- Email: [email protected]
Reperfusion: Novel Insight From Genome-Wide Transcript Profiling
occluding superior mesenteric artery (SMA) for 45 min. Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNFalpha protein (ELISA), histology (Hematoxylin-Eosin stain), iNOS and HO-1 localization (immunohistochemistry), adhesion molecules E-selectin and ICAM-1 (Western blot), NFkappaB activation (EMSA), and polymorphonuclear neutrophil (PMN) tissue accumulation (MPO assay). iNOS and HO-1 were localized in the epithelium and infiltrated PMN. The obtained results indicated that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged WT jejunum. The above changes were significantly attenuated in I/Rchallenged Bach1 deficient jejunum. Taken together these findings indicate that disruption of the Bach1 gene resulted in attenuation of I/R-challenged intestinal injury, indicating that inhibition of Bach1 may be a novel therapeutic strategy for the treatment against I/R-mediated intestinal injury.
Mo2015
Chronic gastrointestinal ischemia (CGI) is increasingly being diagnosed in the elderly population. The first step in diagnosing CGI is visualization of mesenteric artery stenoses with either CT-angiography (CT-A), MRI-angiography, or ultrasonography (US). It is currently unknown which is the preferred imaging modality, as studies on the accuracy of CT-A for mesenteric artery stenoses are lacking, and none of these imaging modalities have ever been compared directly. The aim of the study was to investigate the accuracy of CT-A and US in a head-to-head comparison. Methods: Mesenteric arteries of patients referred to our tertiary center for evaluation of CGI, were visualized with both CT-A and US. Examiners of CT-A and US were blinded to the outcome of other examinations. As US is known to be operator dependent, each sonographist performed 50 ultrasounds of known mesenteric artery stenoses as part of a training program. A ≥ 70%stenosis was defined significant. The Moneta criteria were used for US. Sensitivity (sens.), specificity (spec.), negative and positive predictive value (NPV & PPV) were determined in the comparison of US or CT-A with digital subtraction angiography (DSA; gold standard). Results: Of 67 patients (61% females, median age 65 ±11 yrs, 71% CGI), CT-A could be compared to corresponding individual DSA examinations. Atherosclerosis was the etiology of stenosis in the vast majority of cases (89%). CT-A detected all 18 stenoses in the superior mesenteric artery (SMA), and 36/40 stenoses in the celiac artery (CA). CT-A was false positive in 5/41 (12%) positive cases of CA stenoses (sens. 0.9, spec. 0.81, NPV 0.84, PPV0.87), and false positive in 4/22 (18%) positive cases of SMA stenoses (sens. 1.0, spec. 0.92, NPV 1.0, PPV 0.82). US was performed in 63 patients (age 56 ± 15 yrs, 73% female, 76% CGI), which were matched with 21 individual DSA examinations. US could not be performed in 11/63 (17%) due to interposition of air. US underestimated 3, and overestimated 3 CA stenoses (sens. 0.8, spec. 0.5, NPV 0.5, PPV 0.8). In the SMA, US underestimated 1, and overestimated 3 stenoses (sens. 0.83, spec. 0.78, NPV 0.91, PPV 0.63). A CT-A was also performed in these 21 patients. CT-A detected all 17 stenoses observed at DSA, and overestimated 1 stenosis (sens. 0.94, spec. 0.75, NPV 0.75, PPV 0.94). CT-A detected all 6 stenoses in the SMA, and overestimated 4 stenoses (sens. 1.0, spec. 0.8, NPV 1, PPV 0.66). In this cohort, the positive diagnostic likelihood ratio of CT-A for CA and SMA stenoses was 3.8 and 12,5 respectively, as compared to 1.6 and 3.7 for US. In addition, CT-A led to an alternative diagnosis in 3/21 (14%) cases, compared to none based on US findings. Conclusion: CT-A is a more accurate technique to visualize the mesenteric arteries than US and should be the preferred imaging technique in patients with high suspicion of CGI.
Mo2013 Hypoxia Disrupts Recovery of Injured Non-Transformed Small Intestinal Cells, but Not Transformed Cells Mary A. Brown, Anthony Blikslager Studies suggest that hypoxia inducible factor-1α (HIF-1α) plays a protective role in intestinal epithelial barrier function following a hypoxic insult. However, these studies have been limited to transformed cell lines, most notably T84 cells and Caco-2 cells. We hypothesized that in an In Vitro model of hypoxic injury and recovery, transformed cells would not be affected by reduced oxygen levels, whereas as cells more indicative of normal small intestinal cells would be injured and recover more slowly following hypoxia. Caco-2 cells were used as a differentiated cancer-derived small intestinal model, and IPEC-J2 cells (a porcine small intestinal cell line derived from neonatal jejunum that can be repeatedly passaged) were used as a differentiated non-transformed small intestinal cell line. Caco-2 cells were grown on permeable supports at 37°C and 5% CO2 for 10 days. IPEC-J2 cells were grown under similar conditions for 7 days. Hypoxic injury was induced by incubating cells for 2 hours in a hypoxia chamber (95% N2 and 5% CO2) at 37°C before recovery in 21% O2. The effect of hypoxia on barrier function was monitored by measurements of transepithelial electrical resistance (TER) before and after 0, 1, 2, 4 and 24 hours of hypoxia. Caco-2 cells showed no evidence of any effect on barrier function in response to hypoxia for 2-hours, whereas IPEC-J2 cells had substantially reduced measurements of TER during hypoxia. Furthermore, there was no evidence of any effect of hypoxia during the recovery period in Caco-2 cells. On the other hand, IPEC-J2 Cells showed rapid recovery of TER following hypoxia, after which they underwent a precipitous drop in TER between 2 and 24 hours of recovery. Application of PGE2 did not prevent this effect, and indomethacin had no effect, suggesting that the COX pathway is not involved. The presumption is that transformed cells, such as Caco-2 cells, can express protective factors other than prostanoids such as HIF-1α whereas non-transformed cells do not constitutively express such protective factors. Interestingly, although IPEC-J2 cells were able to recover within 2 hours of hypoxia, the subsequent decline in barrier function suggests a mechanism similar to ischemia/ reperfusion injury. Further studies will be directed at understanding how transformed and non-transformed cells differ in their response to hypoxia, and the mechanism responsible for declining TER in recovering IPEC-J2 cells.
Mo2016 Disruption of Endoplasmic Reticulum Homeostasis in Human Intestine in Response to Ischemia/Reperfusion: Novel Insight From Genome-Wide Transcript Profiling Kaatje Lenaerts, Joep Grootjans, Marco Manca, Bas Boonen, Joep Derikx, Ronald van Dam, Erik A. Biessen, Cornelis H. Dejong, Wim A. Buurman BACKGROUND & AIM: Gastrointestinal complications are frequent in patients undergoing major surgery and often originate from intestinal ischemia/reperfusion (I/R). I/R causes damage to the intestinal mucosa and initiates a pleiotropic cellular response aimed at restoring homeostasis. We sought to identify key regulated biological processes involved in the I/Rinduced tissue damage response of the human intestine to find new targets for therapy. METHODS: Jejunal I/R was studied in 30 patients using a human experimental model in which an isolated part of jejunum, to be removed for surgical reasons, was subjected to ischemia during 30 min (30I, n=10), 45 min (45I, n=10), or 60 min (60I, n=10), followed by reperfusion (0 min [0R], 30 min [30R] and 120 min [120R]). A control sample not exposed to I/R was also taken. Whole genome expression profiles were generated of consecutive specimens harvested during I/R in a subset of 7 patients using Illumina HT-12 expression beadchips. Data analysis was performed using GeneSpring X11. Quantitative PCR was performed to confirm expression changes in all patients included in the study. RESULTS: The signal intensity of 1080 probe sets (representing 832 unique genes) changed significantly in response to I/R in the human intestine, as determined by repeated measures ANOVA (corrected P<0.05) and fold change (FC)≥1.3. Most dramatic changes in gene expression occurred in the 0R to 30R time interval. Gene ontology analysis showed significant enrichment in the category response to unfolded protein, which is indicative of endoplasmic reticulum (ER) stress. Transcript levels of 20 ER stress-related genes were significantly up-regulated at 30R. From this category, expression of heat shock protein Hsp72 (encoded by HSPA1A and HSPA1B) and its cofactor Hsp40 (encoded by DNAJB1), chaperones responsible for protein refolding after cell stress, were among the most highly upregulated genes overall in this time frame. Elevated expression of Hsp72 at 30R was confirmed in 30 patients (average FC is 16.2, 24.9, and 42.1 for 30I, 45I, and 60I group, respectively, P<0.01 compared to control). Furthermore, enhanced levels of spliced XBP1 at 30R (average FC is 1.4, 1.7, and 2.0 for 30I, 45I, and 60I group, respectively, P<0.05 compared to control) revealed activation of IRE1/XBP1 pathway, a highly conserved branch of the ER stress response. CONCLUSION: This is the first study characterizing the temporal transcriptional changes in response to I/ R in the human intestine and demonstrates that I/R disrupts ER homeostasis leading to misfolding of proteins and massive induction of pro-survival chaperone Hsp72 and its cochaperone Hsp40. These newly identified transcriptional changes suggest that therapeutic interventions targeting molecules of the ER stress pathway are promising tools in the prevention or treatment of I/R-induced intestinal injury.
Mo2014 Chronic Gastrointestinal Ischemia Due to Atherosclerotic Narrowing is Related to Classical Risk Factors for Cardiovascular Disease Aria Sana, Désirée van Noord, Stephanie Kooij, Kim van Dijk, Bert Bravenboer, Louis Lieverse, Eric Sijbrands, Janneke Langendonk, Peter Mensink Introduction: Chronic gastrointestinal ischemia (CGI) is most commonly caused by atherosclerotic stenosis of the gastrointestinal arteries. Although CGI is an extracardial manifestation of atherosclerosis, its relationship with classical risk factors of cardiovascular disease (CVD) is unclear. The aim of this study was to determine whether classical risk factors of CVD associate with atherosclerotic CGI (ath-CGI). Methods: A prospective cohort study was performed in patients with unexplained chronic abdominal symptoms referred for evaluation of CGI. A standard work-up was conducted including CTA or MRA for imaging the abdominal vessels and gastrointestinal tonometry. Additionally, an extensive evaluation for atherosclerotic risk factors was performed. Healthy subjects from the DiaGene Study population served as controls. The controls were previously not diagnosed with CGI or diabetes. Results: Between 2006 and 2009 195 patients were evaluated. Ath-CGI was diagnosed in 69 patients: 48 (70%) females, median age 66 (IQR 57-75) years. Weight loss was reported in 74% of ath-CGI patients and was reported median 9 (IQR 6 -12) kg. The controls consisted of 132 subjects: 63 (48%) females, median age 66 (IQR 63-72). The prevalence of hypercholesterolemia (38% vs. 24%, p-value = 0.03), personal history of CVD 51% vs. 8%, p-value <0.01), family history of CVD(51% vs. 33%, p-value < 0.01),smoking 39% vs. 13%, p-value < 0,01), use of statins (46% vs. 17%, p-value < 0.01), and anti-hypertensive agents 52% vs. 30%, p-value < 0.01) were significantly higher in ath-CGI patients compared with controls. Total LDL-cholesterol was lower in ath-CGI vs. controls (median 2.2 (IQR 1.8-2.9) vs. 3.5 (IQR 3.0-3.4), P < 0.01), this can be explained by higher statin use and substantial weight loss. Conclusion: CGI due to atherosclerotic stenosis is associated with classical CVD risk factors. This advocates secondary prevention therapy for these patients. However, the female preponderance is remarkable.
