Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone: A potent,selective, orally active dipeptidyl peptidase IV inhibitor

A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC₅₀ = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.     

Assessing Anticalcification Treatments in Bioprosthetic Tissue by Using the New Zealand Rabbit Intramuscular Model

The objective of this work was to demonstrate that the New Zealand White (NZW) rabbit intramuscular model can be used for detecting calcification in bioprosthetic tissue and to compare the calcification in the rabbit to that of native human valves. The rabbit model was compared with the commonly used Sprague–Dawley rat subcutaneous model. Eighteen rabbits and 18 rats were used to assess calcification in bioprosthetic tissue over time (7, 14, 30, and 90 d). The explanted rabbit and rat tissue discs were measured for calcium by using atomic absorption and Raman spectroscopy. Calcium deposits on the human valve explants were assessed by using Raman spectroscopy. The results showed that the NZW rabbit model is robust for detecting calcification in a shorter duration (14 d), with less infection complications, more space to implant tissue groups (thereby reducing animal use numbers), and a more metabolically and mechanically dynamic environment than the rat subcutaneous model . The human explanted valves and rabbit explanted tissue both showed Raman peaks at 960 cm⁻¹ which is representative of hydroxyapatite. Hydroxyapatite is the final calcium and phosphate species in the calcification of bioprosthetic heart valves and rabbit intramuscular implants. The NZW rabbit intramuscular model is an effective model for assessing calcification in bioprosthetic tissue.Abbreviations: BHV, bioprosthetic heart valve; FET, formaldehyde–ethanol–Tween 80; NZW, New Zealand WhiteBecause of their outstanding durability, mechanical heart valves were 1 of the first replacement valves used in humans. However, drawbacks to mechanical valves include patient complaints regarding the noise the valve makes and the risk of lifelong anticoagulation therapy.^4,20 Currently bioprosthetic heart valves (BHV) are 1 of the most common medical devices used to replace damaged mitral and aortic heart valves in men and women 65 y and older.¹⁷A leading drawback to BHV is dystrophic calcification, which is typically the primary failure mode for these devices.¹⁵ Calcification in BHV is believed to be caused by multiple factors, with glutaraldehyde (used to crosslink the tissue), residual cellular debris, and mechanical stress as the major factors. Patient factors including diabetes, renal failure, atherosclerosis, and calcium metabolism disorders can be important contributors as well.¹⁴ BHV are designed by using bovine pericardium, porcine valve leaflets, and porcine pericardium or dura mater; the most common tissues used are bovine pericardium and porcine valve leaflets.³⁶ Preventing or delaying calcification of BHV is an ongoing research dilemma of biomedical device companies. A 1982 study assessed explanted human valves that had been treated with glutaraldehyde only and found that calcification was the main cause of valve explantation and valves typically lasted only 9 1/2 y on average.³ To increase the durability and delay the onset of calcification of glutaraldehyde-treated valves, researchers and heart valve manufacturers began developing anticalcification treatments. In early studies, chemical compounds like protamine were used to block charged chemical groups and thus reduce calcification in glutaraldehyde-treated BHV.¹³ Other treatments used ethanol, AlCl₃, FeCl₃, L-Hydro, and osteopontin.^11,23,25,35 Many of these treatments were either proven ineffective or were not able to be manufactured. Effective new anticalcification treatments currently available commercially include α-amino oleic acid (Medtronic, Minneapolis, MN), an ethanol-based treatment (Linx, St Jude Medical, St Paul, MN), and a process involving formaldehyde, ethanol, and Tween 80 (ThermaFix, Edwards Lifesciences, Irvine, CA).^10,12 However, young adolescents and patients with calcium metabolic disorders still receive mechanical valves due to rapid calcification of BHV implants in these patient groups.²⁶An animal model is necessary to assess the effects of anticalcification treatments on bioprosthetic tissue in a short and effective timeframe. When assessing anticalcification tissue treatments, the animal models used are typically rats and rabbits. Wistar and Sprague–Dawley rats and New Zealand White (NZW) rabbits are the strains of choice.^5,29 Small animals are chosen because they are inexpensive, the tissue calcifies in a short period of time, the surgical procedure requires minimal pain and discomfort, and the animals are easy to care for.⁹ Typically, the BHV leaflet tissue is cut into discs or is used as whole leaflets and implanted subcutaneously in either rats or rabbits. Typical study duration to assess anticalcification efficacy in the rat model varies from 90 d to 6 mo.^24,41 The Sprague–Dawley rat is one of the most common models used to assess BHV tissue calcification properties.²⁷ The NZW rabbit model is another small animal model available, but to date only the subcutaneous route has been used for tissue biocompatibility and toxicology in drug efficacy studies only.Large animal models used to assess the safety and efficacy of BHV have included pig, sheep, cows, and nonhuman primates, but the primary model currently used is the juvenile sheep.