Controversy surrounds the relations among infertility, fertility drug use, and the risk of ovarian cancer. The authors pooled interview data on infertility and fertility drug use from eight case-control studies conducted between 1989 and 1999 in the United States, Denmark, Canada, and Australia. Odds ratios and 95% confidence intervals were calculated, adjusting for age, race, family history of ovarian cancer, duration of oral contraception use, tubal ligation, gravidity, education, and site. Included in the analysis were 5,207 cases and 7,705 controls. Among nulligravid women, attempts for more than 5 years to become pregnant compared with attempts for less than 1 year increased the risk of ovarian cancer 2.67-fold (95% confidence interval (CI): 1.91, 3.74). Among nulliparous, subfertile women, neither any fertility drug use (odds ratio (OR) = 1.60, 95% CI: 0.90, 2.87) nor more than 12 months of use (OR = 1.54, 95% CI: 0.45, 5.27) was associated with ovarian cancer. Fertility drug use in nulligravid women was associated with borderline serous tumors (OR = 2.43, 95% CI: 1.01, 5.88) but not with any invasive histologic subtypes. Endometriosis (OR = 1.73, 95% CI: 1.10, 2.71) and unknown cause of infertility (OR = 1.19, 95% CI: 1.00, 1.40) increased cancer risk. These data suggest a role for specific biologic causes of infertility, but not for fertility drugs in overall risk for ovarian cancer.

Previous epidemiologic observations consistently suggest that suppression of ovulation, tubal ligation, and hysterectomy reduce the risk of ovarian cancer and that perineal talc use increases the risk. We examined these and other risk factors in the context of a new hypothesis: that inflammation may play a role in ovarian cancer risk. Ovulation entails ovarian epithelial inflammation; talc, endometriosis, cysts, and hyperthyroidism may be associated with inflammatory responses of the ovarian epithelium; gynecologic surgery may preclude irritants from reaching the ovaries via ascension from the lower genital tract. We evaluated these risk factors in a population-based case-control study. Cases 20-69 years of age with a recent diagnosis of epithelial ovarian cancer (767) were compared with community controls (1,367). We found that a number of reproductive and contraceptive factors that suppress ovulation, including gravidity, breast feeding, and oral contraception, reduced the risk of ovarian cancer. Environmental factors and medical conditions that increased risk included talc use, endometriosis, ovarian cysts, and hyperthyroidism. Gynecologic surgery including hysterectomy and tubal ligation were protective. Tubal ligation afforded a risk reduction even 20 or more years after the surgery. The spectrum of associations provides support for the hypothesis that inflammation may mediate ovarian cancer risk.

Angiotensin II (ANG II) promotes vascular inflammation through nuclear factor-kappaB (NF-kappaB)-mediated induction of pro-inflammatory genes. The role of peroxisome proliferator-activated receptors (PPARs) in modulating vascular inflammation and atherosclerosis in vivo is unclear. The aim of the present study was to examine the effects of ANG II on PPARs and NF-kappaB-dependent pro-inflammatory genes in the vascular wall in an in vivo model of atherosclerosis and aneurysm formation. Six-month-old male apolipoprotein E-deficient (apoE-KO) mice were treated with ANG II (1.44 mg/kg per day for 30 days). ANG II enhanced vascular inflammation, accelerated atherosclerosis, and induced formation of abdominal aortic aneurysms. These effects of ANG II in the aorta were associated with downregulation of both PPAR-alpha and PPAR-gamma mRNA and protein and an increase in transcription of monocyte chemotactic protein-1 (MCP-1), macrophage-colony stimulating factor (M-CSF), endothelial-selectin (E-selectin), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) throughout the entire aorta. ANG II also activated NF-kappaB with increases in both p52 and p65 NF-kappaB subunits. In summary, these in vivo results indicate that ANG II, through activation of NF-kappaB-mediated pro-inflammatory genes, promotes vascular inflammation, leading to acceleration of atherosclerosis and induction of aneurysm in apoE-KO mice. Downregulation of PPAR-alpha and -gamma by ANG II may diminish the anti-inflammatory potential of PPARs, thus contributing to enhanced vascular inflammation.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1␣ (MIP-1␣), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K i ranged from 1 nM to 5.5 nM). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca 2؉ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.It is clear that the inappropriate interaction of immune cells, such as T lymphocytes and monocytes, can lead to extensive inflammation and tissue destruction, which is a hallmark of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Immune cells are sent on their destructive journey by chemoattractant molecules known as chemokines, which interact with and signal through specific cell surface chemokine receptors. Chemokine receptors belong to the GPCR 1 superfamily and have been viewed as attractive therapeutic targets by the pharmaceutical industry mainly because of their central role in regulating leukocyte trafficking. The premise that drugs that can inhibit the directed migration and activation of immune cells could be useful therapeutically has prompted the search for specific and highly potent chemokine receptor antagonists.Autoimmune diseases like multiple sclerosis and rheumatoid arthritis are characterized by interactions between invading T lymphocytes and tissue macrophages that result in extensive inflammation, tissue damage, and chronic disease pathologies. Numerous studies have demonstrated CCR1 expression in these cell types, and a variety of evidence provides strong in vivo concept validation for a role of this receptor in animal models of these diseases. For example, Karpus et al. (1, 2) were able to show in a mouse EAE model of multiple sclerosis that antibodies to MIP-1␣ prevented the development of both initial and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment wit...

