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Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans and hyaluronan. However, it is unknown which specific proteoglycans are altered in myxomatous valves. Because proteoglycans perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated proteoglycans, as well as hyaluronan and the hyaluronan receptor for endocytosis, within myxomatous and normal mitral valves.Human mitral posterior leaflets (control, n=6-9; myxomatous, n=14-21; mean age, 61 years for all groups) were histochemically stained for proteoglycan core proteins, hyaluronan, and the hyaluronan receptor for endocytosis. Stain intensity was semiquantitatively graded to determine differences in marker abundance between normal and myxomatous valves. The proteoglycans were localized to different regions of the leaflet by correspondence to parallel Movat-stained sections.The proteoglycans decorin, biglycan, and versican were more abundant in myxomatous valves than in normal controls (P.03). There was a gender effect on proteoglycan presence, but no age-related trends were observed. Hyaluronan and the hyaluronan receptor for endocytosis were distributed throughout all valves. There was no significant difference in hyaluronan between groups, but expression of the hyaluronan receptor for endocytosis was reduced in myxomatous valves compared to normal controls (P.002).Excess decorin, biglycan, and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of the hyaluronan receptor for endocytosis in myxomatous valves suggests that hyaluronan metabolism could be altered in myxomatous mitral valve disease. These findings contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.

Cyclic strain evokes the expression of the small leucine-rich proteoglycans decorin and biglycan in 2-D cultures and native tissues. However, strain dependent expression of these proteoglycans has not been demonstrated in engineered tissues. We hypothesized that the absence of decorin may compromise the effect of cyclic strain on the development of engineered tissues. Thus, we investigated the contribution of decorin to tissue organization in cyclically-strained collagen gels relative to statically-cultured controls. Decorin null (Dcn−/−) and wild-type murine embryonic fibroblasts were seeded within collagen gels and mechanically conditioned using a Flexcell® Tissue Train® culture system. After eight days, the cyclically-strained samples demonstrated greater collagen fibril density, proteoglycan content, and material strength for both cell types. On the other hand, increases in cell density, collagen fibril diameter, and biglycan expression were observed only in the cyclically-strained gels seeded with Dcn−/− cells. Although cyclic strain caused an elevation in proteoglycan expression regardless of cell type, the type of proteoglycan differed between groups: the Dcn−/− cell-seeded gels produced an excess of biglycan not found in the wild-type controls. These results suggest that decorin-mediated tissue organization is strongly dependent upon tissue type and mechanical environment.

A major goal of early childhood special education (ECSE) programs is to promote the entry of young children with handicaps into kindergarten and the elementary school mainstream. The transition from ECSE to the kindergarten classroom and elementary school mainstream may present several challenges for the child, family, and professionals. A model for planning this transition and addressing the challenges is presented. The model, developed through Project TEEM, enables parents, ECSE, and elementary school program staff to collaboratively establish and implement procedures for planning transitions. The model insures that the procedures (a) address the strengths, needs, and characteristics of individual children, families, and school programs; (b) promote the implementation of best practices in transition planning; and (c) result in the successful transition of children and families into the elementary school mainstream.

Although the six-month-old pig is commonly used as a model to study human heart valve biology and various age-specific valve diseases, the correlation of porcine valve biology and development with that of humans has not been thoroughly assessed. Given the important role of the matrix in valve function, the aim of this study was to characterize porcine valve matrix structure and collagen turnover during development and aging.Porcine aortic valves (AV) and mitral valves (MV) were examined throughout fetal development and postnatally at six weeks, six months and six years, using Movat pentachrome staining and immunohistochemistry for collagen III, markers of collagen synthesis (molecular chaperone HSP47, hydroxylating enzyme prolyl-4-hydroxylase (P4H), cross-linking enzyme lysyl oxidase (LOX)) and collagen degradation (matrix metalloproteinase (MMP)-13), and a marker of an 'activated' cellular phenotype, smooth muscle alpha-actin (SMalphaA). An analysis of variance was used to compare the staining intensities.Cell density measurements showed layer differentiation in the first trimester (p0.003), and this decreased ten-fold from the second trimester to six years of age (p0.025). Matrix turnover was identified by the co-localization of P4H, HSP47 and MMP13, and correlated to an 'activated' cellular phenotype. SMalphaA expression was noted on the inflow surface of both valves. P4H and LOX were maximally expressed around mature collagen (p0.001). P4H increased during fetal development (p0.01) and in the six-year-old AV fibrosa (p0.05). Collagen-related markers were consistently higher in the AV than MV (HSP47 in fetal; P4H, Col III, and LOX in six-year-old).The substantial matrix changes shown in this porcine study provide further insight into the role of matrix turnover during development and aging and should be considered when using the porcine animal model to study age-specific human diseases.

