Preclinical Evaluation of the Temporary Drug-Coated Spur Stent System in Porcine Peripheral Arteries.

BACKGROUND: Drug penetration into the deeper arterial wall of heavily calcified lesions is one of the limitations of drug-coated balloons and drug-eluting stents in vascular interventions. The Temporary Spur Stent (TSS) system is characterized by a self-expanding nitinol stent that is uniformly covered in radialspikes, which, when coated, should allow a deeper penetration and longer retention of the drug into the diseased artery walls by penetrating through the calcified plaques. MATERIALS AND METHODS AND RESULTS: Uncoated TSS and paclitaxel (PTX)-coated TSS systems have been deployed in porcine peripheral arteries. Four weeks after the deployment of uncoated TSS systems, no adverse vascular remodeling or neointimal formation in the treated vessel segments were noticed. PTX-coated TSS systems transferred 9%±7% of the drug that was on the device to the targeted vessel area (196±163 ng PTX/mg arterial tissue) and the addition of the fluorescent dye Nile red to the coating showed that the spikes promote the transfer of the coating to the deeper layers of the vessel wall. The PTX-coated TSS systems showed a significant reduction in neointimal proliferation compared to the uncoated TSS systems: quantitative angiography showed a vessel diameter stenosis of 37.2%±11.0% and 16.4%±8.8% 4 weeks after the treatment with uncoated and PTX-coated TSS systems, respectively. CONCLUSION: The treatment with the TSS system was well tolerated and the spikesfacilitate the transfer of the coating into deeper layers of the vessel wall. Moreover, the PTX-coated TSS systems effectively inhibit neointimal proliferation.

Bare Metal Stents on Resveratrol-Coated Balloons in Porcine Coronary and Peripheral Arteries.

Balloon angioplasty and stent implantation are standard techniques to reopen stenotic vessels. Often, balloons or stents coated with cytostatic drugs are used to prevent re-occlusion of the arteries. Resveratrol, which is known for its numerous beneficial effects on cardiovascular health, is used as an antioxidant additive on paclitaxel-coated balloon catheters. What is still unclear is whether resveratrol-only balloon coating in combination with a bare metal stent (BMS) also has positive effects on vascular healing. Here, we analyzed neointimal thickening, fibrin deposition, inflammation, vasa vasorum density, and reendothelialization after implantation of BMS via a resveratrol coated balloon approach in a porcine model. In general, resveratrol treatment did not result in significantly altered responses compared to the control group in peripheral arteries. In coronary arteries, an increase in vasa vasorum density became evident three days after resveratrol treatment compared to the control group and abolished up to day 7. Significant effects of the resveratrol treatment on the fibrin score or intima-media area were transient and restricted to either peripheral or coronary arteries. In conclusion, local single-dose resveratrol treatment via a resveratrol-only coated balloon and BMS approach did not lead to adverse systemic or local effects, but also no significant beneficial effects on vascular healing were detected in the current study.

Resveratrol-Coated Balloon Catheters in Porcine Coronary and Peripheral Arteries.

Angioplasty aiming at vascular dilatation causes endothelial denudation and induces complex inflammatory responses that affect vascular healing, including delayed reendothelialization and excessive neointima proliferation. Resveratrol is known for multiple beneficial effects on the vessel wall after systemic treatment or sustained release from a stent. It is also used as an additive on drug-coated balloon catheters (DCB). In this study, the effect of a single dose of resveratrol, three days to four weeks after administration as a balloon coating during angioplasty, was investigated. Sixteen pigs underwent angioplasty with resveratrol-coated or uncoated balloon catheters in coronary and peripheral arteries. Vessels were overstretched by approximately 20% to enhance vessel wall injury and to produce persistent vessel wall irritation. A significantly reduced number of micro vessels and macrophages in the adventitia, as well as an improved reendothelialization of the vessel lumen, were observed in resveratrol-treated peripheral arteries. The coronaries had a much higher injury score compared to peripheral vessels. Resveratrol-dependent reduction of macrophages, micro vessels or acceleration of reendothelialization was not evident in the coronary vessels. Additionally, no significant effect on neointima proliferation and inflammation score in either vessel territory was observed as a result of resveratrol treatment. In conclusion, the results suggest that resveratrol diminishes the inflammatory response and promotes vascular healing in peripheral arteries. These same effects are absent in more severely injured coronary arteries.

