Search

Increased trunk fat is associated with an elevated risk of breast cancer in normal-weight postmenopausal women. The main objective of this study was to determine whether levels of trunk fat are associated with changes in breast gene expression in normal-weight women. Non-tumorous breast tissue was collected from 32 normal BMI women who underwent mastectomy for breast cancer risk reduction or treatment. Body composition was measured by dual-energy x-ray absorptiometry. High levels of trunk fat were associated with a large number of differentially expressed genes and changes in multiple pathways and processes potentially linked to breast cancer pathogenesis. High levels of trunk fat were also associated with an elevated immune score and increased levels of leptin, CCL2, VEGF-C, IL6, and aromatase. Collectively, these results help to explain why high levels of trunk fat are associated with an increased risk of breast cancer in normal BMI women., (© 2022. The Author(s).)

Excess body fat and sedentary behavior are associated with increased breast cancer risk and mortality, including in normal weight women. To investigate underlying mechanisms, we examined whether adiposity and exercise impact the breast microenvironment (e.g., inflammation and aromatase expression) and circulating metabo-inflammatory factors. In a cross-sectional cohort study, breast white adipose tissue (WAT) and blood were collected from 100 women undergoing mastectomy for breast cancer risk reduction or treatment. Self-reported exercise behavior, body composition measured by dual-energy x-ray absorptiometry (DXA), and waist:hip ratio were obtained prior to surgery. Breast WAT inflammation (B-WATi) was assessed by IHC and aromatase expression was assessed by quantitative PCR. Metabolic and inflammatory blood biomarkers that are predictive of breast cancer risk and progression were measured. B-WATi was present in 56 of 100 patients and was associated with older age, elevated BMI, postmenopausal status, decreased exercise, hypertension and dyslipidemia ( P s < 0.001). Total body fat and trunk fat correlated with B-WATi and breast aromatase levels ( P s < 0.001). Circulating C-reactive protein, IL6, insulin, and leptin positively correlated with body fat and breast aromatase levels, while negative correlations were observed for adiponectin and sex hormone binding globulin ( P < 0.001). Inverse relationships were observed with exercise ( P s < 0.05). In a subgroup of 39 women with normal BMI, body fat levels positively correlated with B-WATi and aromatase expression ( P s < 0.05). In conclusion, elevated body fat levels and decreased exercise are associated with protumorigenic micro- and host environments in normal, overweight, and obese individuals. These findings support the development of BMI-agnostic lifestyle interventions that target adiposity. PREVENTION RELEVANCE: We report that individuals with high body fat and low exercise levels have breast inflammation, higher breast aromatase expression, and levels of circulating metabo-inflammatory factors that have been associated with increased breast cancer risk. These findings support interventions to lower adiposity, even among normal weight individuals, to prevent tumor growth., (©2021 American Association for Cancer Research.)

Obesity is associated with an increased risk of breast cancer in post-menopausal women and decreased risk in pre-menopausal women. Conversely, in BRCA1/2 mutation carriers, pre-menopausal obesity is associated with early-onset breast cancer. Here we show that obese, pre-menopausal BRCA1/2 mutation carriers have increased levels of aromatase and inflammation in the breast, as occurs in post-menopausal women. In a prospective cohort study of 141 women with germline BRCA1 (n = 74) or BRCA2 (n = 67) mutations, leptin, and aromatase expression were higher in the breast tissue of obese versus lean individuals (P < 0.05). Obesity was associated with breast white adipose tissue inflammation, which correlated with breast aromatase levels (P < 0.01). Circulating C-reactive protein, interleukin-6, and leptin positively correlated with body mass index and breast aromatase levels, whereas negative correlations were observed for adiponectin and sex hormone-binding globulin (P < 0.05). These findings could help explain the increased risk of early-onset breast cancer in obese BRCA1/2 mutation carriers.

Obesity is associated with an increased incidence of high-grade prostate cancer (PC) and worse prognosis for PC patients. Recently, we showed in men that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures, was associated with high-grade PC. Possibly, interventions that suppress periprostatic WAT inflammation will improve outcomes for men with PC. Here, we tested the hypothesis that supplemental 17β-estradiol (E2) could decrease periprostatic WAT inflammation in obese male mice. Mice were fed a high-fat diet to induce periprostatic WAT inflammation before being treated with supplemental E2. E2 supplementation suppressed caloric intake, induced weight loss, decreased periprostatic WAT inflammation and downregulated the expression of genes linked to inflammation including Cd68, Mcp1 and Tnf. Similar to the effects of E2 supplementation, treatment with diethylstilbestrol, a synthetic estrogen, also suppressed caloric intake and reduced periprostatic WAT inflammation. To determine whether the observed effects of supplemental estrogen could be reproduced by caloric restriction (CR) alone, obese mice were put on a 30% CR diet. Like estrogen treatment, CR was effective in reducing body weight, periprostatic WAT inflammation and the expression of pro-inflammatory genes. Transcriptomic analyses of periprostatic fat showed that obesity was associated with enrichment in inflammatory response pathways, which were normalized by both supplemental E2 and CR. Taken together, these findings strengthen the rationale for future efforts to determine whether either CR or supplemental estrogen will decrease periprostatic WAT inflammation and thereby improve outcomes for men with PC., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Breast white adipose tissue inflammation (BWATi) is associated with obesity and higher breast cancer risk among non-Hispanic white women. Obesity is prevalent in Hispanic/Latina patients with breast cancer, and the occurrence of BWATi in this population is not well-characterized. The association between BWATi and body mass index (BMI) was evaluated in Hispanic/Latina patients with breast cancer who underwent mastectomy. BWATi was defined as the presence of crown-like structures of the breast (CLS-B), detected by CD68 IHC in nontumor breast tissue. BWATi severity was quantified as number of CLS-B/cm 2 Adipocyte diameter was measured using hematoxylin and eosin-stained breast tissue sections. Preoperative BMI (within 1 week prior to mastectomy) was categorized as normal (18.5-<25.0 kg/m 2 ), overweight (25.0-<30.0 kg/m 2 ), class I obesity (30.0-<35.0 kg/m 2 ), and class II-III obesity (35.0 kg/m 2 or above). Patient charts were abstracted to record clinicopathologic features and liver function tests <90 days before mastectomy. The study included 91 women (mean age 69 years; range 36-96 years). Prevalence of BWATi increased with BMI (24% in normal weight, 34% in overweight, 57% in class I obesity, and 65% in class II-III obesity; P trend <0.01). Severe BWATi (>0.27 CLS-B/cm 2 ) was associated with higher BMI ( P trend = 0.046) and greater adipocyte diameter ( P = 0.04). Adjusting for BMI, neoadjuvant chemotherapy, and elevated alanine aminotransferase were associated with severe BWATi, and current smoking was associated with mild BWATi (all P < 0.05). BWATi was associated with higher BMI in Hispanic/Latina patients with breast cancer, consistent with previously described associations in other populations., (©2018 American Association for Cancer Research.)