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC 50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-L-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd Ϸ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cellbased assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED 50 values of <2 mg͞kg in a rat model of endotoxininduced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.T he mammalian nitric ox ide synthase (NOS) (EC 1.14.13.39) enzyme family comprises three isoforms: inducible (iNOS), neuronal, and endothelial NOS. NOS isoforms are homodimers that catalyze NADPH-dependent oxidation of L-arginine to NO⅐ and citrulline (1-3). NOS monomers consist of an oxidoreductase domain and an oxygenase domain. The reductase domain is homologous to cytochrome P450 reductase and contains binding sites for NADPH, FAD, and FMN (4, 5). The oxygenase domain has binding sites for L-arginine, the heme prosthetic group, and tetrahydrobiopterin (H 4 B). Formation of stable NOS homodimers requires structural elements in the oxygenase domain and is an H 4 B-, substrate-, and heme-dependent process (6 -8). Dimerization of NOS is required for fully coupled enzyme activity because the f low of electrons during catalysis occurs in trans from the reductase domain of one monomer subunit to the oxygenase domain of the other monomer (9). The crystal structures of oxygenase domains of murine iNOS monomer (10), murine and human iNOS dimer (11-13), and human and bovine endothelial NOS dimer (13, 14) indicate a high degree of structural similarity within the critical catalytic center and dimer interface regions between NOS isoforms.NO⅐ plays a pivotal role in the physiology and pathophysiology of the central nervous, cardiovascular, and immune systems (15-17). The reactivity of NO⅐ toward molecular oxygen, thiols, transition metal centers, and other biological targets enables NO⅐ to function both as a rapidly reversible, specific, and local signal transduction molecule as well as a nonspecific mediator of tissue damage (1...

Apolipoprotein E-knockout (ApoE-KO) mice develop advanced atherosclerotic lesions by 1 yr of age and have been well characterized pathologically and morphologically, but little is known regarding their cardiovascular physiology and hemodynamics. We used noninvasive Doppler ultrasound to measure aortic and mitral blood velocity and aortic pulse-wave velocity in 13-mo-old ApoE-KO and wild-type (WT) mice anesthetized with isoflurane. In other mice from the same colony, we measured systolic blood pressure, body weight, heart weight, cholesterol, and hematocrit. Heart rate and blood pressure were comparable (P = not significant) between ApoE-KO and WT mice, but significant decreases (P < 0.001) were found in body weight (-22%) and hematocrit (-11%), and significant increases were found in heart weight (+23%), aortic velocity (+60%), mitral velocity (+81%) (all P < 0.001), and pulse-wave velocity (+13%, P < 0.05). We also found inflections in the aortic arch velocity signal consistent with enhanced peripheral wave reflection. Thus ApoE-KO mice have phenotypic alterations in indexes of peripheral vascular resistance and compliance and significantly elevated cardiac outflow velocities and heart weight-to-body weight ratios.

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA).Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA. Abdominal aortic aneurysm (AAA) is a chronic degenerative disease characterized by segmental weakening and dilation of the vascular wall. Recent estimates indicate that the prevalence of AAA is 4 to 9% in adults older than 65 years of age and is known to be associated with atherosclerosis, aging, hypertension, and cigarette smoking. Continued tissue remodeling results in silent expansion of the AAA with an increased risk of spontaneous rupture. Currently the only available treatments for AAAs are surgical resection and replacement or, more recently, insertion of an endovascular stent. The etiology of AAA is unclear. The extracellular matrix plays an essential role in maintaining the integrity of the vascular wall. Elastin and collagen fibers are the major components of this extracellular matrix. Both plasmin and matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, including collagen, elastin, and fibrin. Urokinase-type plasminogen activator (uPA) hydrolyzes plasminogen to form plasmin, which in turn activates MMPs. The in vivo activity of uPA is also regulated by local concentrations of its major inhibitor, PAI-1. Biochemical studies have demonstrated increased proteolytic activity in the aortic wall of AAA. Schneiderman and colleagues 1 showed that uPA mRNA as well as the tissue-type plasminogen activator (tPA), co-localized with infiltrating macrophages, is significantly increased in human AAA. Increased activities of MMP-2, -3, -9, and -12 in AAA have also been reported. [2][3][4][5] Atherosclerotic aortic lesions from high-cholesterol diet-fed apoE-KO mice show fragmentation of the elastic lamellae and rupture of the media resulting in pseudomicroaneurysm formation. These pathological changes are ...

These data demonstrate that NO modulates vascular compliance independent of blood pressure changes and that an intact endogenous NO system is required to maintain normal vascular compliance.