Protective Function of Ahnak1 in Vascular Healing after Wire Injury.

AIM: Vascular remodeling following injury substantially accounts for restenosis and adverse clinical outcomes. In this study, we investigated the role of the giant scaffold protein Ahnak1 in vascular healing after endothelial denudation of the murine femoral artery. METHODS: The spatiotemporal expression pattern of Ahnak1 and Ahnak2 was examined using specific antibodies and real-time quantitative PCR. Following wire-mediated endothelial injury of Ahnak1-deficient mice and wild-type (WT) littermates, the processes of vascular healing were analyzed. RESULTS: Ahnak1 and Ahnak2 showed a mutually exclusive vascular expression pattern, with Ahnak1 being expressed in the endothelium and Ahnak2 in the medial cells in naïve WT arteries. After injury, a marked increase of Ahnak1- and Ahnak2-positive cells at the lesion site became evident. Both proteins showed a strong upregulation in neointimal cells 14 days after injury. Ahnak1-deficient mice showed delayed vascular healing and dramatically impaired re-endothelialization that resulted in prolonged adverse vascular remodeling, when compared to the WT littermates. CONCLUSION: The large scaffold and adaptor proteins Ahnak1 and Ahnak2 exhibit differential expression patterns and functions in naïve and injured arteries. Ahnak1 plays a nonredundant protective role in vascular healing.

Decellularized amniotic membrane attenuates postinfarct left ventricular remodeling.

BACKGROUND: Placenta and amnion have been suggested as sources of juvenile cells and tissues for use in surgical regenerative medicine. We previously determined the impact of amniotic epithelial cells induced to undergo epithelial-to-mesenchymal transition (EMT) on myocardial remodeling processes and now evaluated the effects of naïve and processed amniotic membrane (AM) on postischemic left ventricular (LV) geometry and function. METHODS: Human AM was used in unmodified form (AM), after EMT induction by transforming growth factor ß (EMT-AM), and after decellularization (Decell-AM). After characterization by histology, electron microscopy, splenocyte proliferation assay, and cytokine release, myocardial infarction was induced in 6-8-week old male BALB/c mice by permanent left anterior descending coronary occlusion, and AM patches were sutured to the anterior LV surface (n = 10 per group). Infarcted hearts without AM or sham-operated mice were used as controls (n = 10 each). After 4 weeks, LV pressure-volume curves were recorded using a conductance catheter before the animals were sacrificed and the hearts analyzed by histology. RESULTS: TGF-ß treatment induced EMT-like changes in amniotic epithelial cells but increased AM xenoreactivity in vitro (splenocyte proliferation) and in vivo (CD4+ cell invasion). Moreover, in vitro interleukin-6 release from AM and from cardiac fibroblasts co-incubated with AM was 300- or 100-fold higher than that of interleukin-10, whereas Decell-AM did not release any cytokines. AM- and Decell-AM-treated hearts had smaller infarct size and greater infarct scar thickness than infarct control hearts, but there was no difference in myocardial capillary density or the number of TUNEL positive apoptotic cells. LV contractile function was better in the AM and EMT-AM groups than in infarcted control hearts, but dP/dt max, dP/dt min, stroke work, and cardiac output were best preserved in mice treated with Decell-AM. Volume-based parameters (LV end-systolic and end-diastolic volume as well as LV ejection fraction) did not differ between AM and Decell-AM. CONCLUSIONS: Decellularized AM supports postinfarct ventricular dynamics independent of the actual regeneration processes. As a cell-free approach to support the infarcted heart, this concept warrants further investigation.

Impact of fetal and neonatal environment on beta cell function and development of diabetes.

