Samuel Asfaha

Exacerbation of Inflammation-associated Colonic Injury in Rat through Inhibition of Cyclooxygenase-2

Cyclooxygenase type 1 is constitutively expressed and accounts for synthesis of prostaglandins in the normal gastrointestinal tract. Cyclooxygenase-2 is expressed at sites of inflammation. Selective inhibitors of cyclooxygenase-2 have been suggested to spare gastrointestinal prostaglandin synthesis, and therefore lack the ulcerogenic effects associated with standard nonsteroidal antiinflammatory drugs. However, the effects of cyclooxygenase-2 inhibitors on inflamed gastrointestinal mucosa have not been examined. We examined cyclooxygenase-2 mRNA and protein expression before and after induction of colitis in the rat, the contribution of cyclooxygenase-2 to colonic prostaglandin synthesis during colitis and the effects of selective inhibitors of cyclooxygenase-2 on colonic injury in this model. Cyclooxygenase-2 mRNA expression increased three to sixfold during the period 24 h to 1 wk after induction of colitis, with marked increases in cyclooxygenase-2 protein expression in the lamina propria and muscularis of the colon during colitis. Cyclooxygenase-1 expression (mRNA and protein) was not affected by the induction of colitis. The prostaglandins produced during colitis were largely derived from cyclooxygenase-2. Treatment with selective cyclooxygenase-2 inhibitors resulted in exacerbation of colitis, with perforation occurring when the compounds were administered for a week. These studies demonstrate that suppression of cyclooxygenase-2 can result in exacerbation of inflammation-associated colonic injury.

Cyclooxygenase 1 contributes to inflammatory responses in rats and mice: Implications for gastrointestinal toxicity

BACKGROUND & AIMS Selective inhibitors of cyclooxygenase (COX)-2 are being developed as gastrointestinal-sparing anti-inflammatory drugs based on the premise that this isoform is solely responsible for prostaglandin synthesis at sites of inflammation, whereas COX-1 produces prostaglandins important for maintenance of mucosal integrity. We investigated the relationship between suppression of inflammation by COX-2 inhibitors (NS-398, nimesulide, DuP697, and etodolac) and their effects on gastric prostaglandin synthesis. METHODS Effects of pretreatment of rats with drugs with a range of in vitro selectivity for COX-2 vs. COX-1 on carrageenan-induced paw inflammation were assessed, along with extent of suppression of COX-1 and COX-2. The role of COX-1 in inflammation was also assessed in COX-2-deficient mice. RESULTS Significant anti-inflammatory effects were only observed at doses of the drugs that inhibited COX-1. At these doses, the drugs also significantly suppressed gastric prostaglandin synthesis and elicited gastric mucosal erosions. The degree of suppression of prostaglandin synthesis at the site of inflammation correlated significantly with inhibition of COX-1 but not COX-2. CONCLUSIONS COX-1 makes an important contribution to inflammatory responses. To achieve desirable anti-inflammatory effects, COX-2 inhibitors needed to be given at doses in which selectivity was lost, leading to suppression of gastric prostaglandin synthesis and to mucosal injury.

Bile Acid and Inflammation Activate Gastric Cardia Stem Cells in a Mouse Model of Barrett-Like Metaplasia

Esophageal adenocarcinoma (EAC) arises from Barrett esophagus (BE), intestinal-like columnar metaplasia linked to reflux esophagitis. In a transgenic mouse model of BE, esophageal overexpression of interleukin-1β phenocopies human pathology with evolution of esophagitis, Barrett-like metaplasia and EAC. Histopathology and gene signatures closely resembled human BE, with upregulation of TFF2, Bmp4, Cdx2, Notch1, and IL-6. The development of BE and EAC was accelerated by exposure to bile acids and/or nitrosamines, and inhibited by IL-6 deficiency. Lgr5(+) gastric cardia stem cells present in BE were able to lineage trace the early BE lesion. Our data suggest that BE and EAC arise from gastric progenitors due to a tumor-promoting IL-1β-IL-6 signaling cascade and Dll1-dependent Notch signaling.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Long-lived intestinal tuft cells serve as colon cancer–initiating cells

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth-sustaining stem cells. DCLK1⁺ tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT-dependent genetic lineage-tracing strategy, we determined that a subpopulation of DCLK1⁺ cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that DCLK1⁺ tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in DCLK1⁺ cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate-induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in DCLK1⁺ cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in DCLK1⁺ cells. Thus, our data define an intestinal DCLK1⁺ tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived DCLK1⁺ cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer.

Aspirin causes rapid up‐regulation of cyclo‐oxygenase‐2 expression in the stomach of rats

Cyclo‐oxygenase‐1 (COX‐1) is believed to produce prostaglandins vital to mucosal defence, whereas cyclo‐oxygenase‐2 (COX‐2) is induced at sites of inflammation. Little is known about the regulation of COX‐2 in the stomach, particularly during the period following mucosal injury. In this study, we examined COX‐1 and COX‐2 expression shortly after administration of NSAIDs or ethanol.