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.

Obesity is associated with an increased incidence of high-grade prostate cancer and poor prognosis for prostate cancer patients. Recently, we showed that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by crown-like structures (CLS) consisting of dead or dying adipocytes surrounded by macrophages, was associated with high-grade prostate cancer. It is possible, therefore, that agents that suppress periprostatic WAT inflammation will alter the development or progression of prostate cancer. Pioglitazone, a ligand of PPARγ, is used to treat diabetes and possesses anti-inflammatory properties. Here, our main objectives were to determine whether pioglitazone inhibited obesity-related periprostatic WAT inflammation in mice and then to elucidate the underlying mechanism. Treatment with pioglitazone reduced the density of CLS in periprostatic fat and suppressed levels of TNFα, TGFβ, and the chemokine monocyte chemoattractant protein-1 (MCP-1). Importantly, the ability of pioglitazone to suppress periprostatic WAT inflammation was abrogated in MCP-1 knockout mice. Pioglitazone caused dose-dependent induction of both adiponectin, an anti-inflammatory adipokine, and its receptor AdipoR2 in cultured 3T3-L1 cells and in periprostatic WAT of obese mice. Pioglitazone blocked TNFα-mediated induction of MCP-1 in 3T3-L1 cells, an effect that was attenuated when either adiponectin or AdipoR2 were silenced. Taken together, pioglitazone-mediated induction of adiponectin suppressed the elevation in MCP-1 levels, thereby attenuating obesity-related periprostatic WAT inflammation. These findings strengthen the rationale for future efforts to determine whether targeting the PPARγ-adiponectin-MCP-1 axis will decrease periprostatic adipose inflammation and thereby reduce the risk of high-grade prostate cancer or improve outcomes for men with prostate cancer. Cancer Prev Res; 11(4); 215-26. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Obesity is associated with white adipose tissue (WAT) inflammation in the breast, elevated levels of the estrogen biosynthetic enzyme, aromatase, and systemic changes that predispose to breast cancer development. We examined whether WAT inflammation and its associated systemic effects correlate with body fat levels in an Asian population where body mass index (BMI) is not an accurate assessment of obesity and cancer risk. We also investigated whether biologic differences could account for the greater proportion of premenopausal estrogen receptor (ER)-positive breast cancer in Asian versus Western countries. Breast WAT and fasting blood were prospectively collected from Taiwanese women undergoing mastectomy for breast cancer treatment. Body composition was measured in a subgroup using bioelectrical impedance analysis. WAT inflammation was defined by the presence of crown-like structures of the breast, which are composed of dead or dying adipocytes surrounded by macrophages. Findings were compared with U.S. Caucasian women. In the Taiwanese cohort ( n = 72), breast WAT inflammation was present in 31 (43%) women and was associated with elevated BMI ( P < 0.01) and increased levels of body fat ( P < 0.01), C-reactive protein ( P = 0.02), triglycerides ( P < 0.01), insulin resistance scores ( P = 0.04), and lower HDL cholesterol ( P < 0.01). ER + tumors were associated with greater body fat versus other subtypes ( P = 0.03). Compared with U.S. Caucasians ( n = 267), Taiwanese women had larger breast adipocytes despite lower BMI after adjusting for BMI and menopausal status ( P = 0.01). A subclinical inflammatory state associated with increased adiposity and metabolic dysfunction could contribute to breast cancer pathogenesis in Asian women. Cancer Prev Res; 11(4); 227-36. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Obesity, a cause of subclinical inflammation, is associated with increased risk of high-grade prostate cancer (PC) and poor outcomes. Whether inflammation occurs in periprostatic white adipose tissue (WAT), and contributes to the negative impact of obesity on PC aggressiveness, is unknown., Methods: In a single-center, cross-sectional design, men with newly diagnosed PC undergoing radical prostatectomy were eligible for study participation. The primary objective was to examine the prevalence of periprostatic WAT inflammation defined by the presence of crown-like structures (CLS-P) as detected by CD68 immunohistochemistry. Secondary objectives were to explore the clinical and systemic correlates of periprostatic WAT inflammation. Tumor characteristics and host factors including BMI, adipocyte diameter, and circulating levels of lipids, adipokines, and other metabolic factors were measured. Wilcoxon rank-sum, Chi-square, or Fisher's exact tests, and generalized linear regression were used to examine the association between WAT inflammation and tumor and host characteristics., Results: Periprostatic fat was collected from 169 men (median age 62 years; median BMI 28.3). Periprostatic WAT inflammation was identified in 49.7% of patients and associated with higher BMI (P=0.02), larger adipocyte size (P=0.004) and Gleason grade groups IV/V tumors (P=0.02). The relationship between WAT inflammation and high Gleason grade remained significant after adjusting for BMI (P=0.04). WAT inflammation correlated with higher circulating levels of insulin, triglycerides, and leptin/adiponectin ratio, and lower high density lipoprotein cholesterol, compared to those without WAT inflammation (P's <0.05)., Conclusion: Periprostatic WAT inflammation is common in this cohort of men with PC and is associated with high-grade PC.