Objectives: Chronic mitral regurgitation (MR) adversely remodels the left ventricle. Although leaflet integrity is important for mitral coaptation, its response to isolated MR remains unknown. In a chronic ovine model, we hypothesize that isolated MR also adversely remodels the mitral leaflet. Methods: 29 sheep were randomized to either control (CTRL, n=11) or experimental groups (HOLE, n=18). In HOLE, a 2.8 – 4.8mm diameter hole was punched in the middle scallop of the posterior mitral leaflet. MR grade was followed by echocardiography. At 12 weeks, immunohistochemistry (IHC) was performed on anterior and posterior mitral leaflet cross-sections to demonstrate active matrix remodeling, including markers for active collagen synthesis synthesis and cross-linking prolyl 4-hydroxylase (P4H), heat shock protein 47 (HSP47), and lysyl oxidase (LOX) and matrix metalloproteinases (MMPs 1, 2, 9, 13) largely responsible for matrix degradation implicated in valvular pathologies. The proteoglycans biglycan (BGN), decorin (DC), and versican (VC), the glycosaminoglycan hyaluronan (HA), as well as fibrillin (F), and collagen III (col 3), all of which regulate matrix organization, were also visualized using IHC. A semi-quantitative intensity and delineation grading scale (from 0 to 3) was used to quantify differences between the HOLE and CTRL leaflets. Results: At 12 weeks, MR grade was greater in HOLE v CTRL (0.4±0.4 v 3.0±0.8, p < 0.001). In HOLE, valve matrix remodeling reduced hole diameter (69.5%32±.8%, p Conclusions: In HOLE, significant remodeling was evident in the posterior leaflet near the site of injury; moreover expression of matrix turnover markers increased in the anterior leaflet due to the chronic low pressure LV volume overload. This adverse remodeling in the anterior leaflet in response to MR may explain how “MR begets MR,” providing insight for reparative surgical techniques.

Background/Aims: Because various regions of the mitral valve contain distinctive extracellular matrix enabling the tissues to withstand diverse mechanical environments, we investigated phenotype and matrix production of porcine valvular interstitial cells (VICs) from different regions. Methods: VICswere isolated from the chordae (MCh), the center of the anterior leaflet (AlCtr), and the posterior leaflet free edge (PlFree), then assayed for metabolic, growth, and adhesion rates; collagen and glycosaminoglycan (GAG) production, and phenotype using biochemical assays, flow cytometry, and immunocytochemistry. Results: The AlCtr VICs exhibited the fastest metabolism but slowest growth. PlFree cells grew the fastest, but demonstrated the least smooth muscle α-actin, vimentin, and internal complexity. AlCtr VICs secreted less collagen into the culture medium but more 4-sulfated GAGs than other cells. Adhesion-based separation resulted in altered secretion of sulfated GAGs by MCh and AlCtr cells but not by the PlFree cells. Conclusions: VICs isolated from various regions of the mitral valve demonstrate phenotypic differences in culture, corresponding to the ability of the mitral valve to accommodate the physical stresses or altered hemodynamics that occur with injury or disease. Further understanding of VIC and valve mechanobiology could lead to novel medical or tissue engineering approaches to treat valve diseases.

Decorin (DCN), a class I member of the small leucine-rich proteoglycan (SLRP) family, is composed of a protein core of approximately 40kDa [1, 2] substituted with a single glycosaminoglycan (GAG) chain of chondroiton/dermatan sulfate on the N-terminal site [3]. DCN has been reported to interact with collagen [4,5] via its core protein, influence collagen fibrillogenesis [6], and inhibit the growth rates of various cell types when added exogenously to cell cultures [5,6]. There has recently been growing interest and studies in DCN related research using the knockout (KO) mice model which provides an excellent example of inherited disorders that stem from deficiencies in decorin expression [7]. Skin and tendon tissues from DCN KO mice have been characterized as being extremely fragile with significantly reduced strength and stiffness [8, 9]. The DCN KO tissues also show potential functional biglycan compensation [9] and at the microscopic level collagen fibrils with highly irregular diameters, abnormal lateral fusion, and loose packing [6] in contrast to wild type (WT) mice. Despite the intensive investigation of the DCN KO mice, the complexity of the animal model makes it difficult to assess the actual influence of decorin. In an attempt to take a more simplistic approach 2D cell phenotypic characterization studies were performed in addition to studying cell growth, contraction, and matrix organization in 3-D models to show the very distinct biochemical responses to type I collagen when compared to WT control cells.

Left Atrium (LA) size has prognostic importance in a variety of cardiac conditions [1] and is known to be enlarged with decreased contractile function in patients with congestive heart failure (CHF) [2]. Nearly 5 million Americans have CHF [3] and a majority of these patients display diastolic dysfunction, which is an abnormality in the left ventricle (LV) myocardial relaxation and/or compliance that alters the ease with which the blood is accepted into the LV from the LA during diastole [4]. Due to abnormal LV filling, the LA experiences intense stress and elevated pressures. In fact, the left atrium is exposed directly to the LV diastolic pressure through the open mitral valve (MV) and because of its thin wall structure it tends to dilate with increasing pressure [5]. This augmented LA size and increased contractility and booster function are some of the mechanisms compensating for decreased early filling in patients with reduced LV compliance [6]. Over time, the LA compensatory contribution decreases, this may lead to intrinsic left atrium dysfunction [7]. This in turn results in a progressive decline in health unless the hearts’ inadequate blood flow is augmented by a left ventricular assist device (LVAD). Although LVAD implantation rest the heart, restores function to the ventricle [8], and improve overall function [9], its effects on the left atrium remain unclear. The purpose of the present study was to use 2D and Doppler echocardiography to define the parameters for assessing LVAD unloading and determine its effect on LA diameter, area, volume, and pressure in patients prior to and following LVAD implantation.Copyright © 2007 by ASME

