Risk-reducing appendectomy and the elimination of BRCA1-associated intraperitoneal cancer.

Risk-reducing bilateral salpingo-oophorectomy (RRBSO) and risk-reducing mastectomy are widely used for BRCA1 and BRCA2 mutation carriers to reduce the risk of ovarian and breast cancer. To our knowledge, no risk-reduction therapy has addressed the BCRA1/2 carrier lifetime risk of intra-abdominal peritoneal carcinoma from an appendix source. We identified a BRCA1 carrier in a hereditary breast and ovarian cancer kindred who developed a low-grade malignant appendiceal mucocele 2 years after risk-reducing salpingo-oophorectomy. Our retrospective meta-analysis assessed the risk of intraperitoneal appendiceal cancer in BRCA1/2 carriers after RRBSO to determine whether elective risk-reduction appendectomy could reduce the incidence of intraperitoneal cancer. Data sources included the case report and 12 reports of BRCA1 and BRCA2 carriers after RRBSO with ovarian, fallopian tube, breast, and peritoneal cancer published from January 1, 1985, through April 30, 2012. Main outcome measures were nonovarian, non-fallopian tube, nonbreast, positive intra-abdominal peritoneal carcinoma in previously cancer-free BRCA1/2 carriers after RRBSO. The source of intraperitoneal cancer in BRCA1/2 carriers after risk-reducing salpingo-oophorectomy is highly likely the appendix. Use of risk-reduction appendectomy with RRBSO in younger BRCA1/2 carriers may reduce lifetime risk of malignant tumor and eliminate intraperitoneal cancer.

Role of endothelial nitric oxide synthase in the development of portal hypertension in the carbon tetrachloride-induced liver fibrosis model.

Portal hypertension (PHT) is a complication of liver cirrhosis and directly increases mortality and morbidity by increasing the propensity of venous hemorrhage. There are two main underlying causations for PHT, increased hepatic resistance and systemic hyperdynamic circulation. Both are related to localized aberrations in endothelial nitric oxide synthase (eNOS) function and NO biosynthesis. This study investigates the importance of eNOS and systemic hyperdynamic-associated hyperemia to better understand the pathophysiology of PHT. Wild-type and eNOS(-/-) mice were given the hepatotoxin CCl(4) for 4-12 wk. Hepatic fibrosis was determined histologically following collagen staining. Portal venous pressure, hepatic resistance, and hyperemia were determined by measuring splenic pulp pressure (SPP), hepatic portal-venous perfusion pressure (HPVPP), abdominal aortic flow (Qao), and portal venous flow (Qpv). Hepatic fibrosis developed equally in wild-type and eNOS(-/-) CCl(4)-exposed mice. SPP, Qao, and Qpv increased rapidly in wild-type CCl(4)-exposed mice, but HPVPP did not. In eNOS(-/-) CCl(4) mice, Qao was not increased, SPP was partially increased, and HPVPP and Qpv were increased nonsignificantly. We concluded that the systemic hyperemia component of hyperdynamic circulation is eNOS dependent and precedes increased changes in hepatic resistance. Alternative mechanisms, possibly involving cyclooxygenase, may contribute. eNOS maintains normal hepatic resistance following CCl(4)-induced fibrosis. Consequently, increased portal pressure following chronic CCl(4) exposure is linked to hyperdynamic circulation in wild-type mice and increased hepatic resistance in eNOS(-/-) mice.

Long-term effects on diet after proctocolectomy for ulcerative colitis.

BACKGROUND: Patients with ulcerative colitis (UC) often report dietary intolerances. Our aim was to assess the effects of proctocolectomy (PC) for UC on dietary intolerances. METHODS: A novel disease-specific questionnaire was used. RESULTS: Eighty-seven percent of patients reported 338 dietary intolerances. Of 225 preoperative dietary intolerances, 151 (67%) resolved/improved, 56 (25%) were unchanged, and 18 (8%) were exacerbated after PC. A total of 113 dietary intolerances developed only after PC. The incidence of specific dietary intolerances in patients 10 years and older post-PC was similar to patients younger than 10 years post-PC except for a lower incidence of caffeinated beverage (P = .01) dietary intolerances 10 years or more post-PC. Intestinal symptoms, bowel function, and activities of daily living largely improved after PC. Extraintestinal UC symptoms worsened or failed to improve in 74%. CONCLUSIONS: PC for UC frequently improves preoperative dietary intolerances. Some patients, however, are at risk for onset of new dietary intolerances after PC. Studies examining traditional symptoms in UC patients pre-PC and post-PC may be enhanced by examining effects on specific dietary intolerances.

Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes.

UNLABELLED: Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31, 56, 84% decrease for 10, 50 and 100 microM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26 and 45% inhibition at 10 and 50 microM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. CONCLUSIONS: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo.

Resveratrol, a polyphenolic phytostilbene, inhibits endothelial monocyte chemotactic protein-1 synthesis and secretion.

BACKGROUND/AIMS: Resveratrol is a naturally occurring polyphenol phytoestrogen and one of several constituents of red wine thought to be cardioprotective. We investigated the effect of resveratrol on the expression of the atherogenic chemokine, monocyte chemotactic protein-1 (MCP-1). METHODS: Human umbilical vein endothelial cells were stimulated with interleukin-1beta (IL-1beta) in the absence or presence of resveratrol. MCP-1 levels were determined by ELISA and MCP-1 mRNA was measured. RESULTS: Resveratrol (1-100 microM) dose-dependently inhibited IL-1beta-stimulated MCP-1 secretion, with approximately 45% inhibition at 50 microM resveratrol. This was a Gi-protein- and NO-dependent effect. Resveratrol also significantly inhibited MCP-1 gene expression in a Gi-protein-dependent but NO-independent manner. While resveratrol had no effect on MCP-1 mRNA degradation, it inhibited MCP-1 promoter activity and reduced nuclear factor kappaB and activator protein-1 binding activity induced by IL-1beta. Moreover, while hemoxygenase-1 (HO-1) expression was induced by resveratrol in human umbilical vein endothelial cells, neither treatment with the HO-1 inhibitor tin-protoporphyrin IX nor siRNA-directed knockdown of HO-1 had any effect on the inhibition of MCP-1 mRNA or protein secretion by resveratrol. CONCLUSION: These data demonstrate an inhibitory effect of resveratrol on MCP-1 synthesis and secretion, mediated via distinct signaling pathways. The inhibition of MCP-1 may represent a novel cardioprotective mechanism of resveratrol.

Ethanol inhibits pulse pressure-induced vascular smooth muscle cell migration by differentially modulating plasminogen activator inhibitor type 1, matrix metalloproteinase-2 and -9.

We investigated the effect of ethanol on the pulse pressure-induced expression of PAI-1 and MMP-2/9 in human smooth muscle cells (SMC). Human SMC were exposed to static or pulse pressure (25 mL/min; pulse pressure 106/50 mm Hg) conditions for 24 h in the absence or presence of ethanol (0.1-100 mM). SMC migration was then measured by Transwell migration assay. SMC exposed to pulse pressure demonstrated a significant increase in PAI-1 mRNA and protein expression (approximately 4-fold and approximately 3-fold) concomitant with a 3- and 8-fold increase in MMP-2 and MMP-9 protein, respectively. Ethanol dose-dependently inhibited the pulse pressure-induced SMC migration with complete inhibition observed at 20 mM. There was no effect of ethanol on basal PAI-1 or MMP-2/9 in SMC under static conditions. However, ethanol significantly enhanced the pulse pressure-induced PAI-1 mRNA and protein expression (2.2 +/- 0.52 fold and 2.5 +/- 0.27 fold, for 10 mM), respectively. In contrast, ethanol dose-dependently inhibited the pulse pressure-induced increases in MMP-9 protein and pro-MMP-9 activity and to a lesser extent MMP-2 mRNA and protein and pro-MMP-2 activity, with significant inhibition observed at 1 mM. These data provide a molecular mechanism mediating the inhibitory effect of ethanol on pulse-pressure-induced SMC migration and may be relevant to the cardioprotective effects of ethanol in vivo.

Ethanol inhibits monocyte chemotactic protein-1 expression in interleukin-1{beta}-activated human endothelial cells.

