3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa.

Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughput in vitro platform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficient in vitro toxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.

Breast Cancer Cells Transition from Mesenchymal to Amoeboid Migration in Tunable Three-Dimensional Silk-Collagen Hydrogels.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

The bio-gripper: a fluid-driven micro-manipulator of living tissue constructs for additive bio-manufacturing.

We previously developed the Bio-Pick, Place, and Perfuse (Bio-P3) instrument to fabricate large perfusable tissue constructs by stacking and aligning scaffold-free living microtissues with integrated lumens. The Bio-P3 required an actuating mechanism to manipulate living microtissues of various sizes and shapes that are fragile, and must remain in an aqueous environment. The optical transparency of the Bio-P3 gripping device was essential to provide unobstructed visuals for accurate alignment of microtissues. We previously engineered a pilot fluid force-driven bio-gripper that can pick-and-place microtissue in planar position without causing cellular damage by pulling culture medium through track-etched membrane-integrated cell culture inserts. In this study, we invented a new flexible bio-gripper design that maximized the bio-gripper utilities. We utilized experimental approaches, multivariate analyzes, and theoretical modeling to elucidate how membrane characteristics (pore size, pore density, membrane thickness, membrane area, and surface chemistry) altered bio-gripper robustness and the flow rate (Q(c)) required for successful gripping. We devised two standardized tests and synthetic parts that mimicked microtissues, to systematically quantify bio-gripper performance. All thirteen syringe pump-driven bio-grippers except one successfully gripped and released synthetic parts with values of Q(c) that coincided with our mathematical simulation of the fluid mechanics of gripping. The bio-gripper could grip synthetic parts of various sizes, shapes and masses, demonstrating the robustness of the actuating mechanism. Multivariate analysis of experimental data indicated that both membrane porosity and thickness modulated Q(c), and in addition, revealed that membrane pore density determined membrane optical transparency. Fabricating large tissue constructs requires repeated stacking of microtissues. We showed that one bio-gripper could pick-and-place living microtissues thirty times with Q(c) corresponding to our simulation. Our bio-gripper was capable of stacking and aligning twenty microtissues. In summary, we successfully engineered a robust controllable fluid-driven bio-gripper to efficiently manipulate living microtissues and micro-objects in an aqueous environment.

Tumor progression locus 2 ablation suppressed hepatocellular carcinoma development by inhibiting hepatic inflammation and steatosis in mice.

BACKGROUND: Tumor progression locus 2 (TPL2), a serine-threonine kinase, functions as a critical regulator of inflammatory pathways and mediates oncogenic events. The potential role of Tpl2 in nonalcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) development remains unknown. METHODS: Both wild-type and Tpl2 knockout male mice were initiated by a hepatic carcinogen (diethylnitrosamine, i.p. with a single dose of 25 mg.kg(-1))at 2 weeks of age, and then were given the high carbohydrate diet feeding to induce hepatic steatosis, inflammation, adenoma and HCC for 24 weeks. RESULTS: Tpl2 knockout mice had significantly lower incidences of liver tumor and developed hepatocellular adenoma only, which is contrast to wild-type mice where they all developed HCC. Tpl2 knockout mice had significantly down-regulated phosphorylation of JNK and ERK, and levels of mRNA expression of pro-inflammatory cytokines (Il-1ß, Il-18, Mcp-1 and Nalp3), which correlated with the reduced incidence and number of hepatic inflammatory foci. Furthermore, Tpl2 ablation resulted in decreased hepatic steatosis and expression of de novo lipogenesis related markers (ACC, SCD1, SREBP1C and AKT phosphorylation), as well as reduction of endoplasmic reticulum stress biomarkers PERK and eIF-2a. CONCLUSION: The study revealed for the first time that Tpl2 plays a significant role in promoting HCC development by its pro-inflammatory effect, which suggested that Tpl2 could be a molecular target for HCC prevention.

Lycopene and apo-10'-lycopenoic acid have differential mechanisms of protection against hepatic steatosis in ß-carotene-9',10'-oxygenase knockout male mice.

BACKGROUND: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10'-lycopenoic acid (APO10LA), a potential oxidation product of apo-10'-lycopenal that is generated endogenously by ß-carotene-9',10'-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. OBJECTIVE: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. METHODS: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. RESULTS: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator-activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death-inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA ß-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. CONCLUSIONS: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids.