The global epidemic of diabetes is a serious threat against health and healthcare expenses. Although genetics is important it does not explain the dramatic increase in incidence, which must involve environmental factors. Two decades ago the concept of the thrifty phenotype was introduced, stating that the intrauterine environment during pregnancy has an impact on the gene expression that may persist until adulthood and cause metabolic diseases like obesity and type 2 diabetes. As the pancreatic beta cells are crucial in the regulation of metabolism this article will describe the influence of normal pregnancy on the beta cells in both the mother and the fetus and how various conditions like diabetes, obesity, overnutrition and undernutrition during and after pregnancy may influence the ability of the offspring to adapt to changes in insulin demand later in life. The influence of environmental factors including nutrients and gut microbiota on appetite regulation, mitochondrial activity and the immune system that may affect beta cell growth and function directly and indirectly is discussed. The possible role of epigenetic changes in the transgenerational transmission of the adverse programming may be the most threatening aspect with regard to the global diabetes epidemics. Finally, some suggestions for intervention are presented.

Repeated Injection of Very Small Superparamagnetic Iron Oxide Particles (VSOPs) in Murine Atherosclerosis: A Safety Study.

Citrate-coated electrostatically stabilized very small superparamagnetic iron oxide particles (VSOPs) have been successfully tested as magnetic resonance angiography (MRA) contrast agents and are promising tools for molecular imaging of atherosclerosis. Their repeated use in the background of pre-existing hyperlipidemia and atherosclerosis has not yet been studied. This study aimed to investigate the effect of multiple intravenous injections of VSOPs in atherosclerotic mice. Taurine-formulated VSOPs (VSOP-T) were repeatedly intravenously injected at 100 µmol Fe/kg in apolipoprotein E-deficient (ApoE KO) mice with diet-induced atherosclerosis. Angiographic imaging was carried out by in vivo MRI. Magnetic particle spectrometry was used to detect tissue VSOP content, and tissue iron content was quantified photometrically. Pathological changes in organs, atherosclerotic plaque development, and expression of hepatic iron-related proteins were evaluated. VSOP-T enabled the angiographic imaging of heart and blood vessels with a blood half-life of one hour. Repeated intravenous injection led to VSOP deposition and iron accumulation in the liver and spleen without affecting liver and spleen pathology, expression of hepatic iron metabolism proteins, serum lipids, or atherosclerotic lesion formation. Repeated injections of VSOP-T doses sufficient for MRA analyses had no significant effects on plaque burden, steatohepatitis, and iron homeostasis in atherosclerotic mice. These findings underscore the safety of VSOP-T and support its further development as a contrast agent and molecular imaging tool.

Drug-Coated Balloons: Drugs Beyond Paclitaxel?

BACKGROUND: Although controversially discussed, paclitaxel is the only clinically proven drug that inhibits restenosis when released from drug-coated balloons (DCBs). Limus drugs are currently being explored as alternatives. The aim of the preclinical studies was to investigate drug candidates beyond paclitaxel considered for balloon coating. METHODS: Drugs were tested with respect to dissolution in organic solvents, coating on balloons, and drug transfer to the vessel wall. Inhibition of neointimal proliferation was tested in the porcine model of coronary in-stent stenosis. Intravascular drug treatment was achieved by DCBs at the time of stent implantation. RESULTS: Coating had to be adjusted for each drug. Doses on the balloons ranged from 1.0 to 8.6 µg/mm2 balloon surface. Satisfactory amounts of drug ranging from 5% to 29% of initial doses were transferred into the vessel wall. Angiographic parameters such as late lumen loss (LLL) at 4 weeks did not show reduction of in-stent neointimal proliferation by treatment with arsenic trioxide (0.87 ± 0.44 mm), betamethasone dipropionate (1.00 ± 0.54 mm), bortezomib (1.74 ± 0.46 mm), green tea extract (1.24 ± 0.51 mm), fantolon, an epothilone (0.86 ± 0.61 mm), methotrexate (1.09 ± 0.72 mm), and thalidomide (1.59 ± 0.55 mm) compared to treatment with uncoated balloons (1.07 ± 0.60 mm), while coatings with paclitaxel reliably reduced in-stent stenosis (LLL = 0.36 ± 0.25 mm). CONCLUSIONS: Despite the proven antiproliferative and/or anti-inflammatory effect of the drugs, none of the coatings significantly reduced LLL compared to uncoated balloons and thus, based on the results presented here, none of the tested coatings may be considered a substitute for the paclitaxel-based coatings currently in clinical use.