Context: Most estrogen-dependent breast cancers occur after menopause, despite low levels of circulating estrogens. Breast expression of the estrogen-biosynthetic enzyme, aromatase, is proposed to drive breast cancer development after menopause. However, the effects of menopause on breast aromatase expression are unknown., Objective: To determine the effect of menopause on breast aromatase expression in relation to body mass index (BMI), white adipose tissue inflammation (WATi), and systemic markers of metabolic dysfunction., Design, Setting, and Participants: Cross-sectional study of 102 premenopausal (age 27 to 56) and 59 postmenopausal (age 45 to 74) women who underwent mastectomy for breast cancer treatment/prevention., Outcome: Breast tissue was assessed for the presence of crown-like structures and the expression and activity of aromatase. Systemic markers examined include interleukin (IL)-6, insulin, glucose, leptin, adiponectin, high-sensitivity C-reactive protein (hsCRP), cholesterol, and triglycerides. Multivariable analysis was performed for aromatase messenger RNA (mRNA) in relation to BMI, WATi, and blood markers., Results: Postmenopausal women had higher BMI and more breast WATi than premenopausal women. Fasting levels of IL-6, glucose, leptin, hsCRP, and homeostatic model assessment 2 insulin resistance score were higher in the postmenopausal group. BMI was positively correlated with aromatase mRNA in both pre- and postmenopausal women. Aromatase levels were higher in breast tissue of postmenopausal women, with levels being higher in inflamed vs noninflamed, independent of BMI. Adipocyte diameter and levels of leptin, hsCRP, adiponectin, and high-density lipoprotein cholesterol were more strongly correlated with aromatase in postmenopausal than premenopausal women., Conclusions: Elevated aromatase in the setting of adipose dysfunction provides a possible mechanism for the higher incidence of hormone-dependent breast cancer in obese women after menopause., (Copyright © 2017 Endocrine Society)

Obesity is associated with breast white adipose tissue (WAT) inflammation, elevated levels of the estrogen biosynthetic enzyme, aromatase, and systemic changes that have been linked to the pathogenesis of breast cancer. Here, we determined whether metabolic obesity, including changes in breast biology and systemic effects, occurs in a subset of women with normal body mass index (BMI). Breast WAT and fasting blood were collected from 72 women with normal BMI (<25 kg/m 2 ) undergoing mastectomy for breast cancer risk reduction or treatment. WAT inflammation was defined by the presence of crown-like structures of the breast (CLS-B) which are composed of dead or dying adipocytes surrounded by macrophages. Severity of inflammation was measured as CLS-B/cm 2 The primary objective was to determine whether breast WAT inflammation is associated with aromatase expression and activity. Secondary objectives included assessment of circulating factors and breast adipocyte size. Breast WAT inflammation was present in 39% of women. Median BMI was 23.0 kg/m 2 (range, 18.4-24.9 kg/m 2 ) in women with breast WAT inflammation versus 21.8 kg/m 2 (range, 17.3-24.6 kg/m 2 ) in those without inflammation ( P = 0.04). Breast WAT inflammation was associated with elevated aromatase expression and activity, which increased with severity of inflammation ( P < 0.05). Breast WAT inflammation correlated with larger adipocytes ( P = 0.01) and higher circulating levels of C-reactive protein, leptin, insulin, and triglycerides ( P ≤ 0.05). A subclinical inflammatory state associated with elevated aromatase in the breast, adipocyte hypertrophy, and systemic metabolic dysfunction occurs in some normal BMI women and may contribute to the pathogenesis of breast cancer. Cancer Prev Res; 10(4); 235-43. ©2017 AACR See related article by Berger, p. 223-25 ., (©2017 American Association for Cancer Research.)

Insulin controls glucose uptake into adipose and muscle cells by regulating the amount of GLUT4 in the plasma membrane. The effect of insulin is to promote the translocation of intracellular GLUT4 to the plasma membrane. The small Rab GTPase, Rab10, is required for insulin-stimulated GLUT4 translocation in cultured 3T3-L1 adipocytes. Here we demonstrate that both insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane are reduced by about half in adipocytes from adipose-specific Rab10 knockout (KO) mice. These data demonstrate that the full effect of insulin on adipose glucose uptake is the integrated effect of Rab10-dependent and Rab10-independent pathways, establishing a divergence in insulin signal transduction to the regulation of GLUT4 trafficking. In adipose-specific Rab10 KO female mice, the partial inhibition of stimulated glucose uptake in adipocytes induces insulin resistance independent of diet challenge. During euglycemic-hyperinsulinemic clamp, there is no suppression of hepatic glucose production despite normal insulin suppression of plasma free fatty acids. The impact of incomplete disruption of stimulated adipocyte GLUT4 translocation on whole-body glucose homeostasis is driven by a near complete failure of insulin to suppress hepatic glucose production rather than a significant inhibition in muscle glucose uptake. These data underscore the physiological significance of the precise control of insulin-regulated trafficking in adipocytes., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Obesity is a risk factor for the development of hormone receptor (HR)-positive breast cancer in postmenopausal women. Obesity causes subclinical inflammation in white adipose tissue (WAT), characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures (CLS). Estrogen synthesis is catalyzed by aromatase. Previously, we demonstrated CLS and elevated levels of proinflammatory mediators and aromatase in the mammary glands of obese mice and breast tissue of obese women. Here, we tested the hypothesis that supplemental estrogen could prevent or reverse WAT inflammation (WATi) and related molecular changes in the mammary gland. C57BL/6J mice were ovariectomized (OVX) to simulate the postmenopausal state. Supplementation with 17β-estradiol (E2) protected against high fat diet (HFD)-induced weight gain and mammary glands WATi. Expression of proinflammatory mediators (Cox-2, TNFα, IL1β) and aromatase were also reduced in the mammary glands of mice that received supplemental E2. Next, to determine whether E2 supplementation can reverse WATi, obese OVX mice were treated with E2 or placebo and then continued on HFD. E2 supplementation induced weight loss, reversed mammary gland inflammation, and downregulated expression of proinflammatory mediators and aromatase. Finally, we determined whether the protective effects of E2 were mediated by estrogen receptor-α (ERα). Knocking out ERα in ovary intact mice fed a HFD led to weight gain, WATi and elevated levels of proinflammatory mediators and aromatase mimicking the effects of OVX. Taken together, our findings indicate that estrogen via ERα protects against weight gain, WATi and associated increases in proinflammatory mediators and aromatase in the mammary gland., (©2015 American Association for Cancer Research.)