Cells respond to changes in mechanical strains by varying their production of extracellular matrix macromolecules. Because differences in strain patterns between mitral valve leaflets and chordae tendineae have been linked to different quantities and types of glycosaminoglycans (GAGs), we investigated the effects of various strain conditions on GAG synthesis by valvular interstitial cells (VICs) using an in vitro 3-dimensional tissue-engineering model. VICs from leaflets or chordae were seeded within collagen gels and subjected to uniaxial or biaxial static tension for 1 week. GAGs synthesized within the collagen gels and secreted into the surrounding medium were analyzed using fluorophore-assisted carbohydrate electrophoresis. In constrained conditions, more 4-sulfated GAGs were retained within the collagen gel, whereas more hyaluronan was secreted into the surrounding medium. Selected GAG classes were found in significantly different proportions in collagen gels seeded with leaflet cells versus chordal cells. The only significant difference between uniaxial and biaxial regions was found for 6-sulfated GAGs in the gels seeded with chordal cells (p

Substantial heart valve research relies on the isolation of valvular interstitial cells (VICs). While a wide variety of conditions have been reported for VIC isolation, the effectiveness of these methods has rarely been compared. It is also likely that valve donor age will influence these valvular tissue dissociation conditions. The study aim was to increase the efficiency and cost-effectiveness of VIC isolation, while taking into account possible differences due to valve donor age.Aortic valves were obtained from six-month-old (n = 24) and six-week-old (suckling) pigs (n = 45) within 24 h of death. After removal of endothelial cells, the tissues were minced and subjected to a variety of enzymatic digestions for variable lengths of time.The optimal concentration of collagenase III was determined as 1 mg/ml for six-week-old pigs, and 2 mg/ml for six-month-old pigs. The optimal duration of digestion was 4 h for both ages. The addition of neutral protease (2 mg/ml) further increased yield, while additional DNAse and hyaluronidase had no effect. Yield was not influenced by the volume of enzyme solution, nor the use of previously frozen enzyme solution.These findings provide age-specific conditions for improving the yield of VIC isolation, which should be of value in experimental studies of valvular cell biology and tissue engineering investigations.

Differently loaded regions of the mitral valve contain distinct amounts and types of proteoglycans (PGs); these PG profiles are altered in abnormal loading and disease conditions. We developed an in vitro three-dimensional model to analyze PGs secreted by valvular interstitial cells (VICs) isolated from distinct regions of porcine mitral valves (leaflet or chordae) and subjected to either biaxial or uniaxial mechanical constraints. In addition, the PGs, DNA and collagen content of the collagen gels was monitored over time. All three PGs previously found in heart valves (decorin, biglycan and versican) were present in the collagen gels and the conditioned medium. Compared to unconstrained gels, the constrained collagen gels (whether biaxially or uniaxially loaded) retained more decorin and biglycan but less versican. However, the conditioned medium from constrained collagen gels contained higher amounts of all three PGs than did medium from unconstrained gels. Constrained collagen gels containing leaflet cells retained more decorin and biglycan than did those containing chordal cells. DNA content was maintained early in the culture period but was reduced by 55–80% after 7 days, whereas PG synthesis increased over time. At the end of the culture period, the cell density was highest in the biaxial region of gels seeded with leaflet cells. In contrast, collagen content in both constrained and unconstrained gels remained consistent over culture duration. This study provides valuable information about the role of applied loading on proteoglycan segregation, which should aid in tissue engineering applications and for understanding valve biology and pathology.

Valvular interstitial cells (VICs) demonstrate a heterogeneous range of phenotypes such as variable expression of smooth muscle alpha-actin (SMalphaA). Myofibroblast-like VICs, expressing high levels of SMalphaA, are thought to be involved in myxomatous degeneration of mitral valves. The inability to isolate specific cell types has restricted potential investigations of valvular disease mechanisms. Thus, investigations were conducted into methods of isolating different cell subpopulations from primary VICs as a preparatory step for cell type-specific evaluations of heart valve disease.VICs were isolated from porcine valves, cultured to 80% confluency, and subdivided using differential detachment or adhesion. The subdivided cells were further cultured and analyzed phenotypically by immunocytochemistry and flow cytometry to characterize SMalphaA expression. Roundness and growth rates were also analyzed.VICs that were relatively sensitive to trypsinization expressed low and heterogeneous levels of SMalphaA (15-35%), whereas more-adherent VICs expressed higher and homogeneous levels (98%) suggestive of a myofibroblast-like phenotype. The more-adherent cells also had lower growth potential and were less round than less-adhesive VICs. Separated cell subtypes were found to maintain their phenotype through several cell passages.VICs are a mixed population of cells, many of which express high levels of SMalphaA. Differential detachment and adhesion can effectively separate cell subpopulations from primary cultures of VICs. The ability to study valve cell subpopulations has substantial implications for future analyses of valvular biology, disease, and tissue engineering.