Improvement of Outcome for Treatment of 'Restenosis-prone' Vascular Lesions? Potential Impact of the Paclitaxel dose on Late Lumen Loss in Porcine Peripheral Arteries.

PURPOSE: Clinical data indicate that the drug density on drug-coated balloons (DCBs) might have a role on treatment effect and durability. The aim of the current study was to investigate inhibition of neointimal formation and potential adverse effects after treatment with a novel double-dose DCB in swine. MATERIAL AND METHODS: A four-week study was performed in peripheral arteries of 12 domestic pigs after vessel injury and stent implantation. The novel double-dose DCB with 6-µg paclitaxel (Ptx)/mm2 balloon surface (1 × 6) was compared to a standard DCB with 3.5 µg Ptx/mm2 (3.5) and uncoated balloons (POBA). Potential adverse effects were stimulated by using three fully overlapping DCBs with 6 µg Ptx/mm2 each (3 × 6). Quantitative angiography, histomorphometry and histopathological analyses were performed. RESULTS: Higher paclitaxel doses per square millimeter of treated arteries were associated with reduced late lumen loss (LLL) in quantitative angiography 4 weeks after treatment (POBA: 0.91 ± 0.75 mm; 3.5: 0.45 ± 0.53 mm; 1 × 6: 0.21 ± 0.41 mm; 3 × 6: - 0.38 ± 0.65 mm). In histomorphometry, maximal neointimal thickness and neointimal area were the lowest for the 1 × 6 group (0.15 ± 0.06 mm/1.5 ± 0.4 mm2), followed by 3 × 6 (0.20 ± 0.07 mm/1.8 ± 0.4 mm2), 3.5 (0.22 ± 0.12 mm/2.2 ± 1.1 mm2) and POBA (0.30 ± 0.07 mm/3.2 ± 0.7 mm2). Downstream tissue showed histopathological changes in all groups including POBA, in larger number and different quality (e.g., edema, inflammation, vessel wall necrosis, vasculitis and perivasculitis) in the 3 × 6 group, which did not cause clinical or functional abnormalities throughout the study. CONCLUSION: Treatment with the double-dose DCB (6 µg Ptx/mm2) tended to increase inhibition of in-stent neointimal formation and to diminish LLL after peripheral intervention in the porcine model compared to a market-approved DCB with 3.5 µg Ptx/mm2.

Role of PGC-1α in exercise and fasting-induced adaptations in mouse liver.

The transcriptional coactivator peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)-1α plays a role in regulation of several metabolic pathways. By use of whole body PGC-1α knockout (KO) mice, we investigated the role of PGC-1α in fasting, acute exercise and exercise training-induced regulation of key proteins in gluconeogenesis and metabolism in the liver. In both wild-type (WT) and PGC-1α KO mice liver, the mRNA content of the gluconeogenic proteins glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) was upregulated during fasting. Pyruvate carboxylase (PC) remained unchanged after fasting in WT mice, but it was upregulated in PGC-1α KO mice. In response to a single exercise bout, G6Pase mRNA was upregulated in both genotypes, whereas no significant changes were detected in PEPCK or PC mRNA. While G6Pase and PC protein remained unchanged, liver PEPCK protein content was higher in trained than untrained mice of both genotypes. The mRNA content of the mitochondrial proteins cytochrome c (Cyt c) and cytochrome oxidase (COX) subunit I was unchanged in response to fasting. The mRNA and protein content of Cyt c and COXI increased in the liver in response to a single exercise bout and prolonged exercise training, respectively, in WT mice, but not in PGC-1α KO mice. Neither fasting nor exercise affected the mRNA expression of antioxidant enzymes in the liver, and knockout of PGC-1α had no effect. In conclusion, these results suggest that PGC-1α plays a pivotal role in regulation of Cyt c and COXI expression in the liver in response to a single exercise bout and prolonged exercise training, which implies that exercise training-induced improvements in oxidative capacity of the liver is regulated by PGC-1α.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes.