Chronic inflammation is recognized as a risk factor for the development of several malignancies. Local white adipose tissue (WAT) inflammation, defined by the presence of dead or dying adipocytes encircled by macrophages that form crown-like structures (CLS), occurs in the breasts (CLS-B) of most overweight and obese women. Previously, we showed that the presence of CLS-B is associated with elevated tissue levels of proinflammatory mediators and aromatase, the rate-limiting enzyme for estrogen biosynthesis. The associated increased levels of aromatase in the breast provide a plausible mechanistic link between WAT inflammation and estrogen-dependent breast cancers. Thus, breast WAT inflammation could be relevant for explaining the high incidence of estrogen-dependent tumors with aging despite diminished circulating estrogen levels after menopause. To explore this possibility, we determined whether menopause in addition to body mass index (BMI) is associated with breast WAT inflammation among 237 prospectively enrolled women. The presence of CLS-B and its severity (CLS-B/cm(2)) as indicators of WAT inflammation correlated with menopausal status (P = 0.008 and P < 0.001) and BMI (P < 0.001 for both). In multivariable analyses adjusted for BMI, the postmenopausal state was independently associated with the presence (P = 0.03) and severity of breast WAT inflammation (P = 0.01). Mean adipocyte size increased in association with CLS-B (P < 0.001). Our findings demonstrate that breast WAT inflammation, which is associated with elevated aromatase levels, is increased in association with the postmenopausal state independent of BMI. Breast WAT inflammation, a process that can potentially be targeted, may help to explain the high incidence of estrogen-dependent tumors in postmenopausal women., (©2015 American Association for Cancer Research.)

The mechanisms by which IL-6 contributes to the pathogenesis of chronic inflammatory diseases and cancer are not fully understood. We previously reported that cyclooxygenase-2 (Cox-2)-dependent PGE2 synthesis regulates macrophage matrix metalloproteinase (MMP)-9 expression, an endopeptidase that participates in diverse pathologic processes. In these studies, we determined whether IL-6 regulates the Cox-2→PGE2→MMP-9 pathway in murine macrophages. IL-6 coinduced Cox-2 and microsomal PGE synthase-1, and inhibited the expression of 15-hydroxyprostaglandin dehydrogenase, leading to increased levels of PGE2. In addition, IL-6 induced MMP-9 expression, suggesting that the observed proteinase expression was regulated by the synthesis of PGE2. However, inhibition of PGE2 synthesis partially suppressed IL-6-mediated induction of MMP-9. In the canonical model of IL-6-induced signaling, JAK activation triggers STAT and MAPK(erk1/2)-signaling pathways. Therefore, the ability of structurally diverse JAK inhibitors to block IL-6-induced MMP-9 expression was examined. Inhibition of JAK blocked IL-6-induced phosphorylation of STAT3, but failed to block the phosphorylation of MAPK(erk1/2), and unexpectedly enhanced MMP-9 expression. In contrast, MEK-1 inhibition blocked IL-6-induced phosphorylation of MAPK(erk1/2) and MMP-9 expression without affecting the phosphorylation of STAT3. Thus, IL-6-induced MMP-9 expression is dependent on the activation of MAPK(erk1/2) and is restrained by a JAK-dependent gene product. Using pharmacologic and genetic approaches, we identified JAK-dependent induction of IL-10 as a potent feedback mechanism controlling IL-6-induced MMP-9 expression. Together, these data reveal that IL-6 induces MMP-9 expression in macrophages via Cox-2-dependent and -independent mechanisms, and identifies a potential mechanism linking IL-6 to the pathogenesis of chronic inflammatory diseases and cancer.

Obesity is a risk factor for the development of hormone receptor-positive breast cancer in postmenopausal women. Estrogen synthesis is catalyzed by aromatase. Recently, we identified an obesity→inflammation→aromatase axis in mouse models and women. In mouse models of obesity, inflammatory foci characterized by crown-like structures (CLS) consisting of dead adipocytes encircled by macrophages were found in the mammary gland. CLS of the breast were found in most overweight and obese women. CLS were associated with adipocyte hypertrophy, activation of NF-κB, elevated levels of proinflammatory mediators and aromatase, and increased expression of the progesterone receptor (PR). Collectively, these findings provide a plausible explanation for the link between obesity, chronic inflammation, and postmenopausal breast cancer. Here, we investigated whether caloric restriction (CR) reversed the inflammatory state and related molecular changes in the mammary gland of obese mice. Obese ovariectomized C57BL/6J mice were subjected to 30% CR for 7 or 14 weeks. Findings in CR mice were compared with the results in mice fed a high-fat diet ad libitum or with control mice fed a low-fat diet. CR was associated with more than a 75% decrease in mammary CLS/cm(2). Reduced histologic inflammation following CR was associated with decreased adipocyte diameter and monocyte chemoattractant protein-1 (MCP-1) levels, reduced NF-κB binding activity, and normalization of levels of proinflammatory mediators, aromatase, and PR. In summary, obesity-related inflammation of the mammary gland and elevated aromatase and PR levels were reversed with CR. Our results provide a rationale for determining whether weight loss can reverse breast inflammation associated with obesity in women.

Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro-nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75(NTR) in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75(NTR) by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75(NTR) in proNGF-expressing mice rescues the phenotype, confirming the importance of p75(NTR)-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75(NTR) limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.

Matrix metalloproteinase (MMP)-9 contributes to the pathogenesis of chronic inflammatory diseases and cancer. Thus, identifying targetable components of signaling pathways that regulate MMP-9 expression may have broad therapeutic implications. Our previous studies revealed a nexus between metalloproteinases and prostanoids whereby MMP-1 and MMP-3, commonly found in inflammatory and neoplastic foci, stimulate macrophage MMP-9 expression via the release of TNF-α and subsequent induction of cyclooxygenase-2 and PGE(2) engagement of EP4 receptor. In the current study, we determined whether MMP-induced cyclooxygenase-2 expression was coupled to the expression of prostaglandin E synthase family members. We found that MMP-1- and MMP-3-dependent release of TNF-α induced rapid and transient expression of early growth response protein 1 in macrophages followed by sustained elevation in microsomal prostaglandin synthase 1 (mPGES-1) expression. Metalloproteinase-induced PGE(2) levels and MMP-9 expression were markedly attenuated in macrophages in which mPGES-1 was silenced, thereby identifying mPGES-1 as a therapeutic target in the regulation of MMP-9 expression. Finally, the induction of mPGES-1 was regulated, in part, through a positive feedback loop dependent on PGE(2) binding to EP4. Thus, in addition to inhibiting macrophage MMP-9 expression, EP4 antagonists emerge as potential therapy to reduce mPGES-1 expression and PGE(2) levels in inflammatory and neoplastic settings.

