Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.United States. Army Research Office (Grant W911NF-12-2-0039

Protection, promotion and support of breast-feeding in Europe: progress from 2002 to 2007.

Objective To assess progress in the protection, promotion and support of breast-feeding in Europe. Design Data for 2002 and 2007 were gathered with the same questionnaire. Of thirty countries, twenty-nine returned data for 2002, twenty-four for 2007. Results The number of countries with national policies complying with WHO recommendations increased. In 2007, six countries lacked a national policy, three a national plan, four a national breast-feeding coordinator and committee. Little improvement was reported in pre-service training; however, the number of countries with good coverage in the provision of WHO/UNICEF courses for in-service training increased substantially, as reflected in a parallel increase in the number of Baby Friendly Hospitals and the proportion of births taking place in them. Little improvement was reported as far as implementation of the International Code on Marketing of Breastmilk Substitutes is concerned. Except for Ireland and the UK, where some improvement occurred, no changes were reported on maternity protection. Due to lack of standard methods, it was difficult to compare rates of breast-feeding among countries. With this in mind, slight improvements in the rates of initiation, exclusivity and duration were reported by countries where data at two points in time were available. Conclusions Breast-feeding rates continue to fall short of global recommendations. National policies are improving slowly but are hampered by the lack of action on maternity protection and the International Code. Pre-service training and standard monitoring of breast-feeding rates are the areas where more efforts are needed to accelerate progress

Heterogeneous distribution of a fatty acid analogue uptake in the myocardium of aged rats

The aim of this study was to determine the extent and location of damaged myocardial areas in senescent rats. The viability of myocardial cells was evaluated in virgin young (4 months old) and aged (29 months old) female Wistar rats by analysing the uptake of a slowly metabolisable radiolabelled fatty acid analogue, 15-p-iodophenyl-β-methylpentadecanoic acid (IMPPA). The biodistribution of IMPPA was measured in various organs, and regional myocardial uptake was specifically assessed using quantitative autoradiography. Myocardial enzymatic activity and DNA content were also evaluated with nitro blue tetrazolium (NBT) and propidium iodide (PÏ) staining, respectively. In senescent rats, cardiac and renal IMPPA uptake showed a significant (50%) reduction compared with young adult rats and the uptake was not significantly changed in the liver, spleen, lungs, and skeletal muscle. Total ventricular NBT staining and IMPPA uptake were almost homogeneous in young adult rats, whereas they were very heterogeneous in aged rats. In the latter, approximately 11% of the total ventricular volume showed a significantly decreased (by 60% or more) IMPPA uptake compared with normal values, and this reduction was greater in ventricle base than in apex. The myocardial areas unlabelled or poorly labelled by IMPPA represented 4, 5, 6, and 21% of the right ventricular, left ventricular epicardial, septal, and left ventricular endocardial volume, respectively, and were poorly stained with NBT. In some of these areas, PI staining indicated the presence of living cells unable to pick up NBT staining. In conclusion, in young adult rats, no myocardial lesions were observed using three different labelling techniques. However, important and significant myocardial lesioned areas were detected in senescent rats and were located preferentially in the left ventricular endocardium, as shown by a decrease in NBT staining and IMPPA uptake. These likely corresponded to a reduced number of cardiomyocytes and (or) a reduced aerobic substrate utilization, along with the development of fibrosis.Key words: senescent female rats, fatty acid analogue uptake, myocyte loss, lesioned and borderline areas. </jats:p

Decreased efficiency of adenovirus-mediated gene transfer in aging cardiomyocytes.

BACKGROUND: Aging is an independent risk factor for the development of cardiovascular disease. Clinical application of myocardial gene transfer may be best suited in the elderly. In vivo gene transfer by adenovirus is less efficient in aging myocardium. METHODS AND RESULTS: When infected with adenovirus containing beta-galactosidase (beta-gal) and green fluorescent protein (GFP) driven by cytomegalovirus promoters in vitro, aging rat cardiac myocytes exhibit significantly lower infectivity and delayed transgene expression compared with adult controls. Abnormalities of viral internalization may be one mechanism accounting for this difference. To investigate this, we studied expression levels of the coxsackievirus and adenovirus receptor (CAR) as well as other potential integrins involved in the internalization of adenoviruses. CAR expression tended to be upregulated whereas among potential integrins, alpha(3)beta(1) was downregulated in aging cardiac myocytes. Blocking the beta(1) component of alpha(3)beta(1) further decreased infectivity, suggesting that the interaction between the penton base of the adenovirus and beta(1) maybe a crucial component of the viral entry mechanism. CONCLUSIONS: These results suggest that it is integrin-stimulated internalization rather than the adenovirus-CAR interaction that plays a vital role in adenoviral entry. The downregulation of integrins observed in senescent cells may be a key mechanism accounting for the decrease in viral infectivity seen in these cells. These findings have implications for the gene therapy treatment of myocardial failure in the elderly

Β1 Integrins Expression in Adult Rat Ventricular Myocytes and Its Role in the Regulation of β-Adrenergic Receptor-Stimulated Apoptosis

We have shown that the stimulation of β-adrenergic receptors (β-AR) increases apoptosis in adult rat ventricular myocytes (ARVMs). Integrins, a family of αβ-heterodimeric cell surface receptors, are postulated to play a role in ventricular remodeling. Here, we show that norepinephrine (NE) increases β1 integrins expression in ARVMs via the stimulation of α1-AR, not β-AR. Inhibition of ERK1/2 using PD 98059, an inhibitor of ERK1/2 pathway, inhibited α1-AR-stimulated increases in β1 integrins expression. Activation of β1 integrins signaling pathway using laminin (LN) inhibited β-AR-stimulated apoptosis as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-staining and flow cytometry. Likewise, ligation of β1 integrins with anti-β1 integrin antibodies prevented β-AR-stimulated apoptosis. Treatment of cells using LN or anti-β1 integrin antibodies activated ERK1/2 pathway. PD 98059 inhibited activation of ERK1/2 by LN, and prevented the anti-apoptotic effects of LN. Thus (1) stimulation of α1-AR regulates β1 integrins expression via the activation of ERK1/2, (2) β1 integrins signaling protects ARVMs from β-AR-stimulated apoptosis, (3) activation of ERK1/2 plays a critical role in the anti-apoptotic effects of β1-integrin signaling. These data suggest that β1 integrin signaling protects ARVMs against β-AR-stimulated apoptosis possibly via the involvement of ERK1/2

Β-Adrenergic Receptor-Stimulated Apoptosis in Cardiac Myocytes Is Mediated by Reactive Oxygen Species/C-Jun NH\u3csub\u3e2\u3c/sub\u3e-Terminal Kinase-Dependent Activation of the Mitochondrial Pathway

Stimulation of β-adrenergic receptors (βARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating βAR-stimulated apoptosis is not known. Stimulation of βARs with norepinephrine (10 μmol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6-fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L) and Euk-134 decreased βAR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 μmol/L) decreased βAR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 μmol/L) decreased βAR-stimulated apoptosis by 62±11%. βAR-stimulated cytochrome c release was inhibited by MnTMPyP. βAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited βAR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway