barrier in a highly dynamic environment. Capable of regulating other cells within the vasculature (eg, smooth muscle cells), ECs govern response to injury and inflammation. MicroRNAs (miR) are emerging as critical regulators of vascular disease, implicating EC miRs in homeostasis maintenance. Increased EC permeability has been documented in atherosusceptible regions of the aorta, suggesting it plays a role in disease development, but whether this is a cause or consequence is unknown. We hypothesized EC barrier dysfunction is a critical event that perpetuates chronic vascular disease via altered miR profiles. Methods: Human aortic endothelial cells (HAEC) cultured in transwells were exposed to thrombin (0.5, 1, 2 U/mL), and permeability was measured by fluorescence flux across monolayers at 90 minutes. Total HAEC RNA was isolated at various time points, with individual miR transcripts counted using nanoString nCounter (n 1⁄4 6 samples). miRs that were altered more than twofold compared to controls were analyzed using nSolver software and Ingenuity Pathway Analysis. Results: Thrombin exposure increased EC permeability to 130% 6 8.4% of untreated controls (2 U/mL thrombin; n 1⁄4 16 transwells; P < .05). Heatmaps from miR counts showed extensive miR profile changes in response to thrombin (n 1⁄4 6 samples; control, 90 minutes, 4 hours, 24 hours). Ingenuity Pathway Analysis showed miRs were clustered based on seed regions and matched with experimentally confirmed mRNA targets. At all time points, mRNA targets were shown to be most involved in cancer and injury disease pathways, with top cellular functions identified as cell movement, proliferation, growth, and survival. Conclusions: In response to a permeability insult, HAEC miR levels are significantly altered. Although our preliminary data need to be further substantiated, they highlight a possible mechanism whereby EC barrier dysfunction is a critical event that perpetuates chronic vascular disease. In this way, we might envision a scenario not unlike cancer (which has been compared to atherosclerosis), where an initial appropriate endothelial repair response becomes dysregulated in the context of ongoing injury (eg, permeability) in atherosusceptible regions.