Jeff Reese

Host cyclooxygenase-2 modulates carcinoma growth

Cyclooxygenase-2 (COX-2; Ptgs2) acts as a tumor promoter in rodent models for colorectal cancer, but its precise role in carcinogenesis remains unclear. We evaluated the contribution of host-derived COX-1 and COX-2 in tumor growth using both genetic and pharmacological approaches. Lewis lung carcinoma (LLC) cells grow rapidly as solid tumors when implanted in C57BL/6 mice. We found that tumor growth was markedly attenuated in COX-2(-/-), but not COX-1(-/-) or wild-type mice. Treatment of wild-type C57BL/6 mice bearing LLC tumors with a selective COX-2 inhibitor also reduced tumor growth. A decrease in vascular density was observed in tumors grown in COX-2(-/-) mice when compared with those in wild-type mice. Because COX-2 is expressed in stromal fibroblasts of human and rodent colorectal carcinomas, we evaluated COX-2(-/-) mouse fibroblasts and found a 94% reduction in their ability to produce the proangiogenic factor, VEGF. Additionally, treatment of wild-type mouse fibroblasts with a selective COX-2 inhibitor reduced VEGF production by 92%.

Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation

Augmentation of endogenous cannabinoid (eCB) signaling represents an emerging approach to the treatment of affective disorders. Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid to form prostaglandins, but also inactivates eCBs in vitro. However, the viability of COX-2 as a therapeutic target for in vivo eCB augmentation has not been explored. Using medicinal chemistry and in vivo analytical and behavioral pharmacological approaches, we found that COX-2 is important for the regulation of eCB levels in vivo. We used a pharmacological strategy involving substrate-selective inhibition of COX-2 to augment eCB signaling without affecting related non-eCB lipids or prostaglandin synthesis. Behaviorally, substrate-selective inhibition of COX-2 reduced anxiety-like behaviors in mice via increased eCB signaling. Our data suggest a key role for COX-2 in the regulation of eCB signaling and indicate that substrate-selective pharmacology represents a viable approach for eCB augmentation with broad therapeutic potential.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

COX-2 compensation in the uterus of COX-1 deficient mice during the pre-implantation period.

Prostaglandins (PGs) produced by cyclooxygenase (COX) participate in many aspects of female reproduction. The two isoforms of cyclooxygenase, COX-1 and COX-2, have distinct expression patterns in the mouse uterus during the peri-implantation period and suggest their independent contribution to uterine PGs. Using wild type and COX-1(-/-) mice, we examined the role of COX-1-derived PGs on day 4 of pregnancy, when its expression is maximal. Uterine vascular permeability was measured by 125I-labeled bovine serum albumin (BSA) uptake, and PG content was measured by gas chromatography-mass spectrometry. Vascular permeability and PG concentrations were reduced in COX-1(-/-) mice, but by less than the expected amount. After ovariectomy, uterine vascular permeability declined in both groups, but returned to baseline in wild type and was exaggerated in COX-1(-/-) females after treatment with ovarian steroids. Most importantly, COX-1(-/-) uteri displayed COX-2 expression on the morning of day 4, when COX-2 is normally absent. This hybridization pattern resembles the native expression of COX-1, and may partially offset the loss of COX-1-derived PGs. These data indicate that COX-1-derived PGs are important during uterine preparation for implantation, and that COX-2 compensation occurs in the absence of COX-1.

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.

Methodologies to Study Implantation in Mice

Pregnancy begins with fertilization of the ovulated oocyte by the sperm. After fertilization, the egg undergoes time-dependent mitotic division while trying to reach the blastocyst stage and the uterus for implantation. Uterine preparation for implantation is regulated by coordinated secretions and functions of ovarian sex steroids. The first sign of contact between the blastocyst and the uterus can be detected experimentally by an intravenous blue dye injection as early as the end of day 4 or the beginning of day 5 of pregnancy. This blastocyst-uterine attachment reaction leads to stromal decidual reaction only at sites of implantation. The process of implantation can be postponed and reinstated experimentally by manipulating ovarian estrogen secretion. Stromal decidualization can also be induced experimentally in the hormonally prepared uterus in response to stimuli other than the embryo. Fundamental biological questions surrounding these essential features of early pregnancy can be addressed through the application of various techniques and manipulation of this period of early pregnancy. This chapter describes the routine laboratory methodologies to study the events of early pregnancy, with special emphasis on the implantation process in mice.