The aim of this study was to determine the effect of ethanol (EtOH) on endothelial monocyte chemotactic protein-1 (MCP-1) expression. IL-1beta increased the production of MCP-1 by human umbilical vein endothelial cells from undetectable levels to approximately 900 pg/ml at 24 h. EtOH dose-dependently inhibited IL-1beta-stimulated MCP-1 secretion as determined by ELISA: 25 +/- 1%, 35 +/- 7%, and 65 +/- 5% inhibition for 1, 10, and 100 mM EtOH, respectively, concomitant with inhibition of monocyte adhesion to activated endothelial cells. Similarly, EtOH dose-dependently inhibited IL-1beta-stimulated MCP-1 mRNA expression. Experiments with actinomycin D demonstrated that EtOH decreased the stability of MCP-1 mRNA. In addition, EtOH significantly reduced NF-kappaB and AP-1 binding activity induced by IL-1beta and inhibited MCP-1 gene transcription. Binding of (125)I-labeled MCP-1 to its receptor (CCR2) on THP-1 human monocytic cells was not affected by EtOH treatment. Modulation of the expression of MCP-1 represents a mechanism whereby EtOH could inhibit atherogenesis by blocking the crucial early step of monocyte adhesion and subsequent recruitment to the subendothelial space. These actions of EtOH may underlie, in part, its cardiovascular protective effects in vivo.

A technique for emergency liver packing.

Control of liver hemorrhage may present a daunting clinical scenario. Use of liver packing techniques is highly effective to control bleeding but can result in significant recurrent bleeding with pack removal. Such bleeding is particularly a problem when large portions of the hepatic parenchymal surface and Glisson's capsule have been disrupted. We describe, herein, our approach to hepatic packing in scenarios where a large component of hepatic capsular disruption has occurred. Use of a non-stick bowel bag is employed on the disrupted liver surface, which, when removed, will not result in liver rebleeding. This technique has been used successfully in the management of five cases of severe liver injury with extensive capsular disruption. Familiarity with such an approach may facilitate management of similar liver injuries.

Pulse pressure-induced transmural fluid flux increases bovine aortic smooth muscle cell apoptosis in a mitogen activated protein kinase dependent manner.

BACKGROUND: Mechanical forces associated with blood flow are critical in the regulation of vascular smooth muscle cell (VSMC) growth, migration, differentiation, and apoptosis as fundamental features in the pathogenesis of vascular disease. We investigated the effect of pulse pressure on VSMC apoptosis. METHODS: Using a perfused transcapillary co-culture system, bovine thoracic aortic SMC (BASMC) were exposed to increases in pulsatile flow (0.3-17 ml/min) hence pulse pressure (amplitude of pulse 6-50 mmHg in the absence or presence of bovine aortic endothelial cells (BAEC). The extent of apoptosis was determined by measuring caspase-3 activity, the levels of pro- and anti-apoptotic Bcl-2 family proteins, FasL and cellular apoptosis susceptibility (CAS) protein expression and the extent of DNA fragmentation. RESULTS: Changes in pulse pressure resulted in a significant force- and time-dependent increase in caspase-3 activity in BASMC. This effect was maximal after 6 h, independent of BAEC presence, and attenuated following inhibition of mitogen-activated protein kinase (MAPK) activity with PD98059. In parallel cultures, there was a significant increase in Bad and Bax expression, concomitant with an increase in DNA fragmentation, and a significant decrease in Bcl-2 and Bcl-XL expression. The pro-apoptotic effects of pulse pressure were specific differentiated cells but independent of p53, in as much as FasL and CAS expression were enhanced in differentiated adult but decrease in de-differentiated embryonic cells in response to flow. CONCLUSIONS: These results suggest that pulse pressure promotes phenotypically distinct VSMC apoptosis in vitro in an endothelial-independent, MAPK-dependent, manner.

Plasminogen activator inhibitor-1 deficiency enhances flow-induced smooth muscle cell migration.