S-699
AGA Abstracts
AGA Abstracts
A Direct Comparison of CT-Angiography With Ultrasonography for the Detection of Mesenteric Artery Stenoses Leon M. Moons, Adriaan Moelker, Trude C. Leertouwer, Aria Sana, Ernst J. Kuipers, Jeoffrey N. Schouten
Mo2015
Chronic gastrointestinal ischemia (CGI) is increasingly being diagnosed in the elderly population. The first step in diagnosing CGI is visualization of mesenteric artery stenoses with either CT-angiography (CT-A), MRI-angiography, or ultrasonography (US). It is currently unknown which is the preferred imaging modality, as studies on the accuracy of CT-A for mesenteric artery stenoses are lacking, and none of these imaging modalities have ever been compared directly. The aim of the study was to investigate the accuracy of CT-A and US in a head-to-head comparison. Methods: Mesenteric arteries of patients referred to our tertiary center for evaluation of CGI, were visualized with both CT-A and US. Examiners of CT-A and US were blinded to the outcome of other examinations. As US is known to be operator dependent, each sonographist performed 50 ultrasounds of known mesenteric artery stenoses as part of a training program. A ≥ 70%stenosis was defined significant. The Moneta criteria were used for US. Sensitivity (sens.), specificity (spec.), negative and positive predictive value (NPV & PPV) were determined in the comparison of US or CT-A with digital subtraction angiography (DSA; gold standard). Results: Of 67 patients (61% females, median age 65 ±11 yrs, 71% CGI), CT-A could be compared to corresponding individual DSA examinations. Atherosclerosis was the etiology of stenosis in the vast majority of cases (89%). CT-A detected all 18 stenoses in the superior mesenteric artery (SMA), and 36/40 stenoses in the celiac artery (CA). CT-A was false positive in 5/41 (12%) positive cases of CA stenoses (sens. 0.9, spec. 0.81, NPV 0.84, PPV0.87), and false positive in 4/22 (18%) positive cases of SMA stenoses (sens. 1.0, spec. 0.92, NPV 1.0, PPV 0.82). US was performed in 63 patients (age 56 ± 15 yrs, 73% female, 76% CGI), which were matched with 21 individual DSA examinations. US could not be performed in 11/63 (17%) due to interposition of air. US underestimated 3, and overestimated 3 CA stenoses (sens. 0.8, spec. 0.5, NPV 0.5, PPV 0.8). In the SMA, US underestimated 1, and overestimated 3 stenoses (sens. 0.83, spec. 0.78, NPV 0.91, PPV 0.63). A CT-A was also performed in these 21 patients. CT-A detected all 17 stenoses observed at DSA, and overestimated 1 stenosis (sens. 0.94, spec. 0.75, NPV 0.75, PPV 0.94). CT-A detected all 6 stenoses in the SMA, and overestimated 4 stenoses (sens. 1.0, spec. 0.8, NPV 1, PPV 0.66). In this cohort, the positive diagnostic likelihood ratio of CT-A for CA and SMA stenoses was 3.8 and 12,5 respectively, as compared to 1.6 and 3.7 for US. In addition, CT-A led to an alternative diagnosis in 3/21 (14%) cases, compared to none based on US findings. Conclusion: CT-A is a more accurate technique to visualize the mesenteric arteries than US and should be the preferred imaging technique in patients with high suspicion of CGI.