Elderly patients would receive substantial benefits from tissue-engineered heart valves (TEHVs), but most TEHV research has not focused on applications for this growing patient population. There will be numerous technical challenges involved in developing TEHVs for the elderly, such as designing tissues to accommodate higher blood pressure and larger aortic roots that may be friable or calcified. Concomitant medications may also affect the biology of the TEHV. Due to the predominantly senescent behavior of cells from older persons, a nonautologous cell source may be required to develop the TEHV. Decellularized heart valve allografts from elderly donors may not be durable enough to use as a scaffold, but several polymer and natural biodegradable scaffolds may provide promising alternatives. The selection of cell sources, scaffolds, and mechanical/biologic conditioning will need to be precisely targeted to meet the diverse physiological, medical, and surgical requirements of elderly patients.

OBJECTIVE: To determine the proportion of televised movie trailers that included images of tobacco use during 1 year and the extent of youth exposure to those trailers. DESIGN: Content analysis combined with Nielsen data measuring media exposure. All movie trailers (N = 216) shown on television from August 1, 2001, through July 31, 2002. MAIN OUTCOME MEASURES: Exposure among youth aged 12 to 17 years to televised movie trailers that included smoking imagery. RESULTS: Of the movie trailers televised during the study period, 14.4% (31 trailers) included images of tobacco use. Tobacco use was shown in 24.0% of the 23 trailers for R-rated (restricted) movies and 7.5% of the 8 trailers for PG-13- and PG-rated (parental guidance) movies. Ninety-five percent of all youth aged 12 to 17 years in the United States saw at least 1 movie trailer depicting tobacco use on television during this 1 year, and 88.8% saw at least 1 of these trailers 3 or more times. CONCLUSIONS: Nearly all US youth aged 12 to 17 years were exposed to images of tobacco use on television in the context of a movie trailer during the study period. Given the relationship between youth exposure to tobacco use in movies and smoking initiation, the public health community should work to enact policy to reduce or eliminate the influence of tobacco use in televised movie trailers.

This study assesses whether a national anti-tobacco campaign for youth could create a social context that would elevate social desirability response bias on surveys, as measured by an increase in under-reporting of smoking. This could give rise to data that falsely suggest a campaign-induced decline in youth smoking, or it could exaggerate campaign effects. Data were obtained from a national sample of 5511 students from 48 high schools that were matched to schools sampled for the 2002 National Youth Tobacco Survey (NYTS). Self-reported smoking was compared with biochemical indicators of smoking, measured using saliva cotinine. The rate of under-reporting detected was 1.3%. Level of truth® exposure was not related to under-reporting. This study suggests that for high school students, anti-tobacco campaigns are not an important cause of social desirability responses on surveys, and that in general under-reporting smoking is not a major source of error in school-based surveys.

Despite a high incidence of calcific aortic valve disease in metabolic syndrome, there is little information about the fundamental metabolism of heart valves. Cell metabolism is a first responder to chemical and mechanical stimuli, but it is unknown how such signals employed in valve tissue engineering impact valvular interstitial cell (VIC) biology and valvular disease pathogenesis. In this study porcine aortic VICs were seeded into three-dimensional collagen gels and analysed for gel contraction, lactate production and glucose consumption in response to manipulation of metabolic substrates, including glucose, galactose, pyruvate and glutamine. Cell viability was also assessed in two-dimensional culture. We found that gel contraction was sensitive to metabolic manipulation, particularly in nutrient-depleted medium. Contraction was optimal at an intermediate glucose concentration (2 g l −1 ) with less contraction with excess (4.5 g l −1 ) or reduced glucose (1 g l −1 ). Substitution with galactose delayed contraction and decreased lactate production. In low sugar concentrations, pyruvate depletion reduced contraction. Glutamine depletion reduced cell metabolism and viability. Our results suggest that nutrient depletion and manipulation of metabolic substrates impacts the viability, metabolism and contractile behaviour of VICs. Particularly, hyperglycaemic conditions can reduce VIC interaction with and remodelling of the extracellular matrix. These results begin to link VIC metabolism and macroscopic behaviour such as cell–matrix interaction.