Lycopene attenuated hepatic tumorigenesis via differential mechanisms depending on carotenoid cleavage enzyme in mice.

Obesity is associated with increased liver cancer risks and mortality. We recently showed that apo-10'-lycopenoic acid, a lycopene metabolite generated by beta-carotene-9',10'-oxygenase (BCO2), inhibited carcinogen-initiated, high-fat diet (HFD)-promoted liver inflammation, and hepatic tumorigenesis development. The present investigation examined the outstanding question of whether lycopene could suppress HFD-promoted hepatocellular carcinoma (HCC) progression, and if BCO2 expression is important using BCO2-knockout (BCO2-KO) and wild-type male mice. Results showed that lycopene supplementation (100 mg/kg diet) for 24 weeks resulted in comparable accumulation of hepatic lycopene (19.4 vs. 18.2 nmol/g) and had similar effects on suppressing HFD-promoted HCC incidence (19% vs. 20%) and multiplicity (58% vs. 62%) in wild-type and BCO2-KO mice, respectively. Intriguingly, lycopene chemopreventive effects in wild-type mice were associated with reduced hepatic proinflammatory signaling (phosphorylation of NK-κB p65 and STAT3; IL6 protein) and inflammatory foci. In contrast, the protective effects of lycopene in BCO2-KO but not in wild-type mice were associated with reduced hepatic endoplasmic reticulum stress-mediated unfolded protein response (ER(UPR)), through decreasing ER(UPR)-mediated protein kinase RNA-activated like kinase-eukaryotic initiation factor 2α activation, and inositol requiring 1α-X-box-binding protein 1 signaling. Lycopene supplementation in BCO2-KO mice suppressed oncogenic signals, including Met mRNA, ß-catenin protein, and mTOR complex 1 activation, which was associated with increased hepatic microRNA (miR)-199a/b and miR214 levels. These results provided novel experimental evidence that dietary lycopene can prevent HFD-promoted HCC incidence and multiplicity in mice, and may elicit different mechanisms depending on BCO2 expression.

High-refined-carbohydrate and high-fat diets induce comparable hepatic tumorigenesis in male mice.

Previous studies demonstrated that diet-induced obese mice fed a semi-purified high-fat diet (HFD) had greater liver tumorigenesis than mice fed a non-semi-purified diet. Because ingredients present in standard unpurified diets may elicit potential chemopreventive properties that are not present in semi-purified diets, the present study evaluated hepatic tumorigenic effects of dietary fat by replacing it with refined carbohydrates [digestible saccharides; high-carbohydrate diet (HCD)] in a semi-purified diet without altering other components. Two-wk-old C57Bl/6J male mice were randomly injected i.p. with either the liver-specific carcinogen diethylnitrosamine (25 mg/kg body weight) to induce liver cancer or saline as the nontumor control. At age 6 wk, mice with or without cancer initiation were further randomly assigned to an HFD (26% and 60% energy from carbohydrates and fat, respectively) or an HCD (66% and 12% energy from carbohydrates and fat, respectively) and consumed food ad libitum for 24 wk. Results showed that HCD-fed mice had a comparable degree of hepatic tumorigenesis (tumor number and volume) as HFD-fed mice, despite having significantly reduced body weights. HCD feeding induced greater hepatic endoplasmic reticulum (ER) stress-mediated protein kinase RNA-activated-like kinase (PERK) activation and oncogenic interleukin-6/signal transducer and activator of transcription 3 signaling than HFD feeding. HCD-stimulated PERK signaling was associated with elevated expression of prosurvival markers in tumors, including induced protein kinase B activation, increased extracellular signal-regulated kinases 1/2 phosphorylation, and elevated cyclin D1 protein expression. However, HCD-mediated PERK activation in tumors was also positively associated with markers of proapoptosis, which included elevated CCAAT/enhancer-binding protein homology protein expression and increased cleaved caspase-3. HCD-fed mice had greater severity in hepatic steatosis than HFD-fed mice. HCD-induced steatosis exacerbation was associated with increased expression in hepatic de novo lipogenic markers that can promote ER stress. Together, these data indicated that chronic HCD consumption by mice can produce comparable severity of hepatic tumorigenesis as HFD consumption, potentially through upregulating PERK-mediated ER stress.

Lycopene metabolite, apo-10'-lycopenoic acid, inhibits diethylnitrosamine-initiated, high fat diet-promoted hepatic inflammation and tumorigenesis in mice.

