Post-Myocardial Infarction T-tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin-2 and Bridging Integrator 1 (BIN-1).

BACKGROUND: Heart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t-tubule (t-t) loss. However, post-MI nano-scale morphological changes to the remaining t-ts are poorly understood. METHOD AND RESULTS: We utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post-MI, after mild left-ventricular dysfunction has developed, t-ts are not only lost in the peri-infarct region, but also the remnant t-ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin-2 (JP2), important for maintaining t-t trajectory, is depressed (-0.5×) in keeping with the t-ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t-ts. The bridging integrator 1 protein (BIN-1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t-ts. Importantly, we have determined that t-ts, in the remote region, are narrowed and also contain dense membrane folds (BIN-1 is up-regulated +3.4×), whereas the t-ts have a radial organization comparable to control JP2 is upregulated +1.7×. CONCLUSIONS: This study reveals previously unidentified remodeling of the t-t nano-architecture in the post-MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t-ts, attenuating the development of hypocontractility post-MI.

Combined MSC and GLP-1 Therapy Modulates Collagen Remodeling and Apoptosis following Myocardial Infarction.

Background. Mesenchymal stem cells (MSCs) and glucagon-like peptide-1 (GLP-1) are being tested as treatment strategies for myocardial infarction (MI); however, their mechanisms in the heart are not fully understood. Methods. We examined the effects of MSCs, either native, or engineered to secrete a GLP-1 fusion protein (MSCs ± GLP-1), on human cardiomyocyte apoptosis in vitro. The effect on cardiac remodeling when encapsulated in alginate beads (CellBeads-MSC and CellBeads-MSC + GLP-1) was also evaluated in a pig MI model, whereby pigs were treated with Empty Beads, CellBeads-MSC, or CellBeads-MSC + GLP-1 and sacrificed at one or four weeks following MI. Results. MSC + GLP-1 conditioned media demonstrated antiapoptotic effects on ischaemic human cardiomyocytes in vitro. In vivo, qRT-PCR revealed large changes in the expression of several genes involved in extracellular matrix remodeling, which were altered following MSC ± GLP treatment. After four weeks, infarcted areas were imaged using atomic force microscopy, demonstrating significant alterations between groups in the structure of collagen fibrils and resulting scar. Conclusions. These data demonstrate that MSCs ± GLP-1 exhibit modulatory effects on healing post-MI, affecting both apoptosis and collagen scar formation. These data support the premise that both MSCs and GLP-1 could be beneficial in MI treatment.

Serum cholesterol selectively regulates glucocorticoid sensitivity through activation of JNK.

Glucocorticoids (Gc) are potent anti-inflammatory agents with wide clinical application. We have previously shown that increased serum concentration significantly attenuates regulation of a simple Gc-responsive reporter. We now find that glucocorticoid receptor (GR) regulation of some endogenous transactivated but not transrepressed genes is impaired, suggesting template specificity. Serum did not directly affect GR expression, activity or trafficking, implicating GR crosstalk with other signalling pathways. Indeed, a JNK inhibitor completely abolished the serum effect. We identified the Gc modulating serum component as cholesterol. Cholesterol loading mimicked the serum effect, which was readily reversed by JNK inhibition. Chelation of serum cholesterol with methyl-ß-cyclodextrin or inhibition of cellular cholesterol synthesis with simvastatin potentiated the Gc response. To explore the effect in vivo we used ApoE(-/-) mice, a model of hypercholesterolaemia. Consistent with our in vitro studies, we find no impact of elevated cholesterol on the expression of GR, or on the hypothalamic-pituitary-adrenal axis, measured by dexamethasone suppression test. Instead we find selective Gc resistance on some hepatic target genes in ApoE(-/-) mice. Therefore, we have discovered an unexpected role for cholesterol as a selective modulator of Gc action in vivo. Taken together these findings reveal a new environmental constraint on Gc action with relevance to both inflammation and cancer.

Encapsulated glucagon-like peptide-1-producing mesenchymal stem cells have a beneficial effect on failing pig hearts.