³⁸ The juvenile sheep is a robust model that performs similarly to humans with regards to hemodynamics, valve annulus sizing, and thrombogenicity.^1,10 A typical safety and efficacy study in juvenile sheep lasts 20 wk.²¹ The drawbacks to using sheep to assess anticalcification treatments are the large sample size needed to demonstrate statistically significant differences, high surgical costs, and high animal-care costs.The age of the animal is important when assessing the calcification potential of BHV tissue. Compared with their older counterparts, juvenile animals demonstrate higher calcium metabolism because of bone-building, organ function, and structural tissues.⁷ Ways to assess calcium metabolism in small animals include measuring skeletal length and bone growth relative to sexual maturation.³² In Wistar rats, bone growth increased from 276 µm/d in 21-d-old weanlings to 330 µm/d in 35-d-old rats.^19,42 The bone growth spurt in the rats began to slow, falling to 85 µm/d by day 80, with full maturity by 24 wk. Compared with rats, rabbits mature more slowly, reaching maturity by 34 wk of age. A study involving 17 male and 12 female NZW rabbits assessed growth of the tibia and femur, assessing the correlation of tibial and femoral lengths and sexual maturity in rabbits.³⁹ This knowledge helps researchers assess the progressive growth and maturity of rabbits as they change from juvenile to adults.²⁸ This type of assessment may be important for understanding how intramuscular implants calcify in juvenile (6- to 8-mo-old) rabbits.The NZW rabbit intramuscular model offers a particular advantage for assessing tissue calcification properties. Epinephrine in rabbits had a higher absorbance and diffusion rate when injected intramuscularly compared with subcutaneously.¹⁶ The intramuscular region in rabbits has a rich vascular supply due to the high density in the latissimus dorsi compared with the subcutaneous of either rats or rabbits. The vascular transport mechanisms of muscle allow it to respond to foreign material (such as BHV tissue) more efficiently than the response to subcutaneous implants.⁴² The intramuscular region also offers a more mechanically dynamic environment than the static subcutaneous region, better mimicking some of the stresses on a tissue that a BHV tissue might endure when implanted in humans.Many analytical methods are used to assess the type of calcification that occurs in arteries or bioprosthetic tissue; currently gaining popularity is near-infrared Fourier transform Raman spectroscopy.³⁴ This method measures light scattered inelastically from photons.⁴⁰ Elastically scattered photons have the same energy (frequency) and therefore wavelength as the incident beam. However, a small fraction of light (approximately 1 in 10⁷ photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. This process of inelastic scattering of photons is called Raman scattering, which can occur with a change in vibrational, or rotational, or electronic energy of the molecule being studied. The difference in energy between the incident photon and the Raman-scattered photon is equal to the energy of a vibration of the scattering molecule, such as calcium or phosphate. A plot of intensity of scattered light versus energy difference is called a Raman spectrum. When used to assess the type of calcification in BHV, Raman spectroscopy has been shown to be effective in analyzing the presence of calcium phosphate species and, when combined with a calcium assay, the relative calcium:phosphate ratios.⁶ The most common type of calcification is the mineral hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂]. Roughly 70% of all bone is composed of hydroxyapatite.¹⁸ By using Raman spectroscopy (830 nm), hydroxyapatite was found at wavelengths of 960 to 1200 cm⁻¹ in human explanted BHV.³³ Other calcium–phosphate combinations found on explanted human heart valves include carbonate apatites, octacalcium, dicalcium, and amorphous calcium phosphates.⁸ Nascent calcification in biologic tissues goes through phase transformations of unstable calcium–phosphate salts to more stable calcium phosphate salts, ultimately maturing into hydroxyapatite.²² Raman spectroscopy can be applied to a variety of different morphologies, giving it a unique advantage when analyzing biologic samples, which are generally mixtures of fluids, tissues, and mineral deposits.³⁰ Raman analysis requires minimal preparation of biologic samples and is nondestructive to the sample. In the current study, near-infrared Fourier transform Raman spectroscopy was used to detect the presence of calcification in bioprosthetic valves explanted from humans and in bioprosthetic valve tissue from intramuscular rabbit explants.The objective of this study was to validate the rabbit intramuscular model for assessing the calcification potential of bioprosthetic tissue. The outcome of this study was to assess the length of study (days) necessary to see significant differences in calcification among 3 test groups and 1 control group, compare the type of calcification seen in the rabbit model with that of human valve explants (Raman spectroscopy), and to correlate the rabbit model to human BHV calcification.     