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

Obesity is a risk factor for the development of hormone receptor-positive breast cancer in postmenopausal women and has been associated with an increased risk of recurrence and reduced survival. In humans, obesity causes subclinical inflammation in visceral and subcutaneous adipose tissue, characterized by necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS). Recently, we found increased numbers of CLS, activation of the NF-κB transcription factor, and elevated aromatase levels and activity in the mammary glands of obese mice. These preclinical findings raised the possibility that the obesity → inflammation axis is important for the development and progression of breast cancer. Here, our main objective was to determine if the findings in mouse models of obesity translated to women. Breast tissue was obtained from 30 women who underwent breast surgery. CLS of the breast (CLS-B) was found in nearly 50% (14 of 30) of patient samples. The severity of breast inflammation, defined as the CLS-B index, correlated with both body mass index (P < 0.001) and adipocyte size (P = 0.01). Increased NF-κB binding activity and elevated aromatase expression and activity were found in the inflamed breast tissue of overweight and obese women. Collectively, our results suggest that the obesity → inflammation → aromatase axis is present in the breast tissue of most overweight and obese women. The presence of CLS-B may be a biomarker of increased breast cancer risk or poor prognosis.

Tumor-associated macrophages and high levels of cyclooxygenase-2 (COX-2) are associated with poor prognosis in breast cancer patients, but their potential interdependence has not been evaluated. The objective of this study was to determine whether macrophages regulate COX-2 expression in breast cancer cells. For this purpose, THP-1 cells were cocultured with HCC1954 breast cancer cells. Coculture led to increased COX-2 expression in the HCC1954 cells and elevated prostaglandin E(2) levels in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 breast cancer cells or when human monocyte-derived macrophages were cocultured with HCC1954 cells. Coculture triggered production of reactive oxygen species (ROS) in HCC1954 cells. COX-2 induction was blocked in cells preincubated with an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or by silencing p67PHOX, a subunit of NADPH oxidase. ROS production triggered activation of Src and mitogen-activated protein kinases (MAPKs). Blocking Src or MAPK activities or antagonizing the activator protein-1 (AP-1) transcription factor attenuated COX-2 induction in HCC1954 cells. Coculture caused rapid induction of interleukin-1β (IL-1β) in both breast cancer cells and macrophages. Increased IL-1β expression was blocked by an interleukin-1 receptor antagonist (IL-1Ra), suggesting autocrine and paracrine effects. Importantly, macrophage-induced COX-2 expression was blocked in HCC1954 cells preincubated with IL-1Ra or anti-IL-1β IgG. Together, these results indicate that macrophage-mediated induction of COX-2 in breast cancer cells is a consequence of IL-1β-mediated stimulation of ROS→Src→MAPK→AP-1 signaling. IL-1β-dependent induction of COX-2 in breast cancer cells provides a mechanism whereby macrophages contribute to tumor progression and potential therapeutic targets in breast cancer.

Matrix metalloproteinase (MMP)-9 (gelatinase B) participates in a variety of diverse physiologic and pathologic processes. We recently characterized a cyclooxygenase-2 (COX-2)-->PGE(2)-->EP4 receptor axis that regulates macrophage MMP-9 expression. In the present studies, we determined whether MMPs, commonly found in inflamed and neoplastic tissues, regulate this prostanoid-EP receptor axis leading to enhanced MMP-9 expression. Results demonstrate that exposure of murine peritoneal macrophages and RAW264.7 macrophages to MMP-1 (collagenase-1) or MMP-3 (stromelysin-1) lead to a marked increase in COX-2 expression, PGE(2) secretion, and subsequent induction of MMP-9 expression. Proteinase-induced MMP-9 expression was blocked in macrophages preincubated with the selective COX-2 inhibitor celecoxib or transfected with COX-2 small interfering RNA (siRNA). Likewise, proteinase-induced MMP-9 was blocked in macrophages preincubated with the EP4 antagonist ONO-AE3-208 or transfected with EP4 siRNA. Exposure of macrophages to MMP-1 and MMP-3 triggered the rapid release of TNF-alpha, which was blocked by MMP inhibitors. Furthermore, both COX-2 and MMP-9 expression were inhibited in macrophages preincubated with anti-TNF-alpha IgG or transfected with TNF-alpha siRNA. Thus, proteinase-induced MMP-9 expression by macrophages is dependent on the release of TNF-alpha, induction of COX-2 expression, and PGE(2) engagement of EP4. The ability of MMP-1 and MMP-3 to regulate macrophage secretion of PGE(2) and expression of MMP-9 defines a nexus between MMPs and prostanoids that is likely to play a role in the pathogenesis of chronic inflammatory diseases and cancer. These data also suggest that this nexus is targetable utilizing anti-TNF-alpha therapies and/or selective EP4 antagonists.

Multiple tobacco smoke-related premalignant and malignant lesions develop synchronously or metachronously in various organ sites, including the oral cavity. Both field cancerization and clonal migration seem to contribute to the occurrence of multiple tumors. Although the importance of endogenous factors (e.g., oncogenes) in regulating clonal migration is well established, little is known about the role of exogenous factors. Hence, the main objective of this study was to elucidate the mechanism by which tobacco smoke stimulated the migration of cells through extracellular matrix (ECM). Treatment of MSK-Leuk1 cells with a saline extract of tobacco smoke induced the migration of cells through ECM. Tobacco smoke induced the expression of urokinase-type plasminogen activator (uPA), resulting in plasmin-dependent degradation of ECM and increased cell migration. AG1478, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, a neutralizing antibody to EGFR, or an antibody to amphiregulin, an EGFR ligand, also blocked tobacco smoke-mediated induction of uPA and cell migration through ECM. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase activity, caused similar inhibitory effects. Taken together, these results suggest that tobacco smoke activated the EGFR-->extracellular signal-regulated kinase 1/2 MAPK pathway, causing induction of uPA. This led, in turn, to increased plasmin-dependent degradation of matrix proteins and enhanced cell migration through ECM. These data strongly suggest that chemicals in tobacco smoke can mimic the effects of oncogenes in regulating uPA-dependent cell invasion through ECM. These findings also strengthen the rationale for determining whether inhibitors of EGFR tyrosine kinase reduce the risk of tobacco smoke-related second primary tumors.

COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1-4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors.

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.

Monocytes and macrophages participate in a wide variety of host defense mechanisms. Annexin II, a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator (t-PA) independently at the cell surface, thereby enhancing the catalytic efficiency of plasmin production. We demonstrated previously that annexin II on the surface of both cultured monocytoid cells and monocyte-derived macrophages promotes their ability to remodel extracellular matrix. Here, we demonstrate that human peripheral blood monocytes represent the major circulating annexin II-expressing cell. Annexin II supported t-PA-dependent generation of cell surface plasmin and the matrix-penetrating activity of human monocytes. Compared to polymorphonuclear leukocytes, monocytes supported a 12.9-fold greater rate of plasmin generation in the presence of exogenous t-PA, and this activity was largely attributable to annexin II. Likewise, anti-annexin II IgG directed against the t-PA-binding tail domain inhibited plasminogen-dependent, cytokine-directed monocyte migration through extracellular matrix. On differentiation of monocytes to macrophages, there was a 2.4-fold increase in annexin II-specific mRNA, and a 7.9-fold increase in surface annexin II. Thioglycolate-elicited peritoneal macrophages, furthermore, displayed an additional 3.8-fold increase in annexin II surface expression compared with resident cells. Thus, annexin II-mediated assembly of plasminogen and t-PA on monocyte/macrophages contributes to plasmin generation, matrix remodeling, and directed migration.

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that alpha1:Ser(2091)-Arg(2108), a peptide derived from the alpha1-chain of laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that alpha1:Ser(2091)-Arg(2108) is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages' proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the alpha1-chain including alpha1:Ser(2091)-Arg(2108) and the globular domain. A peptide from the first loop of the globular domain (alpha1:Ser(2179)-Ser(2198)) triggered the phosphorylation of MAPK(erk1/2) and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of alpha1-chain and stimulated macrophages' proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the alpha1-chain of laminin are exposed by proteolysis and stimulate macrophages' proteinase expression.

Background: Myocardial fibrosis is common in patients with chronic aortic regurgitation (AR). Experimentally, fibrosis with disproportionate noncollagen extracellular matrix (ECM) elements precedes and contributes to heart failure in AR., Methods and Results: We assessed [3H]-glucosamine and [3H]-proline incorporation in ECM, variations in cardiac fibroblast (CF) gene expression, and synthesis of specific ECM proteins in CF cultured from rabbits with surgically induced chronic AR versus controls. To determine whether these variations are primary responses to AR, normal CF were exposed to mechanical strain that mimicked that of AR. Compared with normal CF, AR CF incorporated more glucosamine (1.8:1, P=0.001) into ECM, showed fibronectin gene upregulation (2.0:1, P=0.02), and synthesized more fibronectin (2:1 by Western blot, P<0.06; 1.5:1 by affinity chromatography, P=0.02). Proline incorporation was unchanged by AR (1.1:1, NS); collagen synthesis was unaffected (type I, 0.9:1; type III, 1.0:1, NS). Normal CF exposed to cyclical mechanical strain during culture showed parallel results: glucosamine incorporation increased with strain (2.1:1, P<0.001), proline incorporation was unaffected (1.1:1, NS), fibronectin gene expression (1.6:1, P=0.07) and fibronectin synthesis (Western analysis, 1.3:1, P<0.01; chromatography, 1.9:1, NS) were upregulated., Conclusions: In AR, CF produce abnormal proportions of noncollagen ECM, specifically fibronectin, with relatively little change in collagen synthesis. At least in part, this is a primary response to strain imposed on CF by AR. Further study must relate these findings to the pathogenesis of heart failure in AR.

In response to vascular injury, smooth muscle cells migrate from the media into the intima, where they contribute to the development of neointimal lesions. Increased matrix metalloproteinase (MMP) expression contributes to the migratory response of smooth muscle cells by releasing them from their surrounding extracellular matrix. MMPs may also participate in the remodeling of extracellular matrix in vascular lesions that could lead to plaque weakening and subsequent rupture. Neurotrophins and their receptors, the Trk family of receptor tyrosine kinases, are expressed in neointimal lesions, where they induce smooth muscle cell migration. We now report that nerve growth factor (NGF)-induced activation of the TrkA receptor tyrosine kinase induces MMP-9 expression in both primary cultured rat aortic smooth muscle cells and in a smooth muscle cell line genetically manipulated to express TrkA. The response to NGF was specific for MMP-9 expression, as the expression of MMP-2, MMP-3, or the tissue inhibitor of metalloproteinase-2 was not changed. Activation of the Shc/mitogen-activated protein kinase pathway mediates the induction of MMP-9 in response to NGF, as this response is abrogated in cells expressing a mutant TrkA receptor that does not bind Shc and by pretreatment of cells with the MEK-1 inhibitor, U0126. Thus, these results indicate that the neurotrophin/Trk receptor system, by virtue of its potent chemotactic activity for smooth muscle cells and its ability to induce MMP-9 expression, is a critical mediator in the remodeling that occurs in the vascular wall in response to injury.

It has become increasingly evident that the generation of cell surface proteases including plasmin is fundamental to a wide variety of in vivo biological processes. Cell surface receptors allow for specific controlled proteolysis, provide protection from inhibitors, and enhance catalytic efficiency. Here we describe one such receptor, annexin II, which serves as a coreceptor for tissue plasminogen activator and plasminogen and is found on a wide variety of cell types including endothelial cells, some tumor cells, monocytes and macrophages, and neuronal cells. Evidence indicates that annexin II may be crucial to the efficient generation of cell surface plasmin, endothelial cell formation of new blood vessels, and maintenance of vascular patency. Additionally, it has been shown that annexin II expression in acute promyelocytic leukemia contributes to the bleeding diathesis seen in this disease and that inhibition of annexin II may be an important mechanism in the formation of atherosclerotic plaque. Furthermore, emerging evidence reveals the importance of annexin II on the surface of monocytes and macrophages, where it may contribute to the cells' ability to degrade extracellular matrix proteins and migrate to sites of injury or inflammation.