OBJECTIVE: Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. METHODS: We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. RESULTS: Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. CONCLUSION: In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Nitric oxide sensitive guanylyl cyclase activity decreases during cerebral postnatal development because of a reduction in heterodimerization.

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co-precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.

Dual lipolytic control of body fat storage and mobilization in Drosophila.

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. We generated a Drosophila mutant lacking the receptor (AKHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. Our data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis.

In vivo biocompatibility study of degradable homo- versus multiblock copolymers and their (micro)structure compared to an established biomaterial.

Copolyetheresterurethane (PDC) is a biodegradable, shape-memory biomaterial, which has been shown to be of low toxicity and pro-angiogenic in vitro. In the present study we examined the in vivo compatibility of PDC as a compression molded film and as electrospun scaffolds and its well established constituent, the homopolymer poly(p-dioxanone) (PPDO), which were compared with the clinically used poly[(vinylidene fluoride)-co-hexafluoropropene] (PVDF) as reference material. The materials were implanted in the subcutaneous tissue of mice and the host responses were analyzed histologically 7 and 28 days after implantation.All materials induced a foreign body response (FRB) including the induction of foreign body giant cells and a peripheral fibrous capsule. PDC, PPDO and PVDF films showed no signs of degradation after 28 days. PDC films showed a significantly reduced associated macrophage layer and fibrous capsule on their surface. Few fragments of PDC and PPDO scaffolds were present at the implantation site, while PVDF scaffolds were still present in large amounts at day 28. Especially aligned electrospun PDC scaffold induced a significantly thinner fibrous and a slightly reduced inflammatory response after 28 days of implantation. In addition, only PDC aligned fibrous scaffold structures induced a significant increase in angiogenesis.In summary, PDC films outperformed PPDO and PVDF films in terms of compatibility, especially in capsule and macrophage layer thickness. Through microstructuring of PDC and PPDO into scaffolds an almost complete degradation was observed after 28 days, while their respective films remained almost unchanged. However, the capsule thickness of all scaffolds was comparable to the films after 28 days. Finally, the parallel arrangement of PDC fibers enabled a strong enhancement of angiogenesis within the scaffold. Hence, material chemistries influence overall compatibility in vivo, while angiogenesis could be influenced more strongly by microstructural parameters than chemical ones.

SLCV-a supervised learning-computer vision combined strategy for automated muscle fibre detection in cross-sectional images.

Muscle fibre cross-sectional area (CSA) is an important biomedical measure used to determine the structural composition of skeletal muscle, and it is relevant for tackling research questions in many different fields of research. To date, time consuming and tedious manual delineation of muscle fibres is often used to determine the CSA. Few methods are able to automatically detect muscle fibres in muscle fibre cross-sections to quantify CSA due to challenges posed by variation of brightness and noise in the staining images. In this paper, we introduce the supervised learning-computer vision combined pipeline (SLCV), a robust semi-automatic pipeline for muscle fibre detection, which combines supervised learning (SL) with computer vision (CV). SLCV is adaptable to different staining methods and is quickly and intuitively tunable by the user. We are the first to perform an error analysis with respect to cell count and area, based on which we compare SLCV to the best purely CV-based pipeline in order to identify the contribution of SL and CV steps to muscle fibre detection. Our results obtained on 27 fluorescence-stained cross-sectional images of varying staining quality suggest that combining SL and CV performs significantly better than both SL-based and CV-based methods with regards to both the cell separation- and the area reconstruction error. Furthermore, applying SLCV to our test set images yielded fibre detection results of very high quality, with average sensitivity values of 0.93 or higher on different cluster sizes and an average Dice similarity coefficient of 0.9778.

Discerning the spatio-temporal disease patterns of surgically induced OA mouse models.