INTRODUCTION: We determined the role of smooth muscle cell (SMC)-derived plasminogen activator inhibitor-1 (PAI-1) in the flow-induced SMC migratory response. MATERIALS AND METHODS: Wild type (wt) or PAI-1 knockout SMC were cultured in the absence or presence of endothelial cells (EC) under static or pulsatile flow conditions in a perfused culture system. SMC migration was then assessed by Transwell assay. RESULTS: Pulsatile flow significantly increased SMC PAI-1 mRNA and protein levels, approximately 4- and 3-fold respectively (n = 4, p < 0.05). In the absence, but not in the presence of EC, pulsatile flow significantly increased ( approximately 2.4-fold) the migration of wt SMC when compared to wt SMC cultured under static conditions. PAI-1 -/-SMC migration was significantly increased under flow conditions as compared to wild-type controls (334 +/- 22% vs. 237 +/- 11%, n = 6, p < 0.05). This flow-induced migration was significantly attenuated, but not completely inhibited, when PAI-1 -/-SMC were cultured in the presence of EC (147 +/- 13%, n = 6, p < 0.05). The flow-induced PAI-1 -/-SMC migratory response was partially inhibited by an anti-urokinase plasminogen activator (uPA) antibody (#1189), and completely inhibited by both 1189 and the matrix metalloproteinase (MMP) inhibitor BB3103. In parallel PAI-1 -/-SMC cells, there was a greater flow-induced increase in proMMP-2 activity as compared to wild-type control cells. Moreover, under both static and flow conditions, tissue inhibitors of matrix metalloproteinases (TIMP)-2 activity was reduced in these PAI-1-deficient cells as compared to wild-type controls. CONCLUSIONS: These results suggest that SMC PAI-1 plays a role in limiting flow-induced SMC migration and thus may be an important mechanism for controlling the process of vascular remodelling.

Two third-year medical student-level laboratory shock exercises without large animals.

BACKGROUND: Historically, medical schools have taught principles of hemodynamic shock using large animal models. Such exercises are infrequent today due to the increasing aversion of students and the wider community to the use of large animals in teaching. Herein, we describe two alternative exercises that communicated basic science and clinical principles of shock effectively. METHODS: We developed two complementary, distinct single-afternoon laboratory exercises for third-year medical students. The first exercise (lab) demonstrated three principles: (1) in vitro cytokine-induced apoptosis (illustrating mechanisms and consequences of sepsis), (2) the hemodynamic manifestations of hypovolemia and septic shock in rats, and (3) the effects of fluid resuscitation or vasopressor administration in these same rat models. In the second exercise, students managed the diagnosis, initial resuscitation, surgical treatment, and ICU care of a "patient" with abdominal sepsis, using a manikin-based patient simulator and actual patient test data. Current basic science and clinical literature were incorporated. RESULTS: Efficacy was evaluated by polling students in one of four rotations (n = 25). Educational value of the lab exercise was rated 3.70 (1, worst rating; 5, best rating), whereas its applicability to clinical care was rated 4.35. Educational value and clinical applicability of the patient simulator were rated 4.52 and 4.76, respectively. CONCLUSIONS: These exercises combining laboratory demonstrations of the pathophysiologic mechanisms and manifestations of shock with simulation were judged effective and clinically relevant while fulfilling the National Institutes of Health (NIH) mandate to reduce use of experimental animals.

Eicosanoids in cirrhosis and portal hypertension.

In the last decade, the knowledge of the pathogenesis of portal hypertension and cirrhosis has increased dramatically. In portal hypertension, almost all the known vasoactive systems/substances are activated or increased and the most recent studies have stressed the importance of the endothelial factors, in particular, prostaglandins. Prostaglandins are formed following the oxygenation of arachidonic acid by the cyclooxygenase (Cox) pathway. An important consideration in portal hypertension and cirrhosis in the periphery is the altered hemodynamic profile and its contributory role in controlling endothelial release of these vasoactive substances. Prostaglandins are released from the endothelium in response to both humoral and mechanical stimuli and can profoundly affect both intrahepatic and peripheral vascular resistance. Within the liver, intrahepatic resistance is altered due to a diminution in sinusoidal responsiveness to vasodilators and an increase in prostanoid vasoconstrictor responsiveness. This review will examine the contributory role of both hormonal and/or hemodynamic force-induced changes in prostaglandin production and signaling in cirrhosis and portal hypertension and the consequence of these changes on the structural and functional response of both the vasculature and the liver.

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice.