Genetic evidence demonstrates the importance of plasminogen activation in the migration of macrophages to sites of injury and inflammation, their removal of necrotic debris, and their clearance of fibrin. These studies identified the plasminogen binding protein annexin II on the surface of macrophages and determined its role in their ability to degrade and migrate through extracellular matrices. Calcium-dependent binding of annexin II to RAW264.7 macrophages was shown using flow cytometry and Western blot analysis of EGTA eluates. Ligand blots demonstrated that annexin II comigrates with one of several proteins in lysates and membranes derived from RAW264.7 macrophages that bind plasminogen. Preincubation of RAW264.7 macrophages with monoclonal anti-annexin II IgG inhibited (35%) their binding of 125I-Lys-plasminogen. Likewise, plasmin binding to human monocyte-derived macrophages and THP-1 monocytes was inhibited (50% and 35%, respectively) when cells were preincubated with anti-annexin II IgG. Inhibition of plasminogen binding to annexin II on RAW264.7 macrophages significantly impaired their ability to activate plasminogen and degrade [3H]-glucosamine-labeled extracellular matrices. The migration of THP-1 monocytes through a porous membrane, in response to monocyte chemotactic protein-1, was blocked when the membranes were coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG inhibited (60%) their plasminogen-dependent chemotaxis through the extracellular matrix. These studies identify annexin II as a plasminogen binding site on macrophages and indicate an important role for annexin II in their invasive and degradative phenotype.

Components of the extracellular matrix contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules. The disparate cellular response to extracellular matrix fragments and parent intact molecules suggests differential recognition and signaling pathways. In experiments reported here, we demonstrate that urokinase and matrix metalloproteinase-9 expression by RAW264.7 macrophages is stimulated by a synthetic laminin peptide derived from the alpha1-chain (SRARKQAASIKVAVSADR), whereas intact laminin-1 has no effect on proteinase expression by macrophages. Incubation of macrophages with alpha1:SRARKQAASIKVAVSADR stimulates tyrosine phosphorylation of several proteins including mitogen-activated protein kinase (MAPK)(erk1/2). In contrast, neither intact laminin-1 nor the beta1-chain peptide CDPGYIGSR stimulated protein tyrosine phosphorylation in these cells. Inhibition of tyrosine kinases or protein kinase C blocked alpha1-chain peptide-induced phosphorylation of MAPK(erk1/2) and the up-regulation of steady state levels of urokinase mRNA and matrix metalloproteinase-9 activity. A MAPK kinase inhibitor blocked alpha1-chain-induced phosphorylation of MAPK(erk1/2) and the induction of proteinase expression. Intact laminin-1, which was unable to induce macrophage proteinase expression, failed to stimulate the phosphorylation of MAPK(erk1/2). These data demonstrate that incubation of macrophages with alpha1:SRARKQAASIKVAVSADR, but not intact laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of proteinase expression.

Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin directly or in cooperation with tumor-associated macrophages. In studies reported here, we determined whether angiostatin is generated in mice under non-neoplastic settings. Utilizing murine RAW264.7 macrophages and thioglycollate-elicited peritoneal macrophages, we demonstrate that angiostatin-like fragments are generated as a byproduct of the proteolytic regulation of membrane-bound plasmin. Plasmin proteolysis and subsequent loss in membrane-bound plasmin activity requires active plasmin but was unaffected by inhibitors of metalloproteinases. Lysine binding fragments of plasmin, isolated from macrophage-conditioned media utilizing affinity chromatography, appeared as a major (48 kDa) and two minor bands (42 and 50 kDa) in SDS-polyacrylamide gel electrophoresis and were immunoreactive with anti-kringle 1-3 IgG. Each peptide begins with Lys77 and contains the entire sequence of angiostatin. The affinity isolated plasmin fragments inhibited bFGF-induced endothelial cell proliferation. Lavage fluid recovered from the peritoneal cavities of mice previously injected with thioglycollate contained angiostatin-like plasmin fragments similar to those generated in vitro. This is the first demonstration that angiostatin-like plasmin fragments are generated in a non-neoplastic inflammatory setting. Thus, in addition to regulating pericellular plasmin activity, proteolysis of plasmin generates inactive kringle-containing fragments expressing angiostatic properties.

Macrophage scavenger receptor-type A (MSR-A) has been implicated in the transmission of cell signals and the regulation of diverse cellular functions (Falcone, D. J., and Ferenc, M. J. (1988) J. Cell. Physiol. 135, 387-396; Falcone, D. J., McCaffrey, T. A., and Vergilio, J. A. (1991) J. Biol. Chem. 266, 22726-22732; Palkama, T. (1991) Immunology 74, 432-438; Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601-637); however, the signaling mechanisms are unknown. In studies reported here, we demonstrate that binding of both lipoprotein and non-lipoprotein ligands to MSR-A induced protein tyrosine phosphorylation and increased protein kinase C (PKC) activity leading to up-regulated urokinase-type plasminogen activator (uPA) expression. Specifically, the binding of acetylated low density lipoprotein and fucoidan to MSR-A in human THP-1 macrophages triggered tyrosine phosphorylation of many proteins including phospholipase C-gamma1 and phosphatidylinositol-3-OH kinase. Inhibitors of tyrosine kinase dramatically reduced MSR-induced protein tyrosine phosphorylation and PKC activity. Moreover, inhibitors of tyrosine kinase and PKC reduced uPA activity expressed by THP-1 macrophages exposed to MSR-A ligands. The intracellular signaling response for tyrosine phosphorylation following ligand binding was further demonstrated by using the stable MSR-transfected Bowes cells that express surface MSR-A. These findings establish for the first time a signaling pathway induced by ligand binding to MSR-A and suggest a molecular model for the regulation of macrophage uPA expression by specific ligands of the MSR-A.

Urokinase-type plasminogen activator (uPA) and 92-kDa matrix metalloproteinase (MMP-9) expression by RAW264.7 macrophages were up-regulated when plated on extracellular matrices. Collagen IV, fibronectin, and tenascin stimulated macrophages' MMP-9 expression. In contrast, laminin stimulated both uPA and MMP-9 expression in a dose- and time-dependent manner. The increase in macrophage uPA activity was preceded by an increase in their steady state levels of uPA mRNA. Laminin-induced uPA expression was most pronounced in RAW264.7 macrophages followed by THP-1 monocytes, J774A.1 macrophages, and bone marrow-derived macrophages. Neither laminin nor matrix induced alterations in THP-1 monocyte expression of plasminogen activator inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-2. Synthetic laminin peptides were utilized to identify the laminin domain(s) responsible for induction of uPA expression. Peptides derived from the beta1 chain of laminin had no effect on macrophage uPA expression, whereas SIKVAV, derived from alpha1 chain, stimulated uPA expression 20-fold. Preincubation of THP-1 monocytes with a monoclonal antibody directed against the alpha6 subunit of the alpha6beta1 laminin receptor blocked matrix induction of uPA without affecting the induction of MMP-9. These results demonstrate that macrophage binding to laminin plays an important role in the regulation of their degradative phenotype via the up-regulation of uPA and MMP-9.