Molecular determinants of lung development.

Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

Prostaglandins are essential for cervical ripening in LPS-Mediated preterm birth but not term or antiprogestin-driven preterm ripening

Globally, an estimated 13 million preterm babies are born each year. These babies are at increased risk of infant mortality and life-long health complications. Interventions to prevent preterm birth (PTB) require an understanding of processes driving parturition. Prostaglandins (PGs) have diverse functions in parturition, including regulation of uterine contractility and tissue remodeling. Our studies on cervical remodeling in mice suggest that although local synthesis of PGs are not increased in term ripening, transcripts encoding PG-endoperoxide synthase 2 (Ptgs2) are induced in lipopolysaccharide (LPS)-mediated premature ripening. This study provides evidence for two distinct pathways of cervical ripening: one dependent on PGs derived from paracrine or endocrine sources and the other independent of PG actions. Cervical PG levels are increased in LPS-treated mice, a model of infection-mediated PTB, consistent with increases in PG synthesizing enzymes and reduction in PG-metabolizing enzymes. Administration of SC-236, a PTGS2 inhibitor, along with LPS attenuated cervical softening, consistent with the essential role of PGs in LPS-induced ripening. In contrast, during term and preterm ripening mediated by the antiprogestin, mifepristone, cervical PG levels, and expression of PG synthetic and catabolic enzymes did not change in a manner that supports a role for PGs. These findings in mice, supported by correlative studies in women, suggest PGs do not regulate all aspects of the parturition process. Additionally, it suggests a need to refocus current strategies toward developing therapies for the prevention of PTB that target early, pathway-specific processes rather than focusing on common late end point mediators of PTB.

Overexpression of angiopoietin-2 impairs myocardial angiogenesis and exacerbates cardiac fibrosis in the diabetic db/db mouse model

The angiopoietins/Tie-2 system is essential for the maintenance of vascular integrity and angiogenesis. The functional role of angiopoietin-2 (Ang-2) in the regulation of angiogenesis is dependent on other growth factors such as VEGF and a given physiopathological conditions. This study investigates the potential role of Ang-2 in myocardial angiogenesis and fibrosis formation in the diabetic db/db mouse. Diabetic db/db mice received intramyocardial administration of either adenovirus Ang-2 (Ad-CMV-Ang-2) or Ad-β-gal. The levels of Tie-2, VEGF, caspase-3, Wnt7b, fibroblast-specific protein-1 (FSP-1), and adhesion molecules (ICAM-1 and VCAM-1) expression were measured. Apoptosis, capillary density, and cardiac fibrosis were also analyzed in the db/db mouse hearts. Overexpression of Ang-2 suppressed Tie-2 and VEGF expression in db/db mouse hearts together with significant upregulation of Wnt7b expression. Overexpression of Ang-2 also sensitizes ICAM-1 and VCAM-1 expression in db/db mouse hearts. Immunohistochemical analysis revealed that overexpression of Ang-2 resulted in a gradual apoptosis as well as interstitial fibrosis formation, these leading to a significant loss of capillary density. Data from these studies were confirmed in cultured mouse heart microvascular endothelial cells (MHMEC) exposed to excessive Ang-2. Exposure of MHMEC to Ang-2 resulted in increased caspase-3 activity and endothelial apoptosis. Knockdown of Ang-2 attenuated high glucose-induced endothelial cell apoptosis. Further, counterbalance of Ang-2 by overexpression of Ang-1 reversed loss of capillary density and fibrosis formation in db/db mouse hearts. Our data demonstrate that Ang-2 increases endothelial apoptosis, sensitizes myocardial microvascular inflammation, and promotes cardiac fibrosis and thus contributes to loss of capillary density in diabetic diseases.

Histamine Directly and Synergistically with Lipopolysaccharide Stimulates Cyclooxygenase-2 Expression and Prostaglandin I2 and E2 Production in Human Coronary Artery Endothelial Cells

Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE2, prostacyclin (PGI2), and thromboxane A2 in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI2 and PGE2 with no significant change in the expression of COX-1. Histamine-induced increases in PGI2 and PGE2 production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H1 receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE2 and PGI2 production. In contrast, histamine did not stimulate thromboxane A2 production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE2 and PGI2 were associated with increased expression of mRNA encoding PGE2 and PGI2 synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.