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10'-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9',10'-double bond by carotene-9',10'-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)-promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation.

Aggravation of nonalcoholic steatohepatitis by moderate alcohol consumption is associated with decreased SIRT1 activity in rats.

Chronic alcohol intake decreases adiponectin and sirtuin 1 (SIRT1) expressions, both of which have been implicated in various biological processes including inflammation, apoptosis and metabolism. We have previously shown that moderate consumption of alcohol aggravates liver inflammation and apoptosis in rats with pre-existing nonalcoholic steatohepatitis (NASH). This study investigated whether moderate alcohol intake alters SIRT1 activity, adiponectin/Adiponectin receptor (AdipoR)-related signaling and lipid metabolism in a pre-existing NASH status. Sprague-Dawley rats were fed with a high-fat diet (71% energy from fat) for 6 weeks to induce NASH then subsequently divided into 2 sub-groups: fed either a modified high-fat diet (HFD, 55% energy from fat) or a modified high-fat alcoholic diet (HFA, 55% energy from fat and 16% energy from ethanol) for an additional 4 weeks. We observed in comparison to HFD group, HFA increased hepatic nuclear SIRT1 protein but decreased its deacetylase activity. SREBP-1c protein expression and FAS mRNA levels were significantly upregulated, while DGAT1/2 and CPT-I mRNA levels were downregulated in the livers of HFA compared to HFD. Although hepatic AdipoR1 decreased, HFA did not alter AdipoR2 and their downstream signaling. There were no significant changes in plasma adiponectin and free fatty acids (FFA), as well as adiponectin expression in adipose tissue between the two groups. The present study indicates that suppression in SIRT1 deacetylase activity contributes to alcohol-exacerbated hepatic inflammation and apoptosis in rats with pre-existing NASH. In addition, moderate alcohol intake did not modulate adiponectin/AdipoR signaling axis in this model.

Non-alcoholic steatohepatitis and hepatocellular carcinoma: implications for lycopene intervention.

Increased prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the consequences of the current obesity epidemic. NAFLD is a major form of chronic liver disease that is highly prevalent in obese and overweight adults and children. Nonalcoholic steatohepatitis (NASH) is the severe form of NAFLD, and uncontrolled inflammation as displayed in NASH has been identified as one of the key events in enhancing hepatic carcinogenesis. Lycopene is a non-provitamin A carotenoid and the pigment principally responsible for the characteristic deep-red color of ripe tomato and tomato products, as well as some fruits and vegetables. Lycopene's innate antioxidant and anti-inflammatory properties have generated research interests on its capacity to protect against human diseases that are associated with oxidative stress and inflammation. In addition, differential mechanisms of lycopene metabolism including endogenous cleavage by carotenoid cleavage oxygenases (BCOs), generate lycopene metabolites that may also have significant impact on human disease development. However, it remains to be elucidated as to whether lycopene or its metabolites apolycopenoids have protective effects against obesity-related complications including inflammation and tumorigenesis. This article summarizes the in vivo experiments that elucidated molecular mechanisms associated with obesity-related hepatic inflammation and carcinogenesis. This review also provides an overview of lycopene metabolism, and the molecular pathways involved in the potential beneficial properties of lycopene and apolycopenoids. More research is clearly needed to fully unravel the importance of BCOs in tomato carotenoid metabolism and the consequence on human health and diseases.

Using email reminders to engage physicians in an Internet-based CME intervention.

BACKGROUND: Engaging practicing physicians in educational strategies that reinforce guideline adoption and improve the quality of healthcare may be difficult. Push technologies such as email offer new opportunities to engage physicians in online educational reinforcing strategies. The objectives are to investigate 1) the effectiveness of email announcements in engaging recruited community-based primary care physicians in an online guideline reinforcement strategy designed to promote Chlamydia screening, 2) the characteristics of physicians who respond to email announcements, as well as 3) how quickly and when they respond to email announcements. METHODS: Over a 45-week period, 445 recruited physicians received up to 33 email contacts announcing and reminding them of an online women's health guideline reinforcing CME activity. Participation was defined as physician log-on at least once to the website. Data were analyzed to determine participation, to compare characteristics of participants with recruited physicians who did not participate, and to determine at what point and when participants logged on. RESULTS: Of 445 recruited physicians with accurate email addresses, 47.2% logged on and completed at least one module. There were no significant differences by age, race, or specialty between participants and non-participants. Female physicians, US medical graduates and MDs had higher participation rates than male physicians, international medical graduates and DOs. Physicians with higher baseline screening rates were significantly more likely to log on to the course. The first 10 emails were the most effective in engaging community-based physicians to complete the intervention. Physicians were more likely to log on in the afternoon and evening and on Monday or Thursday. CONCLUSIONS: Email course reminders may enhance recruitment of physicians to interventions designed to reinforce guideline adoption; physicians' response to email reminders may vary by gender, degree, and country of medical training. Repetition of email communications contributes to physician online participation.