Mo2013 Hypoxia Disrupts Recovery of Injured Non-Transformed Small Intestinal Cells, but Not Transformed Cells Mary A. Brown, Anthony Blikslager Studies suggest that hypoxia inducible factor-1α (HIF-1α) plays a protective role in intestinal epithelial barrier function following a hypoxic insult. However, these studies have been limited to transformed cell lines, most notably T84 cells and Caco-2 cells. We hypothesized that in an In Vitro model of hypoxic injury and recovery, transformed cells would not be affected by reduced oxygen levels, whereas as cells more indicative of normal small intestinal cells would be injured and recover more slowly following hypoxia. Caco-2 cells were used as a differentiated cancer-derived small intestinal model, and IPEC-J2 cells (a porcine small intestinal cell line derived from neonatal jejunum that can be repeatedly passaged) were used as a differentiated non-transformed small intestinal cell line. Caco-2 cells were grown on permeable supports at 37°C and 5% CO2 for 10 days. IPEC-J2 cells were grown under similar conditions for 7 days. Hypoxic injury was induced by incubating cells for 2 hours in a hypoxia chamber (95% N2 and 5% CO2) at 37°C before recovery in 21% O2. The effect of hypoxia on barrier function was monitored by measurements of transepithelial electrical resistance (TER) before and after 0, 1, 2, 4 and 24 hours of hypoxia. Caco-2 cells showed no evidence of any effect on barrier function in response to hypoxia for 2-hours, whereas IPEC-J2 cells had substantially reduced measurements of TER during hypoxia. Furthermore, there was no evidence of any effect of hypoxia during the recovery period in Caco-2 cells. On the other hand, IPEC-J2 Cells showed rapid recovery of TER following hypoxia, after which they underwent a precipitous drop in TER between 2 and 24 hours of recovery. Application of PGE2 did not prevent this effect, and indomethacin had no effect, suggesting that the COX pathway is not involved. The presumption is that transformed cells, such as Caco-2 cells, can express protective factors other than prostanoids such as HIF-1α whereas non-transformed cells do not constitutively express such protective factors. Interestingly, although IPEC-J2 cells were able to recover within 2 hours of hypoxia, the subsequent decline in barrier function suggests a mechanism similar to ischemia/ reperfusion injury. Further studies will be directed at understanding how transformed and non-transformed cells differ in their response to hypoxia, and the mechanism responsible for declining TER in recovering IPEC-J2 cells.
Mo2016 Disruption of Endoplasmic Reticulum Homeostasis in Human Intestine in Response to Ischemia/Reperfusion: Novel Insight From Genome-Wide Transcript Profiling Kaatje Lenaerts, Joep Grootjans, Marco Manca, Bas Boonen, Joep Derikx, Ronald van Dam, Erik A. Biessen, Cornelis H. Dejong, Wim A. Buurman BACKGROUND & AIM: Gastrointestinal complications are frequent in patients undergoing major surgery and often originate from intestinal ischemia/reperfusion (I/R). I/R causes damage to the intestinal mucosa and initiates a pleiotropic cellular response aimed at restoring homeostasis. We sought to identify key regulated biological processes involved in the I/Rinduced tissue damage response of the human intestine to find new targets for therapy. METHODS: Jejunal I/R was studied in 30 patients using a human experimental model in which an isolated part of jejunum, to be removed for surgical reasons, was subjected to ischemia during 30 min (30I, n=10), 45 min (45I, n=10), or 60 min (60I, n=10), followed by reperfusion (0 min [0R], 30 min [30R] and 120 min [120R]). A control sample not exposed to I/R was also taken. Whole genome expression profiles were generated of consecutive specimens harvested during I/R in a subset of 7 patients using Illumina HT-12 expression beadchips. Data analysis was performed using GeneSpring X11. Quantitative PCR was performed to confirm expression changes in all patients included in the study. RESULTS: The signal intensity of 1080 probe sets (representing 832 unique genes) changed significantly in response to I/R in the