Extracellular matrix (ECM) provides several structural and functional characteristics to tissues including cell support, mechanical integrity and biological signaling. In cardiovascular tissues, cells produce various ECM components such as collagen, elastin, proteoglycans, matrix metalloproteinases, growth factors and signaling molecules. The cardiovascular cells (cardiac fibroblasts, cardiomyocytes, endothelial cells, and vascular smooth muscle cells) sense the changes in mechanical strains applied to them, through cell-surface receptors such as integrins and ion channels, and adjust their expression and synthesis of ECM molecules in order to adapt their environment to these changes. ECM changes due to altered mechanics are evident in numerous pathological situations including hypertension, cardiac hypertrophy, myocardial infarction, myxomatous heart valve disease, and atherosclerosis. In hypertrophic conditions, for example, increased mechanical loading is involved with enhanced collagen synthesis, whereas in myxomatous and atherosclerotic conditions reduced mechanical strains are accompanied by an accumulation of proteoglycans. Therefore, investigating the effects of various strain patterns on cardiovascular cells can enhance our understanding of ECM regulation and pathologies. This review focuses on the in vitro modulation of the synthesis of various ECM molecules through static or cyclic stretching of cardiovascular cells.

In order to test the hypothesis that CD8 + cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8 + lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8 + T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8 + T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8 + T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8 + T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.

Galactosemia is detected by newborn screening in New South Wales and managed by the metabolic team at the Children's Hospital at Westmead. Infants with the Duarte variant are not treated. Management is based on the Handbook for Galactosemia prepared in 1998. This handbook provides information for the family on the dietary management, inheritance and ovarian function. The major dietary sources of galactose are milk and milk products. Breastfeeding must be ceased and replaced with a soy formula. Once solid foods are commenced certain foods should be avoided. Other foods, which may contain some free galactose are recommended in limited quantities only. There is no restriction on other fruits and vegetables. An ongoing issue with dietary management is adequate nutrient intake, particularly of calcium. Intake of milk substitutes and calcium supplements is often inadequate.

s from the 13th Biennial Meeting of the International Society for Applied Cardiovascular Biology September 12–15, 2012 University College, London

Using a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that bind to the human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified PRP4, a serine-threonine protein kinase. Specific interaction of PRP4 and HIV-2 Gag was confirmed in in vitro and in vivo assays. The interacting region of HIV-2 Gag is located in the conserved matrix and capsid domains, while both the RS (arginine-serine-rich) domain and the KS (kinase) domain of PRP4 are able to bind to HIV-2 Gag. PRP4 is not incorporated into virus particles. HIV-2 Gag is able to inhibit PRP4-mediated phosphorylation of the splicing factor SF2. This is also observed with Gag from simian immunodeficiency virus, a closely related virus, but not with Gag from human T-cell lymphotropic virus type 1. Our results provide evidence for a novel interaction between Gag and a cellular protein kinase involved in the control of constitutive splicing in two closely related retroviruses. We hypothesize that as Gag accumulates in the cell, down regulation of splicing occurs through reduced phosphorylation of SF2. At late stages of infection, this interaction may replace the function of the early viral regulatory protein Rev.

Data suggest that 12%-22% of women smoke during pregnancy. The link between smoking during pregnancy and adverse health and reproductive outcomes has been well documented. Great Start is a public education and smoking cessation program developed especially for pregnant women. Launched in December 2001, Great Start was the first national program focused on providing free and confidential smoking cessation counseling to pregnant women through a toll-free quitline. Great Start consisted of a media campaign to raise awareness and direct women to telephone counseling tailored for the pregnant smoker, and educational materials designed to support pregnant women through cessation counseling. The program was evaluated to assess the ability of the television ads to reach pregnant smokers and the effectiveness of a quitline for increasing cessation rates among pregnant women. Great Start demonstrates that it is possible to reach pregnant smokers through television ads that provide information about the consequences of smoking while pregnant, are affirming in tone, and provide direction for women to take action. Initial response to the program indicates that pregnant women want to quit and confirms the need for programs designed specifically to address the needs of this population.

Organ cultures maintain cells within their native microstructural environment, and thus offer greater potential for studying tissue disease and remodeling than do monolayer cell cultures or pathological examinations of diseased tissue. To validate an in-vitro heart valve organ culture model, cell viability was examined within valve tissues over sustained culture periods.Following culture of blocks of valve tissue for 1 to 49 days, cross-sections were cut with a vibratome, stained with a LIVE/DEAD kit, and imaged with confocal microscopy to quantify the number of live and dead cells present.In numerous organ cultures, valvular interstitial cells were found to be viable beyond 30 days. Live cells were abundant in the central region of the valve, but more sparse in the deepest central regions. Dead cells were found mainly on the surface of both fresh tissues and tissues after prolonged culture, with few dead cells occurring centrally.This is the first reported mapping of cell viability within heart valve organ cultures, and results suggest that extended organ culture of valve leaflets is indeed possible. The derived viability staining methods have wide applicability for organ cultures of other tissues as well as tissue-engineered matrices.