Chromosomal inversion polymorphism in Drosophila mediopunctata: seasonal, altitudinal, and latitudinal variation

The most polymorphic chromosome for inversions in Drosophila mediopunctata is the chromosome II, where 17 inversions have been found, eight of which occurring in the distal region and nine in the proximal region. We present an analysis of the chromosome II inversion polymorphism with respect to seasonal, altitudinal and latitudinal variation. In D. mediopunctata from the Parque Nacional do Itatiaia (southeastern Brazil), the frequencies of three of the distal inversions (namely DA, DS, and DP) vary seasonally. These inversions also show altitudinal clines in their frequencies. This microgeographic pattern was not observed on a macrogeographic scale. D. mediopunctata from Porto Alegre are less polymorphic for inversions than other populations, the most remarkable reduction occurring in the proximal region of chromosome II. There is a considerable difference between D. mediopunctata from Campinas and specimens from Serra do Japi, which are separated by only 50 km. In contrast, D. mediopunctata from Serra do Japi are much more similar to specimens from the Parque Nacional do Itatiaia, which is 200 km far.

Does total orthotopic heart transplantation offer improved hemodynamics during cellular rejection events?

BACKGROUND: We hypothesized that total orthotopic heart transplantation (TOHT) improves hemodynamics during cellular rejection compared with biatrial transplantation (SOHT). METHODS: We reviewed 1942 biopsies from 134 patients (pts) and right heart catheterization data obtained at endomyocardial biopsy. Biopsies that displayed cellular rejection grade 1B as classified according to International Society for Heart and Lung Transplantation (ISHLT) criteria were analyzed. Pts with pacemakers, atrial fibrillation, or beta-blocker therapy at the time of biopsy were excluded. Twenty-three pts after TOHT and 38 after SOHT were identified to match these criteria. RESULTS: Demographic data and pretransplant hemodynamics were similar. TOHT pts had a higher mean cardiac index than SOHT recipients (3.3 +/- 0.8 vs 2.7 +/- 0.5 L/min/m(2); P =.002). Right atrial mean pressure was lower after TOHT (8 +/- 4 vs 11 +/- 4 mm Hg; P =.006). Pulmonary pressures, pulmonary vascular resistance, and heart rate were similar. CONCLUSIONS: TOHT offers improved hemodynamics during cellular rejection grade 1B as evidenced by higher cardiac output and index with lower right atrial pressures. Future studies must examine the potential benefits of TOHT during more severe rejection events.

Combined heart-kidney transplantation with single-donor allografts.

OBJECTIVES: Combined heart-kidney transplantation with allografts from the same donor has been long proved to be a feasible approach for selected patients with coexisting end-stage cardiomyopathy and renal disease. The purpose of this retrospective study is to analyze our long-term results and compare these results with heart-only transplantation over a 7-year period. METHODS: Between June 1992 and April 1999, 10 patients underwent combined heart-kidney transplantation at Cedars-Sinai Medical Center. They were all men from 44 to 70 years old (mean age, 59 +/- 8.3 years) who had a mean left ventricular ejection fraction of 19.4% +/- 5.0% (range, 9%-25%) and a mean creatinine clearance of 25.4 mL/min (range, 10-39 mL/min). Four patients underwent pretransplantation dialysis. RESULTS: There was no operative mortality. The actuarial survival at 1, 2, and 5 years was 100%, 88% +/- 11.7%, and 55% +/- 20.1%, respectively. By comparison, the operative mortality of 169 patients who underwent heart-only transplantation during the same time interval was 2.4%, with an actuarial survival at 1, 2, and 5 years of 92% +/- 2.1%, 84% +/- 2.8%, and 71% +/- 3.9%, respectively (P =.37). Eight patients showed no evidence of significant (> or =1B) cardiac allograft rejection postoperatively, and the actuarial freedom from rejection at 30 days, 1 year, and 2 years was 90% +/- 9%, 80% +/- 13%, and 80% +/- 13%, respectively. Renal allograft survival was 90% at 1 and 2 years. CONCLUSIONS: Combined heart-kidney transplantation yields satisfactory long-term results similar to those for heart-only transplantation, with a low incidence of cardiac allograft rejection and renal allograft survival when both allografts are from the same donor. This approach effectively expands the selection criteria for heart-only and kidney-only transplantation in potential candidates with coexisting end-stage cardiac and renal disease.