BACKGROUND & AIMS: Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. METHODS: The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. RESULTS: In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 +/- 0.37 vs 17.57 +/- 0.51 cmH(2)O, at 14 days) concomitant with a significant increase in Qao (0.12 +/- 0.003 vs 0.227 +/- 0.005 mL/min/g) and portosystemic shunt (0.47% +/- 0.01% vs 84.13% +/- 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 +/- 0.22 vs 8.07 +/- 0.4 cmH(2)0) or Qao (0.111 +/- 0.01 vs 0.14 +/-.023 mL/min/g). However, eNOS (-/-) mice did develop a substantial portosystemic shunt (0.33% +/- 0.005% vs 84.53% +/- 0.19% shunt), comparable to that seen in wild-type animals after PVL. CONCLUSIONS: These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.

Can ethanol therapies injure the bile ducts?

Administration of ethanol, whether applied directly to tissue or via an intra-arterial route, has been widely used to treat a variety of hepatic disorders, most importantly hepatocellular carcinoma. Animal studies, however, have demonstrated that intravascular hepatic ethanol therapy causes significant bile duct injury, and therefore, many centers have used intravascular ethanol applications with caution. A case of extensive bile duct injury in a 35-year-old female with a symptomatic hepatic hemangioma treated with ethanol embolization is presented. Although a seemingly rare complication, the possibility of bile duct injury should be considered prior to planned ethanol liver treatment, and a high index of suspicion should be maintained should signs of hepatic dysfunction subsequently arise.

Pulsatile flow-induced angiogenesis: role of G(i) subunits.

OBJECTIVE: Angiogenesis plays a key role in the growth and function of normal and pathological tissues. We investigated the effect of pulsatile flow on endothelial cell (EC) in vitro angiogenic activity. METHODS AND RESULTS: Bovine aortic ECs were exposed to "static" or "flow" (1.2 to 67.0 mL/min, shear stress 1.4 to 19.2 dyne/cm2) conditions for 2 to 24 hours. After exposure, angiogenesis was measured as tubule formation on Matrigel, and EC migration was assessed by filter migration assay. Pulsatile flow increased angiogenesis and EC migration in a temporal and force-dependent manner, with a maximal effect at 16 hours (13.2 dyne/cm2). Pertussis toxin completely inhibited the effect of pulsatile flow on angiogenesis and migration. Transfection of ECs with inhibitory mutants of the alpha subunit of G(i)1 or G(i)3, but not G(i)2, inhibited the flow-induced angiogenic response by 61+/-2% and 32+/-6%, respectively, whereas transfection with constitutively activated mutants of the alpha subunit of G(i)1 or G(i)3, but not G(i)2, increased the flow-induced response by 202+/-23% and 70+/-4%, respectively. In contrast, inhibition of Gbetagamma by the carboxy terminal fragment of beta-adrenergic receptor kinase overexpression increased the flow-induced response by 82+/-8%. CONCLUSIONS: These results suggest that pulsatile flow stimulates angiogenesis and that this effect is mediated by activation of G(ialpha)1 or G(ialpha)3, but not Gbetagamma, subunits.

Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma.

Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P <.05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% +/- 9.4% vs. control, n = 4, P <.05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% +/- 8% increase vs. control, n = 4, P <.05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.

Ethanol differentially modulates the expression and activity of cell cycle regulatory proteins in rat aortic smooth muscle cells.

The aim of this study was to determine the effect of ethanol on cell cycle events during the G(1) and S phases in cultured vascular smooth muscle cells (VSMC). Flow cytometric analysis for the DNA content in rat aortic VSMC indicated that following ethanol treatment, the cell population in the G(0)/G(1) phase increased; 57.8+/-1.6% vs. 72.3+/-1.2%, concomitant with a decrease in cells in the S phase; 12.7+/-1.4% vs. 3.67+/-0.6%, for control vs. ethanol, respectively. Western blot analysis on VSMC lysates demonstrated that ethanol (10-160 mmol/l) dose-dependently inhibited serum-induced retinoblastoma (pRb) hyperphosphorylation. While having no effect on Cdk2 protein expression, ethanol dose-dependently decreased (IC(50) approximately 60 mmol/l) Cdk2 activity, assessed by histone H1 phosphorylation. Furthermore, ethanol induced the expression of the cyclin-dependent kinase (Cdk) inhibitor p21(waf1/cip1), and inhibited the induction of cyclin A. These data demonstrate that modulation of the expression and activity of key cell cycle regulatory molecules may be a mechanism by which ethanol inhibits VSMC proliferation. These actions of ethanol may be relevant to its cardiovascular protective effect in vivo.