This study was designed to identify the specific proteoglycans and glycosaminoglycans (GAGs) in the leaflets and chordae of the mitral valve and to interpret their presence in relation to the tensile and compressive loads borne by these tissues. Leaflets and chordae from normal human mitral valves (n = 31, obtained at autopsy) were weighed and selected portions digested using proteinase K, hyaluronidase, and chondroitinases. After fluorescent derivatization, fluorophore-assisted carbohydrate electrophoresis was used to separate and quantify the derivatized saccharides specific for each GAG type. In addition, the lengths of the chondroitin/dermatan sulfate chains were determined. Proteoglycans were identified by western blotting. The regions of the valve that experience tension, such as the chordae and the central portion of the anterior leaflet, contained less water, less hyaluronan, and mainly iduronate and 4-sulfated N-acetylgalactosamine with chain lengths of 50-70 disaccharides. These GAGs are likely associated with the small proteoglycans decorin and biglycan, which were found in abundance in the tensile regions. The valve regions that experience compression, such as the posterior leaflet and the free edge of the anterior leaflet, contained significantly more water, hyaluronan, and glucuronate and 6-sulfated N-acetylgalactosamine with chain lengths of 80-90 disaccharides. These GAGs are likely components of water-binding versican aggregates, which were abundant in the compressive loading regions. The relative amounts and distributions of these GAGs are therefore consistent with the tensile and compressive loads that these tissues bear. Finally, the concentrations of total GAGs and many different chondroitin/dermatan sulfate subclasses were significantly decreased with advancing age.

This study assessed apparently normal mitral valves from patients with congestive heart failure (CHF) using biochemical and echocardiographic measures of extracellular matrix (ECM) and anatomy.Mitral regurgitation (MR) is frequently found in patients with CHF. This MR is considered purely functional, yet animal studies suggest that altered left ventricular (LV) function leads to increased cellularity and fibrosis of the mitral valve. Therefore, we hypothesized that patients with CHF might have partly organic MR, via dysfunctional valvular remodeling.Mitral valves from transplant recipient hearts of patients with CHF (23 dilated, 14 ischemic) were analyzed for deoxyribonucleic acid (DNA), collagen, glycosaminoglycan (GAG), and water concentrations and compared with autopsy controls. Cardiac dimensions and functional parameters (measured from recent echocardiograms) were compared with biochemical parameters using a repeated measures generalized linear model.The mitral valves in CHF had up to 78% more DNA (p0.03), 59% more GAGs (p0.02), and 15% more collagen (p0.007), but 7% less water (p0.05) than normal. The absence of anterior leaflet redundancy was associated with these deranged biochemical measures (p0.03). Associations were found between leaflet thickness and DNA concentration (+, p=0.003), annular diameter and chordal collagen (+, p=0.03), and water concentration and both left atrial diameter (-, p=0.008) and LV collagen concentration (-, p=0.04).Mitral valves in CHF are biochemically different from normal, with ECM changes that are influenced by the altered cardiac dimensions. This remodeling suggests that MR in patients with CHF may not be purely functional, and that these valves are not "normal."

Recent findings have implicated the 5-hydroxytryptamine 2B (5-HT2B) serotonin receptor in mediating the heart valve fibroplasia [valvular heart disease (VHD)] and primary pulmonary hypertension observed in patients taking the now-banned appetite suppressant fenfluramine (Pondimin, Redux). Via large-scale, random screening of a portion of the receptorome, we have discovered that the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") and its N-demethylated metabolite 3,4-methylenedioxyamphetamine (MDA) each preferentially bind to and activate human recombinant 5-HT2B receptors. We also demonstrate that MDMA and MDA, like fenfluramine and its N-deethylated metabolite norfenfluramine, elicit prolonged mitogenic responses in human valvular interstitial cells via activation of 5-HT2B receptors. We also report that pergolide and dihydroergotamine, two drugs recently demonstrated to induce VHD in humans, potently activate 5-HT2B receptors, thus validating this assay system for its ability to predict medications that might induce VHD. Our discovery that MDMA and a major metabolite, MDA, induce prolonged mitogenic responses in vitro similar to those induced by fenfluramine and norfenfluramine in vivo (i.e., valvular interstitial cell fibroplasia) predict that long-term MDMA use could lead to the development of fenfluramine-like VHD. Because of the widespread abuse of MDMA, these findings have major public health implications. These findings also underscore the necessity of screening current and future drugs at h5-HT2B receptors for agonist actions before their use in humans.

Rupture of chordae tendineae is the main cause of mitral valve insufficiency, and often requires corrective surgery. The precise mechanisms of chordal rupture, however, are unknown.Failure mechanics were measured in porcine mitral valve chordae (37 anterior marginal, 40 anterior basal, 35 posterior marginal, and 38 posterior basal). Full-length chordae were weighed, measured, and stretched to failure in an Instron tensile testing machine. The ruptured ends were characterized under a dissecting microscope.Marginal chordae had 68% thinner cross-sectional areas and failed at 68% less load and 28% less strain than basal chordae. Chordae from the posterior leaflet were 35% thinner and failed at 43% less load and 22% less strain than anterior leaflet chordae. Failure strength was lowest for posterior marginal chordae. Chordae most frequently tore just below the leaflet insertion, in what was often their narrowest section.Overall, the marginal chordae and posterior leaflet chordae were thinner and required less strain and load to fail than basal chordae and anterior leaflet chordae, respectively. These results support previous reports of decreased extensibility in marginal chordae. The high incidence of ruptures in the posterior marginal chordae of diseased mitral valves may be due to an inherent weakness in these chordae.