A rounded evaluation of the national health insurance proposals that now seem to be taken seriously by political elites requires conceptual organization. This article adopts a typology that describes each major proposal as a social, mixed or a private insurance scheme depending on the source(s) of funding, method of compensating hospitals and physicians, the unit of payment, and mechanism for financing capital. Not surprisingly, the analysis suggests that the social insurance model, closely resembling the Canadian system, is more likely to control inflation and redress distributional inequities than are other approaches. Why, then, has this approach not been adopted? The answer may be found in the widespread acceptance of disjointed incrementalism as a valid description of the policy process which yields an ideological orientation that can be termed "prescriptive incrementalism." This orientation is closely related to a belief in an "American exceptionalism," a belief that is not warranted by a cross-sectional examination of the political culture infusing issues about the proper role of government in health care financing and delivery. Unfortunately for advocates, the truly exceptional factor restricting the United States' ability to effect national health reform is a quite delberately obstruction-oriented political structure.

Using discriminant analyses of data on 916 returned questionnaires from a mailing to 1,650 administrators in 82 South Carolina hospitals, this study examines the allocation of interpersonal, informational, decisional, and treatment roles among executive, administrative, and clinical directors. Educational attainment, years of experience, and gender were found to influence respondents' positions. Results also indicate that executive directors assume responsibility for the organization and its relation to the environment. As expected, those in clinical and administrative positions assume more responsibility for interpersonal and treatment roles than do executive directors.

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor-beta 1 (TGF-beta 1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-beta 1 induction of uPA expression by THP-1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF-beta 1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF-beta 1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-beta 1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF-beta 1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity. Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-beta 1. The increase in membrane-uPA activity expressed by TGF-beta 1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-beta 1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF-beta 1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-beta 1-treated cells. However, no change in immunoreactive membrane-bound plasmin(ogen) was observed. In addition, binding of 125I-Lys-plasminogen to THP-1 cells was not affected by TGF-beta 1 treatment. We conclude that TGF-beta 1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression.

Restenosis occurs in 35% of patients within months after balloon angioplasty, due to a fibroproliferative response to vascular injury. These studies describe a combined fibrosuppressive/antiproliferative strategy on smooth muscle cells cultured from human primary atherosclerotic and restenotic coronary arteries and from normal rat aortas. L-Mimosine suppressed the posttranslational hydroxylation of the precursors for collagen and for eukaryotic initiation factor-5A (eIF-5A) by directly inhibiting the specific protein hydroxylases involved, prolyl 4-hydroxylase (E.C. 1.14.11.2) and deoxyhypusyl hydroxylase (E.C. 1.14.99.29), respectively. Inhibition of deoxyhypusyl hydroxylation correlated with a dose-dependent inhibition of DNA synthesis. Inhibition of prolyl hydroxylation caused a dose-dependent reduction in the secretion of hydroxyproline-containing protein and decreased the formation of procollagen types I and III. The antifibroproliferative action could not be attributed to nonspecific or toxic effects of mimosine, appeared to be selective for the hydroxylation step in the biosynthesis of the procollagens and of eIF-5A, and was reversible upon removal of the compound. The strategy of targeting these two protein hydroxylases has important implications for the pathophysiology of restenosis and for the development of agents to control fibroproliferative diseases.

The activation of plasminogen by macrophage is regulated by their expression of receptors for urokinase and plasmin(ogen). In these studies we have examined plasmin(ogen) binding to adherent human THP-1 macrophage. Plasmin bound to the THP-1 cells in a time- and dose-dependent manner (Kd 15.8 +/- 6.2 nM; Bmax 1.4 +/- 0.3 x 10(6)/cell). The lysine analog epsilon-aminocaproic acid competitively inhibited plasmin binding. The fraction of membrane-bound plasmin, however, became increasingly resistant to displacement with epsilon-aminocaproic acid. Over a 24-h period, membrane-bound plasmin activity fell 80% despite the presence of catalytically active plasmin in the incubation media. The loss of receptor-bound plasmin activity was not due to proteolytic alterations of its receptor since 125I-Lys-plasminogen bound to THP-1 cells pretreated with plasmin with similar affinity as to untreated cells. Following a 24-h incubation of 125I-Lys-plasminogen or 125I-plasmin with THP-1 cells, several degradative fragments were apparent in their conditioned media. The smaller degradative fragments (28 and 36 kDa) lacked cell binding activity and were demonstrated to be active by casein-zymography. A 48-kDa fragment bound to cells in a lysine-dependent manner but was not active. In contrast, phenylmethylsulfonyl fluoride-inactivated 125I-plasmin retained its binding activity over 24 h, and degradative fragments were not present in the conditioned media. The binding of 125I-Lys-plasmin(ogen) to THP-1 cells was also examined in the presence of excess alpha 2 plasmin inhibitor. Despite the absence of fluid-phase plasmin activity, membrane-bound 125I-Lys-plasmin(ogen) decreased over 24 h. At 24 h a radiolabeled 48-kDa fragment was observed in the conditioned media and together with 125I-Lys-plasmin(ogen) was bound to cells. Unlike 125I-Lys-plasmin, the 48-kDa fragment did not form a complex with alpha 2 plasmin inhibitor. Thus, autoproteolysis of receptor-bound plasmin results in fragments with truncated physiologic properties that possess either cell binding or catalytic activities. We propose that autoproteolysis is a mechanism for regulating membrane-bound plasmin activity.

Over 10,000 Oklahomans die each year from coronary artery disease or stroke. This study examined the behavioral risk factors for these illnesses present in Oklahomans. Oklahomans are at considerably higher risk than the desirable national goals for such risk factors. For example, too many Oklahomans smoke and too few exercise. The data to support these findings are included herein, and some steps to reverse these trends are recommended.