Volume 133, Issue 6, Pages 1938-1947.e1 (December 2007) Aging Gastropathy—Novel Mechanisms: Hypoxia, Up-regulation of Multifunctional Phosphatase PTEN, and Proapoptotic Factors  Andrzej Tarnawski, Rama Pai, Xiaoming Deng, Amrita Ahluwalia, Tetyana Khomenko, Tetsuya Tanigawa, Tomohiko Akahoshi, Zsuzsanna Sandor, Sandor Szabo  Gastroenterology  Volume 133, Issue 6, Pages 1938-1947.e1 (December 2007) DOI: 10.1053/j.gastro.2007.08.037 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Photomicrographs of gastric mucosa in young and aging rats. H&E staining. (A) Lower magnification (100×) upper row and higher magnification (500×) lower row. In gastric mucosa of aging rats, there is a partial atrophy of gastric glands in the basal mucosa and their replacement with connective tissue (*). (B) Quantification of connective tissue in the lower one third of the gastric mucosa shows in aging rats a significant increase in connective tissue replacing glandular cells. (C) Quantification of the number of inflammatory cells in gastric mucosa shows no inflammation (only minimal number of inflammatory cells) and no difference between young and aging rats, indicating that atrophic changes are not accompanied by an inflammation. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Gastric mucosal blood flow measured with BLF21 Laser-Doppler flow meter and tissue hypoxia visualized by Hypoxy probe-1. (A) In gastric mucosa of aging rats at baseline, mucosal blood flow, expressed as perfusion units,17 is significantly reduced by ∼60% (vs young rats; P = .0015). Such a dramatic reduction in blood flow likely leads to chronic hypoxia. (B) Photomicrographs of rat gastric mucosa. Gastric mucosal hypoxia is visualized by immunohistochemical staining utilizing a small molecular marker pimonidazole HCl, which after IV injection binds selectively to oxygen-starved cells.18 In young rats, Hypoxy probe-1 staining in gastric mucosa in both connective tissue and epithelial cells is negative, demonstrating the absence of hypoxia. In aging rats, positive staining is strongly expressed (dark brown staining arrows) in the upper and middle mucosa, mainly in the progenitor and parietal cell zone, reflecting severe hypoxia in these cells. As a positive control, we used gastric mucosa of young rats that had all major gastric arteries ligated for 1 hour. A strong accumulation of Hypoxy probe-1 is present in the majority of cells (dark brown staining), reflecting critical cell hypoxia. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Increased expression of Egr-1 mRNA and protein and increased Egr-1 transcriptional activity in gastric mucosa of aging and young rats. (A) Expression of Egr-1 mRNA by RT-PCR using specific rat primers. (B) Egr-1 mRNA expression by real-time PCR. In gastric mucosa of aging rats, Egr-1 mRNA is significantly up-regulated vs young rats. (C) Representative Western blots demonstrate increased Egr-1 protein expression in gastric mucosa of aging (vs young rats). (D) Quantification of Western blotting from additional experiments demonstrates significant increase in Egr-1 protein expression in gastric mucosa of aging (vs young) rats. (E) Assessment of Egr-1 transcriptional activity in gastric mucosa of young and aging rats was performed using TranSignal TF-TF Interaction Array (Panomics, Redwood City, CA). Egr-1 cis-element is spotted in duplicate: the first row is DNA spotted without dilution; in the second row, DNA is diluted 10 times (1:10). (F) In gastric mucosa of aging rats, there is a significant 2.7-fold increase (vs that of young rats; *P < .02) in binding of Egr-1 protein to its GC-rich cis elements that are highly expressed on PTEN gene promoter. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Increased expression of PTEN mRNA and protein in gastric mucosa of aging and young rats. (A) Real-time PCR and (B) representative Western blots showing a significant increase in PTEN mRNA and protein expression in gastric mucosa of aging vs young rats. (C) Immunohistochemical staining of gastric mucosa for PTEN. In gastric mucosa of aging rats, expression of PTEN (brown staining) is increased (vs young rats), predominantly in endothelial cells lining superficial mucosal capillaries and collecting venules (arrows), but is also increased in the epithelial cells (arrowheads). Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 TUNEL staining for apoptosis and expression of cleaved caspase-3, caspase-9, and survivin protein levels by Western blotting in gastric mucosa of young and aging rats. (A) The photomicrographs of gastric mucosa of young and aging rats at baseline. In situ cell death (apoptosis) detection by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) demonstrating brown staining in positive cells. (B) Quantification of the number of positively labeled cells demonstrated that gastric mucosa of aging rats exhibits ∼6-fold significantly increased number of apoptotic cells vs mucosa of young rats. In gastric mucosa of aging rats, there is a significant increase in protein expression levels of cleaved caspase-3 (C) and caspase-9 (D) and a significant decrease in survivin (E) compared with gastric mucosa of young rats. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 (A) The extent of ethanol-induced gastric mucosa injury in gastric mucosa of young and aging rats. Three hours after intragastric administration of 8 mL/kg of 50% ethanol, gastric mucosa injury in aging rats is significantly increased vs young rats. (B) Intragastric administration of ZnSO4 for 4 hours (2 mL 0.5% solution) down-regulates PTEN protein expression in gastric mucosa of aging rats similar to that previously demonstrated in human and rat lungs and (C) completely reverses increased susceptibility of gastric mucosa of aging rats to ethanol injury. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 (A) Photomicrographs of representative sections of human gastric mucosa of young and aging patients immunostained for PTEN using same antibody and procedure as for rat gastric mucosa. Gastric mucosa of aging patients has significantly increased PTEN expression (brown staining) and also increased staining intensity. (B) Quantification of PTEN-positive cells in gastric mucosal sections demonstrated significantly increased area of positively stained cells, mainly epithelial cells. The threshold of positive staining was set at level 75 on scale 0–155. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 (A) Photomicrographs of representative sections of human gastric mucosa of young and aging patients immunostained for survivin using same antibody and procedure as in our previous study.30 In gastric mucosa of young patients (40 years of age and younger), survivin expression is strong (brown staining) in the nuclei of the progenitor cells. (B) Quantification of the number of positively stained cells for survivin demonstrated in gastric mucosa of aging patients (70 years of age or older) reduced expression of survivin as reflected by significantly fewer cells positively stained for survivin. Gastroenterology 2007 133, 1938-1947.e1DOI: (10.1053/j.gastro.2007.08.037) Copyright © 2007 AGA Institute Terms and Conditions