Disparities in tobacco use and smoking cessation by race/ethnicity, education, income, and mental health status remain despite recent successes in reducing tobacco use. It is unclear to what extent media campaigns promote cessation within these population groups. This study aims to (1) assess whether exposure to antitobacco advertising is associated with making a quit attempt within a number of population subgroups, and (2) determine whether advertisement type differentialy affects cessation behavior across subgroups. We used data from the New York Adult Tobacco Survey (NY-ATS), a cross-sectional, random-digit-dial telephone survey of adults aged 18 or older in New York State conducted quarterly from 2003 through 2011 (N = 53,706). The sample for this study consists of 9,408 current smokers from the total NY-ATS sample. Regression methods were used to examine the effect of New York State’s antismoking advertising, overall and by advertisement type (graphic and/or emotional), on making a quit attempt in the past 12 months. Exposure to antismoking advertising was measured in two ways: gross rating points (a measure of potential exposure) and self-reported confirmed recall of advertisements. This study yields three important findings. First, antismoking advertising promotes quit attempts among racial/ethnic minority smokers and smokers of lower education and income. Second, advertising effectiveness is attributable in part to advertisements with strong graphic imagery or negative emotion. Third, smokers with poor mental health do not appear to benefit from exposure to antismoking advertising of any type. This study contributes to the evidence about how cessation media campaigns can be used most effectively to increase quit attempts within vulnerable subgroups. In particular, it suggests that a general campaign can promote cessation among a range of sociodemographic groups. More research is needed to understand what message strategies might work for those with poor mental health.

Objective: The treatment of choice for aortic valve insufficiency due to root dilatation has become root replacement with aortic valve sparing. However, root replacement with a synthetic graft may result in altered valve stresses. The purpose of this study was to compare the stress/strain patterns in the spared aortic valve in different root replacement procedures by means of finite element modeling. Methods: Our finite element model of the normal human root and valve was modified to simulate and evaluate three surgical techniques: (1) "cylindrical" graft sutured below the valve at the anulus, (2) "tailored" graft sutured just above the valve, and (3) "pseudosinus" graft, tailored and sutured below the valve at the anulus. Simulated diastolic pressures were applied, and stresses and strains were calculated for the valve, root, and graft. Leaflet coaptation was also quantified. Results: All three root replacement models demonstrated significantly altered leaflet stress patterns as compared with normal patterns. The cylindrical model showed the greatest increases in stress (16%-173%) and strain (10%-98%), followed by the tailored model (stress +10%-157%, strain +9%-36%). The pseudosinus model showed the smallest increase in stress (9%-28%) and strain (2%-31%), and leaflet coaptation was closest to normal. Conclusion: Valve-sparing techniques that allow the potential for sinus space formation (tailored, pseudosinus) result in simulated leaflet stresses that are closer to normal than the cylindrical technique. Normalized leaflet stresses in the clinical setting may result in improved longevity of the spared valve. (J Thorac Cardiovasc Surg 2000;119:753-63)

Purpose: To evaluate pentagalloyl-glucose (PGG) stabilized elastinderived vascular grafts (EDVGs) in terms of their suitability as vascular substitutes. Methods: EDVGs were prepared by alkaline decellularization (0.1 M NaOH at 37°C for 3 h), rinsing and sterilization in 0.1% peracetic acid. Batches of EDVG scaffolds were treated with sterile 0.1% PGG (pH 5.5) containing 20% isopropanol for 24 h, rinsed and stored in sterile PBS. A subset of PGG and non-PGG samples were additionally covalently heparinized via diamine coupling and reductive amination using nitrous acid degraded heparin. Grafts were implanted in a rat infrarenal aortic model for 4 and 8 weeks. Remodeling, patency, endothelialization and healing of the explants were determined by gross and histological evaluation. Results: While PGG treatment did not significantly affect the denaturation temperature (DT) of the tissue, heparinization resulted in significant increases in DT (from 52°C to 81–82°C) and heparin content (from baseline noise levels of b10 mg/g to N100 mg/g). Overall the nonheparinized groups showed strong evidence of remodeling and recellularization, with a high patency rate of 82%. At 8 weeks, only small fragments of the original grafts were preserved with good neovessel formation and moderate intimal hyperplasia (IH). The heparinized groups showed 100% patency, but with little remodeling, presumably due to the increased crosslink density resulting from the amination of the tissue prior to heparin attachment. Some surface endothelialization was observed in all treatment groups. Conclusions: EDVGs are promising candidates as vascular grafts, either as substrates for remodeling, or in more highly cross-linked and heparinized forms.

