Hitoshi Ogawa

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model.

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.

Mechanisms of Endotoxin Tolerance in Human Intestinal Microvascular Endothelial Cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6–60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-κB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24–48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-κB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.

Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsAs effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection.

Cyclosporine A Enhances Leukocyte Binding by Human Intestinal Microvascular Endothelial Cells through Inhibition of p38 MAPK and iNOS PARADOXICAL PROINFLAMMATORY EFFECT ON THE MICROVASCULAR ENDOTHELIUM

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-α/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFκB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.

Cellular Redistribution of Inducible Hsp70 Protein in the Human and Rabbit Heart in Response to the Stress of Chronic Hypoxia: Role of protein kinases

Many infants who undergo cardiac surgery have a congenital cyanotic defect where the heart is chronically perfused with hypoxemic blood. Infant hearts adapt to chronic hypoxemia by activation of intracellular protein kinase signal transduction pathways. However, the involvement of heat shock protein 70 in adaptation to chronic hypoxemia and its role in protein kinase signaling pathways is unknown. We determined expression of message and subcellular protein distribution for inducible (Hsp70i) and constitutive heat shock protein 70 (Hsc70) in chronically hypoxic and normoxic infant human and rabbit hearts and their relationship to protein kinases. In chronically hypoxic human and rabbit hearts message levels for Hsp70i were elevated 4- to 5-fold compared with normoxic hearts, Hsp70i protein was redistributed from the particulate to the cytosolic fraction. In normoxic infants Hsp70i protein was distributed almost equally between the cytosolic and particulate fractions. Hsc70 message and subcellular distribution of Hsc70 protein were unaffected by chronic hypoxia. We then determined if protein kinases influence Hsp70i protein subcellular distribution. In rabbit hearts SB203580 and chelerythrine reduced Hsp70i message levels, whereas SB203580, chelerythrine, and curcumin reversed the subcellular redistribution of Hsp70i protein caused by chronic hypoxia, with no effect in normoxic hearts, indicating regulation of Hsp70i message and subcellular distribution of Hsp70i protein in chronically hypoxic rabbit hearts is influenced by protein kinase C and mitogen-activated protein kinases, specifically p38 MAPK and JNK. We conclude the Hsp70 signal transduction pathway plays an important role in adaptation of infant human and rabbit hearts to chronic hypoxemia.

Antiangiogenic Treatment of Mesenteric Desmoid Tumors With Toremifene and Interferon Alfa-2b: Report of Two Cases

PURPOSE:nDesmoid tumors are uncommon, benign, fibrous lesions occurring sporadically and in association with familial adenomatous polyposis. Typical clinical features include a locally aggressive behavior, an unpredictable course, and a high propensity for recurrence after surgical resection. There are no standard medical or surgical approaches, and no markers for monitoring medical therapy of desmoid tumors.nMETHODS:nWe report two cases of mesenteric desmoid tumors treated with interferon alfa-2b and toremifene, a novel regimen devised to block angiogenesis. Pre- and posttreatment desmoid tumor tissues were obtained in one patient during a repeat resection for recurrent stenosing Crohn’s disease and examined for mean vessel count and cellular proliferation levels by immunostaining for the endothelial surface antigen CD31 and the proliferation associated nuclear antigen, Ki-67, respectively. We assessed plasma D-dimers, a potential marker of angiogenic activity, and followed this throughout the course of antiangiogenic therapy in our two patients.nRESULTS:nExamination of posttreatment tissue revealed a significant decrease in microvessel density (P < 0.02) and Ki-67-positive nuclei (P < 0.0001) compared with pretreatment tissue. Both patients demonstrated a prompt and sustained drop in previously elevated plasma D-dimer levels, which correlated clinically with lesion regression and sustained remission.nCONCLUSIONS:nTreatment with toremifene and interferon alfa-2b was successful and well tolerated in our two patients. Our data suggest a combined antiangiogenic and antiproliferative mechanism of action. Furthermore, normalization of previously elevated plasma D-dimers may emerge as a strategy to monitor treatment efficacy in mesenteric desmoid tumors.

Sodium butyrate inhibits angiogenesis of human intestinal microvascular endothelial cells through COX-2 inhibition.

We examined the effect of sodium butyrate on in vitro angiogenesis and cyclooxygenase (COX) expression using primary cultures of human intestinal microvascular endothelial cells (HIMEC). Butyrate inhibited VEGF‐induced cellular proliferation, transmigration and tube formation of HIMEC. Butyrate also inhibited COX‐2 expression as well as prostaglandin (PG)E2 and PGI2 production, and administration of PGI2 analog partially reversed the effect of butyrate on HIMEC angiogenesis. These results indicate that sodium butyrate inhibits HIMEC angiogenesis through down‐regulation of COX‐2 expression and PG production, and suggest that anti‐angiogenic mechanisms may also be involved in the inhibitory effect of sodium butyrate on tumor growth.

Laparoscopic Surgery for Severe Ulcerative Colitis

Ulcerative colitis is occasionally exacerbated by fulminant manifestation of colitis. Severe ulcerative colitis is usually defined based on Trulove and Witts’ criteria (Table 1) (Truelove & Witts, 1955). The incidence of severe colitis in ulcerative colitis is 5 to 15 percent (Chen et al., 1998). If the patient is not improving despite intensive medical therapy, emergency colectomy is mandatory. In such a case, the patient is often malnourished and anemic, and has received high dose of steroids; therefore, the usual option in patients with severe ulcerative colitis is subtotal colectomy and ileostomy with preservation of the rectum (Gurland & Wexner, 2002). Restorative proctectomy can be done at a later time after the patient has recovered fully and steroids have been withdrawn (Fig. 1).

Moderate-to-Severe Refractory Ulcerative Colitis: Focus on Tacrolimus

Tacrolimus, a 23-member macrolide lactone discovered in 1984 from the actinomycete Streptomyces tsukubaensis, was the first macrolide immunosuppressant agent. Tacrolimus was first used for patients after liver transplantation to reduce the activity of the patients immune system and the risk of organ rejection. Oral tacrolimus was recently approved for use in steroid-refractory moderate-to-severe ulcerative colitis in Japan. This review focuses on the clinical efficacy and safety data of tacrolimus in moderate-to-severe refractory ulcerative colitis.

A Case Report: Repeat Ileal Pouch-anal Anastomosis in a Patients with Severe Ulcerative Colitis Complicated with Intractable Perianastomotic Abscess

回腸肛門吻合術後に難治性吻合部膿瘍を合併したため, 回腸J嚢を再造設し, 回腸肛門再吻合を行った重症潰瘍性大腸炎の1例を経験し, 良好な結果を得たので報告する. 症例は32歳の男性で, 重症潰瘍性大腸炎に対し分割的回腸肛門吻合術を施行したが, ストーマ閉鎖前の回腸.造影にて吻合部周囲に筋筒内膿瘍(cuff abscess)形成が認められた. 難治性のため吻合部の掻爬, 膿瘍ドレナージ, 筋筒部分切除, 再吻合を3度にわたり行ったが, 改善が見られないため, 吻合部を含む回腸J嚢切除と新J嚢回腸肛門再吻合を行った. 術後経過は良好で回腸ストーマ閉鎖後は, 夜間のsoilingはあるもののほぼ満足すべき排便機能を保っている.