Osteoarthritis (OA) is the most common cause of disability in ageing societies, with no effective therapies available to date. Two preclinical models are widely used to validate novel OA interventions (MCL-MM and DMM). Our aim is to discern disease dynamics in these models to provide a clear timeline in which various pathological changes occur. OA was surgically induced in mice by destabilisation of the medial meniscus. Analysis of OA progression revealed that the intensity and duration of chondrocyte loss and cartilage lesion formation were significantly different in MCL-MM vs DMM. Firstly, apoptosis was seen prior to week two and was narrowly restricted to the weight bearing area. Four weeks post injury the magnitude of apoptosis led to a 40-60% reduction of chondrocytes in the non-calcified zone. Secondly, the progression of cell loss preceded the structural changes of the cartilage spatio-temporally. Lastly, while proteoglycan loss was similar in both models, collagen type II degradation only occurred more prominently in MCL-MM. Dynamics of chondrocyte loss and lesion formation in preclinical models has important implications for validating new therapeutic strategies. Our work could be helpful in assessing the feasibility and expected response of the DMM- and the MCL-MM models to chondrocyte mediated therapies.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated ß-cell maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing ß-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat ß-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E ß-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E ß-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal ß-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate ß-cell maturation. This study indicates a functional role for TFF3 in pancreatic ß-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

In vivo biocompatibility assessment of poly (ether imide) electrospun scaffolds.

Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28 days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7-28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7 days after implantation and remained stable until 28 days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing. Copyright © 2015 John Wiley & Sons, Ltd.

Heterodimerization with the ß1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a ß subunit. Two different α subunits (α1 and α2) give rise to two heterodimeric enzymes α1/ß1 and α2/ß1. Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α2/ß1 form is generally much lower and approaches levels similar to the α1/ß1 form in the brain only. In the present paper, we show that the α2/ß1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α2/ß1 form, but not the α1/ß1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α2 subunit was sufficient for cell-cell contact localization. For the full length α2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the ß1 subunit, but not as isolated α2 subunit. We conclude that the PDZ binding motif of the α2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α2/ß1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective.

Skeletal Muscle to Pancreatic ß-Cell Cross-talk: The Effect of Humoral Mediators Liberated by Muscle Contraction and Acute Exercise on ß-Cell Apoptosis.

CONTEXT: Mechanisms explaining exercise-induced ß-cell health are unknown. OBJECTIVE: This study aimed to define the role of muscle contraction and acute exercise-derived soluble humoral mediators on ß-cell health. DESIGN: In vitro models were used. SETTING: University. PARTICIPANTS: Healthy subjects. INTERVENTION(S): Conditioned media (CM) were collected from human skeletal muscle (HSkM) cells treated with or without electrical pulse stimulation (EPS). Antecubital and femoral venous blood serum were collected before and after an exercise bout. CM and sera with or without IL-6 neutralization were used to incubate insulin-producing INS-1 cells and rat islets for 24 h in the presence or absence of proinflammatory cytokines (IL-1ß+IFN-γ). MAIN OUTCOME MEASURE(S): INS-1 and islet apoptosis and accumulated insulin secretion. RESULTS: IL-1ß+IFN-γ increased INS-1 and islet apoptosis and decreased insulin secretion. EPS-treated HSkM cell CM did not affect these variables. Exercise-conditioned antecubital but not femoral sera prevented IL-1ß+IFN-γ-induced INS-1 and islet apoptosis. Femoral sera reduced insulin secretion under normal and proinflammatory conditions in INS-1 but not islet cells. EPS increased HSkM cell IL-6 secretion and exercise increased circulating IL-6 levels in antecubital and femoral serum. IL-6 neutralization demonstrated that muscle-derived IL-6 prevents INS-1 and islet apoptosis in the absence of IL-1ß+IFN-γ, but augments apoptosis under proinflammatory conditions, and that muscle-derived IL-6 supports islet insulin secretion in the absence of IL-1ß+IFN-γ. CONCLUSIONS: Unidentified circulating humoral mediators released during exercise prevent proinflammatory cytokine-induced ß-cell apoptosis. Muscle-derived mediators released during exercise suppress ß-cell insulin secretion. Furthermore, muscle-derived IL-6 seems to prevent ß-cell apoptosis under normal conditions but contributes to ß-cell apoptosis under proinflammatory conditions.