Synthesis and SAR of novel histamine H3 receptor antagonists

The synthesis and biological evaluation of novel tetrahydroisoquinoline, tetrahydroquinoline, and tetrahydroazepine antagonists of the human and rat H(3) receptors are described. The substitution around these rings as well as the nature of the substituent on nitrogen is explored. Several compounds with high affinity and selectivity for the human and rat H(3) receptors are reported.     

Backbone resonance assignments of an artificially engineered TEM-1/PSE-4 Class A β-lactamase chimera

The rapid evolution of Class A β-lactamases, which procure resistance to an increasingly broad panel of β-lactam antibiotics, underscores the urgency to better understand the relation between their sequence variation and their structural and functional features. To date, more than 300 clinically-relevant β-lactamase variants have been reported, and this number continues to increase. With the aim of obtaining insights into the evolutionary potential of β-lactamases, an artificially engineered, catalytically active chimera of the Class A TEM-1 and PSE-4 β-lactamases is under study by kinetics and NMR. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments for the 30 kDa chimera cTEM-17m. Despite its high molecular weight, the data provide evidence that this artificially-evolved chimeric enzyme is well folded. The hydrolytic activity of cTEM-17m was determined using the chromogenic substrate CENTA, with K _M = 160 ± 35 μM and k _cat = 20 ± 4 s^?1, which is in the same range as the values for TEM-1 and PSE-4 β-lactamases.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

The role of tower height and guy wires on avian collisions with communication towers

Every year an estimated 4–5 million migratory birds collide with communication towers in the United States. We examined the relative risks that tower support systems and tower height pose to migrating and other birds. We collected data comparing tower support systems (guyed vs. unguyed) and tower height categories in Michigan during 20 days of the peak of songbird migration at 6 towers in September–October 2003, 23 towers in May 2004, 24 towers in September 2004, and 6 towers in both May and September 2005. We systematically and simultaneously searched for bird carcasses under each tower and measured carcass removal and observer detection rates each season. Of those towers, 21 were between 116 and 146 m above ground level (AGL, medium) and 3 were >305 m AGL (tall). During the five 20-day sample periods we found a mean of 8.2 bird carcasses per guyed medium tower and a mean of 0.5 bird carcasses under unguyed medium towers. During four 20-day sample periods we detected a mean of 34.7 birds per guyed tall tower. Using both parametric and nonparametric tests (Mann–Whitney U-test, Kruskal–Wallis test, and Tukey's Honestly Significant Difference multiple comparison procedure) we determined that unguyed medium towers were involved in significantly fewer fatalities than guyed medium towers. We detected 54–86% fewer fatalities at guyed medium towers than at guyed tall towers. We found 16 times more fatalities at guyed medium towers than at unguyed medium towers. Tall, guyed towers were responsible for 70 times as many bird fatalities as the unguyed medium towers and nearly five times as many as guyed medium towers. These findings will provide managers and regulators, such as the US Fish and Wildlife Service, with quantitative data; thereby, allowing them to effectively work with the Federal Communications Commission in siting and authorizing tower placement. © 2011 The Wildlife Society.     

The Early Activation of Toll-Like Receptor (TLR)-3 Initiates Kidney Injury after Ischemia and Reperfusion

Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.