Wound collagen deposition in rats: effects of an NO-NSAID and a selective COX-2 inhibitor

Selective cyclo‐oxygenase (COX)‐2 inhibitors and nitric oxide‐releasing nonsteroidal anti‐inflammatory drugs (NSAIDs) exhibit reduced toxicity in the gastrointestinal tract, but may affect wound healing in other tissues. In this study, we have compared the effects of a selective COX‐2 inhibitor (celecoxib), a nitric‐oxide releasing derivative of naproxen (HCT‐3012) and naproxen in a model of wound collagen deposition in the rat. Polyvinyl alcohol sponges were implanted subcutaneously in rats. The rats were treated daily for 5 days with the test drugs at equieffective anti‐inflammatory doses. Naproxen (10 mg kg−1) significantly decreased (45%) collagen deposition at the wound site relative to the vehicle‐treated control group. In contrast, HCT‐3012 (14.5 mg kg−1) significantly increased (62%) collagen deposition, while celecoxib (10 mg kg−1) had no effect. Naproxen and HCT‐3012 suppressed prostaglandin (PG) E2 levels at the wound site and whole blood thromboxane synthesis to similar degrees. Celecoxib had no significant effect on wound fluid PGE2 levels, but slightly reduced whole blood thromboxane synthesis (by 17%). COX‐1 mRNA and protein were expressed in the wound exudate, the skin surrounding the wound and in normal skin. In contrast, COX‐2 mRNA, but not protein, was expressed in wound and normal skin. These results demonstrate that HCT‐3012 can significantly enhance collagen deposition at a wound site, despite inhibiting prostaglandin synthesis to the same extent as the parent drug. Nitric oxide‐releasing NSAIDs may represent a safer alternative to standard NSAIDs for use as anti‐inflammatory and analgesic agents by post‐surgery patients.

Induction of Cyclooxygenase 1 and 2 in the Rat Stomach During Endotoxemia: Role in Resistance to Damage

BACKGROUND & AIMS Prostaglandins and nitric oxide are key mediators of gastric mucosal defense. Endotoxemia alters gastric resistance to damage, but little is known of the effects of chronic endotoxemia on the expression of prostaglandin and nitric oxide synthases (NOSs). The effects of short- vs. long-term administration of endotoxin on gastric resistance to damage and on expression of NOS and prostaglandin synthesis were compared. METHODS Rats were treated with short- or long-term bacterial endotoxin, after which susceptibility to ethanol-induced damage was assessed. The effects of various inhibitors of prostaglandin and NOS were examined. Expression of gastric NOS and cyclooxygenase (COX) messenger RNA (mRNA) were examined. RESULTS Repeated administration of endotoxin increased gastric resistance to ethanol- but not indomethacin-induced injury. Indomethacin, but not a highly selective COX-2 inhibitor or an inducible NOS inhibitor, abolished long-term endotoxin-induced gastric resistance to injury. Expression of mRNA for both COX-1 and -2, but not for endothelial or inducible NOS, were significantly increased after long-term endotoxin administration. CONCLUSIONS Repeated exposure to endotoxin resulted in increased resistance of the gastric mucosa to injury through a prostaglandin-dependent pathway. These prostaglandins were produced via COX-1, which like COX-2, is induced by endotoxin administration.

Selective cyclo-oxygenase-2 inhibition with celecoxib elevates blood pressure and promotes leukocyte adherence

Selective inhibitors of cyclo‐oxygenase‐2 have been shown to be effective anti‐inflammatory drugs with reduced gastrointestinal toxicity relative to conventional nonsteroidal anti‐inflammatory drugs (NSAIDs). In the present study, we examined the possibility that selective COX‐2 inhibition, by blocking prostacyclin synthesis, would increase blood pressure and cause leukocyte adherence and platelet aggregation. Normal rats and rats with hypertension induced by chronic administration of Nω‐nitro‐L‐arginine methylester were given celecoxib (10 mg kg−1) daily for 3 weeks. Celecoxib significantly elevated of blood pressure in both the normal and hypertensive rats (mean increase of >33 mm Hg after 3 weeks). In normal rats, celecoxib had no effect on serum 6‐keto prostaglandin (PG)F1α levels. Hypertensive rats exhibited a significant increase (82%) in serum 6‐keto PGF1α levels, and this was reduced to the levels of normal rats by treatment with celecoxib. Rats treated with celecoxib exhibited significant increases in weight gain (20%), plasma arginine‐vasopressin levels (148%) and plasma urea (69%) relative to vehicle‐treated controls. Plasma creatinine levels were unaffected by treatment with celecoxib, while plasma renin levels were significantly decreased (30%) relative to controls. Superfusion of mesenteric venules with celecoxib (3 μM) in vivo resulted in significant increases in leukocyte adherence to the endothelium in both normal and hypertensive rats. These studies suggest that suppression of COX‐2 significantly influences vascular and/or renal function, leading to elevated blood pressure and leukocyte adherence.

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b + Ly6G + immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b+Ly6G+ immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b+Ly6G+ cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b+Ly6G+ IMCs in early cancer development.