This review addresses various methods of integrating proteoglycans (PGs) into the design of engineered tissues and provides insight for designing tissue-engineered disease models that leverage current knowledge of PG biology. Even though PGs show immense possibilities in tissue-engineering applications, they have seldom been used to their full potential. The most common tissue-engineering application of PGs has been in scaffolds (matrigels and collagen-chondroitin sulfate matrices), in which PGs or their glycosaminoglycan (GAG) chains are incorporated into the scaffold to promote cell growth, tissue remodeling, and intracellular signaling. In addition, many studies have reported the total amount of PGs synthesized within engineered tissues but have not delineated which specific PGs or GAG classes are involved in engineered tissue development. In native tissues, various PGs are dynamically and differentially regulated to achieve specific biophysical and biological functions, such as compressibility and transparency. Therefore, the targeted modulation of specific PGs (via exogenous addition, endogenous stimulation with growth factors, or mechanical stimulation) may help engineered tissues to achieve native tissue properties. The PG composition of engineered tissues could also be modified to achieve disease models in vitro and thus provide a way to study the effect of external agents on PG-related disease mechanisms.

The interdisciplinary science of genetic toxicology emerged in the 1960s as a result of a unique cross-fertilization of genetics and radiation biology. The talented, committed scientists who instituted this field of study were aided by new money, new laboratories, new environmental social activism, and new governmental interest. In his first book, Chemical consequences: environmental mutagen, scientist activism, and the rise of genetic toxicology, Scott Frickel, an assistant professor of sociology at Tulane University, explores the many factors that led to the establishment and development of this field through the late 1970s. With relevant and enlightening examples, the author illustrates how science and activism came together to establish what is now a major component of environmental health science. Genetic toxicology today is a standard, although not always straightforward, component of regulatory toxicology testing required for FDA approvals of drugs and animal health products. It is also a regulatory tool used by the Environmental Protection Agency for evaluation of the hazards of pesticides and other environmental exposures. Its inclusion in standard toxicology testing programs is relatively recent, compared to testing for carcinogenicity, reproductive toxicology, and general toxicology. In addition, subdisciplines of genetic toxicology, involving, for example, DNA repair, infidelity of DNA replication, and mutation of specific genes are now recognized as having clinical importance in areas ranging from cardiovascular pathology to cancer. Frickel writes from the perspective of a sociologist, not a historian or scientist. Accordingly, the material presented is a fascinating insight into how larger cultural factors were critical to the development of the field. Attaining a critical mass of scientific knowledge would have been useless in establishing a new field without the societal impetus. The book examines the establishment and interaction of interdisciplinary knowledge, funding, and careers. The emergence of genetic toxicology in the 1960s, a decade of massive transformation in American biology and in American culture as a whole, is well explicated. Frickel tracks how the field quickly moved from limited interactions among a handful of biologists to a full-fledged interdisciplinary science encompassing vigorous professional societies, scientific journals and publications, international conferences, and academic courses, which continue to this day. Of particular importance was the Oak Ridge National Laboratory, which provided an intellectual and physical home for much of the early work. The first portion of the book succinctly discusses the scientific basis of genetic toxicology and nicely illustrates why this interdisciplinary field did not develop earlier, despite the fact that the guiding scientific principles had been clearly established. Frickel also explains why the initial emphasis was on environmental mutagenesis. That historical emphasis is reflected even today in the name of the premiere scientific association for genetic toxicology: Environmental Mutagen Society. Frickel goes on to describe how the founding scientists as well as new recruits developed a type of environmental activism designed to be effective where it could have the strongest impact: within the research areas studied by the scientists on a daily basis. To explain the relevance of environmental mutagenesis to the public and to officials overseeing public health programs, they initially emphasized the need to preserve the integrity of the genetic code of future generations. Both scientific symposia and testimony before governmental agencies on the possible hazards of altering the gene pool gradually became less frequent. Eventually, carcinogenicity became a stronger, more common concern in genetic toxicology. Frickel provides a coherent description of this change in emphasis and its consequences. While the writing style and presentation of data may appeal more to sociologists than to scientists, Frickel’s insights regarding what is actually needed to develop a new scientific discipline are extremely interesting.

Three mutants of Escherichia coli K-12 which form filaments during 42 C incubation have been characterized. The mutant strains AX621, AX629, and AX655 continued to grow and to synthesize deoxyribonucleic acid at 42 C for 150 to 180 min, after which time growth ceased. When cultures of the mutants were transferred from 42 to 28 C, septation of the filaments began after a 25- to 30-min period and continued at a greater than normal rate until no filaments remained. Addition of chloramphenicol at the time of transfer from 42 to 28 C prevented cell division in strain AX655 and caused lysis of strains AX621 and AX629. The temperature sensitivity mutation in each strain mapped near leu . For strain AX621, the mutation was specifically located between leu and nadC by P1 transduction. Properties of these strains are compared with those of other cell division mutants.

The Academic Self-Concept Scale (ASCS) was developed as a measure of an academic facet of general self-concept in college stu dents. The initial item pool consisted of 59 items worded to conform to a 4-point Likert-type response format. On the basis of responses from 427 college students, the final form of the ASCS was con structed consisting of 40 items with an estimated internal con sistency reliability of .91. Validity was established by correlating the ASCS with grade point averages (GPAs) of students and with their scores on the Rosenberg Self-Esteem Scale. A multiple regression analysis of the ASCS with GPA and Rosenberg scores as predictor variables resulted in a multiple correlation of .64. These and other data lend support to the reliability and validity of the ASCS as a measure of academic self-concept.