human intestine, as determined by repeated measures ANOVA (corrected P<0.05) and fold change (FC)≥1.3. Most dramatic changes in gene expression occurred in the 0R to 30R time interval. Gene ontology analysis showed significant enrichment in the category response to unfolded protein, which is indicative of endoplasmic reticulum (ER) stress. Transcript levels of 20 ER stress-related genes were significantly up-regulated at 30R. From this category, expression of heat shock protein Hsp72 (encoded by HSPA1A and HSPA1B) and its cofactor Hsp40 (encoded by DNAJB1), chaperones responsible for protein refolding after cell stress, were among the most highly upregulated genes overall in this time frame. Elevated expression of Hsp72 at 30R was confirmed in 30 patients (average FC is 16.2, 24.9, and 42.1 for 30I, 45I, and 60I group, respectively, P<0.01 compared to control). Furthermore, enhanced levels of spliced XBP1 at 30R (average FC is 1.4, 1.7, and 2.0 for 30I, 45I, and 60I group, respectively, P<0.05 compared to control) revealed activation of IRE1/XBP1 pathway, a highly conserved branch of the ER stress response. CONCLUSION: This is the first study characterizing the temporal transcriptional changes in response to I/ R in the human intestine and demonstrates that I/R disrupts ER homeostasis leading to misfolding of proteins and massive induction of pro-survival chaperone Hsp72 and its cochaperone Hsp40. These newly identified transcriptional changes suggest that therapeutic interventions targeting molecules of the ER stress pathway are promising tools in the prevention or treatment of I/R-induced intestinal injury.
Mo2014 Chronic Gastrointestinal Ischemia Due to Atherosclerotic Narrowing is Related to Classical Risk Factors for Cardiovascular Disease Aria Sana, Désirée van Noord, Stephanie Kooij, Kim van Dijk, Bert Bravenboer, Louis Lieverse, Eric Sijbrands, Janneke Langendonk, Peter Mensink Introduction: Chronic gastrointestinal ischemia (CGI) is most commonly caused by atherosclerotic stenosis of the gastrointestinal arteries. Although CGI is an extracardial manifestation of atherosclerosis, its relationship with classical risk factors of cardiovascular disease (CVD) is unclear. The aim of this study was to determine whether classical risk factors of CVD associate with atherosclerotic CGI (ath-CGI). Methods: A prospective cohort study was performed in patients with unexplained chronic abdominal symptoms referred for evaluation of CGI. A standard work-up was conducted including CTA or MRA for imaging the abdominal vessels and gastrointestinal tonometry. Additionally, an extensive evaluation for atherosclerotic risk factors was performed. Healthy subjects from the DiaGene Study population served as controls. The controls were previously not diagnosed with CGI or diabetes. Results: Between 2006 and 2009 195 patients were evaluated. Ath-CGI was diagnosed in 69 patients: 48 (70%) females, median age 66 (IQR 57-75) years. Weight loss was reported in 74% of ath-CGI patients and was reported median 9 (IQR 6 -12) kg. The controls consisted of 132 subjects: 63 (48%) females, median age 66 (IQR 63-72). The prevalence of hypercholesterolemia (38% vs. 24%, p-value = 0.03), personal history of CVD 51% vs. 8%, p-value <0.01), family history of CVD(51% vs. 33%, p-value < 0.01),smoking 39% vs. 13%, p-value < 0,01), use of statins (46% vs. 17%, p-value < 0.01), and anti-hypertensive agents 52% vs. 30%, p-value < 0.01) were significantly higher in ath-CGI patients compared with controls. Total LDL-cholesterol was lower in ath-CGI vs. controls (median 2.2 (IQR 1.8-2.9) vs. 3.5 (IQR 3.0-3.4), P < 0.01), this can be explained by higher statin use and substantial weight loss. Conclusion: CGI due to atherosclerotic stenosis is associated with classical CVD risk factors. This advocates secondary prevention therapy for these patients. However, the female preponderance is remarkable.
S-699
AGA Abstracts
AGA Abstracts
A Direct Comparison of CT-Angiography With Ultrasonography for the Detection of Mesenteric Artery Stenoses Leon M. Moons, Adriaan Moelker, Trude C. Leertouwer, Aria Sana, Ernst J. Kuipers, Jeoffrey N. Schouten