Twenty-year comparison of tissue and mechanical valve replacement.

OBJECTIVE: We sought to compare outcomes with tissue and St Jude Medical mechanical valves over a 20-year period. METHODS: Valve-related events and overall survival were analyzed in 2533 patients 18 years of age or older undergoing initial aortic, mitral, or combined aortic and mitral (double) valve replacement with a tissue valve (Hancock, Carpentier-Edwards porcine, or Carpentier-Edwards pericardial) or a St Jude Medical mechanical valve. Total follow-up was 13,390 patient-years. There were 666 St Jude Medical aortic valve replacements, 723 tissue aortic valve replacements, 513 St Jude Medical mitral valve replacements, 402 tissue mitral valve replacements, 161 St Jude Medical double valve replacements, and 68 tissue double valve replacements. The mean age was 68 +/- 13.3 years (St Jude Medical valve, 64.5 +/- 12.9; tissue valve, 72.0 +/- 12.6). RESULTS: There were no overall differences in survival between tissue and mechanical valves. Multivariable analysis indicated that the type of valve did not affect survival. Analysis by age less than 65 years or 65 years or older and presence or absence of coronary disease revealed similar long-term survival in all subgroups. The risk of hemorrhage was lower in patients receiving tissue aortic valve replacements but was not significantly different in patients receiving mitral valve or double valve replacements. Thromboembolism rates were similar for tissue and mechanical valve recipients. However, reoperation rates were significantly higher in patients receiving both aortic and mitral tissue valves. The reoperation hazard increased progressively with time both in patients receiving aortic and in those receiving mitral tissue valves. Overall valve complications were initially higher with mechanical aortic valves but not with mechanical mitral valves. However, valve complication rates later crossed over, with higher rates in tissue valve recipients after 7 years in patients undergoing mitral valve replacement and 10 years in those undergoing aortic valve replacement. CONCLUSIONS: Tissue and mechanical valve recipients have similar survival over 20 years of follow-up. The primary tradeoff is an increased risk of hemorrhage in patients receiving mechanical aortic valve replacements and an increased risk of late reoperation in all patients receiving tissue valve replacements. The risk of tissue valve reoperation increases progressively with time.

Milrinone echocardiographic viability test: a pilot study.

We assessed the utility of milrinone to predict recovery of function after surgical myocardial revascularization in patients with severe baseline left ventricular systolic dysfunction caused by coronary artery disease (CAD). Prediction of viable myocardial segments that will regain function after revascularization may help in the selection of patients who will benefit from coronary artery bypass graft surgery (CABG) as well as aid in the choice of target sites for coronary revascularization. We investigated 20 consecutive patients with CAD and left ventricular ejection fraction < or = 40% who had evidence of myocardial viability by either thallium scan or dobutamine viability test and were candidates for elective CABG. Left ventricular regional wall motion and global ejection fraction were assessed by transesophageal echocardiography in the operating room. Measurements were done before and 10 minutes after milrinone infusion, and immediately after CABG. Left ventricular wall motion score was derived by means of a 12-segment model. Functional improvement for each segment was defined as a wall motion change > 1. Baseline ejection fraction was 27% +/- 5% (mean +/- SD). Ejection fraction increased to 35% +/- 5% after milrinone infusion (P < .0001) and to 36% +/- 6% after CABG (P < .0001). Post-CABG ejection fraction was significantly correlated with postmilrinone ejection fraction (r = 0.65, P < .0001). Milrinone infusion resulted in augmentation of contraction in 98 of the 209 abnormal segments (wall motion score > or = 2); 91 (92.9%) of these improved after CABG. One hundred nine of the 111 segments that showed no improvement with milrinone did not improve after revascularization (98.2%). Seventy-three segments were akinetic or dyskinetic at baseline; 46 (63.0%) of these improved with milrinone. Improvement in regional wall motion after revascularization was detected in 84.8% of the segments that improved with milrinone versus only 3.7% of the segments that did not improve with milrinone. In patients with ischemic cardiomyopathy, improvement in left ventricular function (segmental wall motion and global ejection fraction) during milrinone infusion is highly predictive of improvement after CABG.

Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines.

Hypoxia is an important regulator of vascular endothelial growth factor (VEGF) expression, and VEGF is associated with poor prognosis in bladder cancer. To investigate further the mechanisms of VEGF regulation, we examined VEGF expression by mRNA and protein analysis in four human bladder cancer cell lines, showing a progression from well to poorly differentiated phenotypes under varying conditions of confluence and hypoxia (0.1% O(2)) and with chemical mimics of hypoxia. Hypoxia significantly increased VEGF protein expression in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia inducibility at confluence, whereas inducibility was maintained in the invasive cell lines 253J and EJ28. This pattern of VEGF expression in the invasive cell lines correlated with the expression of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible factor-2 alpha (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease.

Radial artery harvesting for coronary bypass operations: neurologic complications and their potential mechanisms.

OBJECTIVES: We sought to determine the incidence of self-reported neurologic hand complications after radial artery harvest for coronary artery bypass grafting. METHODS: Between February 20, 1996, and December 31, 1999, 615 patients underwent coronary bypass operations with radial arteries. A scripted telephone interview was performed, collecting data on perceived thumb weakness and sensation abnormalities in the distribution of the radial nerve in 560 patients. The average time to follow-up interview was 14.5 +/- 9 months. RESULTS: Neurologic complications were reported in 30.1%, decreased thumb strength in 5.5%, and any sensation abnormality in 18.1% of patients. There was a high rate of symptom improvement over an average of 8.7 +/- 7.5 months, such that only 12.1% of patients reported symptoms without any improvement. Associations between thumb weakness and sensory abnormalities imply median nerve damage in some patients. There were statistically significant associations between neurologic complications and diabetes, peripheral vascular disease, elevated creatinine levels, smoking, and number and site of radial artery harvest. CONCLUSIONS: The overall rate of self-reported neurologic complications after radial artery harvest was higher than previously reported. These symptoms may be attributable to radial and median nerve injury caused by trauma and devascularization. These data have important implications not only in attempting to improve harvesting techniques but also in guiding informed consent before coronary artery bypass grafting.

Heart transplantation in patients seventy years of age and older: A comparative analysis of outcome.

OBJECTIVE: Advanced age has traditionally been considered a contraindication for heart transplantation because of the reported adverse effect of increased age on long-term survival. However, as the field of transplantation continues to evolve, the criteria regarding the recipient's upper age limit have been expanded and older patients are being considered as potential candidates. We analyzed the outcome of heart transplantation in patients 70 years of age and older and compared these results with those in younger patients (<70 years) over a 4-year period. METHOD: We retrospectively analyzed the results of 15 patients 70 years of age and older who underwent heart transplantation between November 1994 and May 1999 and compared them with results in 98 younger patients undergoing transplantation during the same period RESULTS: The older age group had a higher preoperative left ventricular ejection fraction (P =.02), higher incidence of female donors (P =.02), and longer cardiac allograft ischemic time (P =.01). No differences were found regarding incidence of diabetes mellitus, donor age, donor/recipient weight ratio, and mismatch (<0.80). The 30-day or to-discharge operative mortality was similar in both groups (0% in the older vs 5.1% in younger patients). Actuarial survival at 1 year and 4 years was not statistically different between the older and younger patients (93.3% +/- 6.4% vs 88.3% +/- 3.3% and 73.5% +/- 13.6% vs 69.1% +/- 5.8%, respectively). The length of intensive care unit stay and total post-transplantation hospital stay, incidence of rejection, and incidence of cytomegalovirus infection were similar between the groups. CONCLUSIONS: Heart transplantation in selected patients 70 years of age and older can be performed as successfully as in younger patients (<70 years of age) with similar morbidity, mortality, and intermediate-term survival. Advanced age as defined (> or =70 years) should not be an exclusion criterion for heart transplantation. The risks and benefits of transplant surgery should be applied individually in a selective fashion.