Stem cell therapy is an exciting and emerging treatment option to promote post-myocardial infarction (post-MI) healing; however, cell retention and efficacy in the heart remain problematic. Glucagon-like peptide-1 (GLP-1) is an incretin hormone with cardioprotective properties but a short half-life in vivo. The effects of prolonged GLP-1 delivery from stromal cells post-MI were evaluated in a porcine model. Human mesenchymal stem cells immortalized and engineered to produce a GLP-1 fusion protein were encapsulated in alginate (bead-GLP-1 MSC) and delivered to coronary artery branches. Control groups were cell-free beads and beads containing unmodified MSCs (bead-MSC), n = 4-5 per group. Echocardiography confirmed left ventricular (LV) dysfunction at time of delivery in all groups. Four weeks after intervention, only the bead-GLP-1 MSC group demonstrated LV function improvement toward baseline and showed decreased infarction area compared with controls. Histological analysis showed reduced inflammation and a trend toward reduced apoptosis in the infarct zone. Increased collagen but fewer myofibroblasts were observed in infarcts of the bead-GLP-1 MSC and bead-MSC groups, and significantly more vessels per mm(2) were noted in the infarct of the bead-GLP-1 MSC group. No differences were observed in myocyte cross-sectional area between groups. Post-MI delivery of GLP-1 encapsulated genetically modified MSCs provided a prolonged supply of GLP-1 and paracrine stem cell factors, which improved LV function and reduced epicardial infarct size. This was associated with increased angiogenesis and an altered remodeling response. Combined benefits of paracrine stem cell factors and GLP-1 were superior to those of stem cells alone. These results suggest that encapsulated genetically modified MSCs would be beneficial for recovery following MI.

C-Myb function in the vessel wall.

C-Myb is a DNA-binding transcription factor that functions in apoptosis, proliferation and differentiation. The role of c-Myb in vascular injury has been investigated previously both in vitro and in vivo, where knock-down of c-Myb is known to lead to a reduction in proliferation and an increase in apoptosis of vascular smooth muscle cells (VSMCs). Reduction of c-Myb activity has also been shown to decrease neointimal formation in vivo, by reducing VSMC proliferation. In contrast, over-expression of c-Myb in vivo leads to increased survival rates in certain cell types. This review will look mainly at studies investigating c-Myb function in the vasculature, and evidence of signalling interactions which may be considered with regard to c-Myb as a possible target in the treatment of vasculoproliferative diseases.

Controlled delivery of antisense oligonucleotides: a brief review of current strategies.

Antisense therapy has been investigated extensively over the past two decades, either experimentally for gene functional research or clinically as therapeutic agents owing to the conceptual simplicity, ease of design and low cost. The concept of this therapeutic approach is promising because short antisense oligonucleotides (ASOs) can be delivered into target cells for specific hybridisation with target mRNA, resulting in the inhibition of the expression of pathogenic genes. However, the efficient delivery of the ASO molecules into target cells remains challenging; this bottleneck together with several other technical hurdles need to be overcome before this approach becomes effective and widely adopted. A variety of vectors such as lipids, polymers, peptides and nanoparticles have been explored. This review outlines the recent advances of the non-viral ASO delivery strategies. Several recent scientific studies, including authors' contributions, have been selected to highlight the technical aspects of ASO delivery.

Controlled delivery of anti-sense oligodeoxynucleotide from multilayered biocompatible phosphorylcholine polymer films.

Fabrication of polymeric multilayered films based on the electrostatic self-assembly of polycations and polyanions is a promising approach for controlled loading and release in gene delivery. In this study, we have fabricated a series of multilayered films based on alternate deposition between positively-charged cationic phosphorylcholine copolymer (PC copolymer) and negatively-charged c-myc anti-sense oligodeoxynucleotide (AS-ODN). The growth of film thickness and increase of ODN loading capacity were monitored by spectroscopic ellipsometry (SE) and confocal laser scanning microscopy (CLSM). After elution into PBS buffer under physiological conditions, the elution profile was monitored by UV spectrometry and gel electrophoresis. Employing a secondary transgenic vector, the cellular uptake of the eluted AS-ODN into HeLa cells was evaluated by fluorescent microscopy and FACS analysis. The biological effect of eluted AS-ODN was evaluated by cell growth inhibition. The results showed that AS-ODN loading capacity increased almost linearly with the number of PC polymer/ODN bilayers and was also strongly dependent upon the cationic charge density. Through swelling, a non-degradable release mechanism, the AS-ODN release was characterized by two distinguishable release regimes: a fast release regime during the first 6 hour period and a slow release regime from 6 hour to the 8th day, both of which were characterized by zero-order kinetics. Gel electrophoresis showed excellent DNA integrity and strong transfection was observed when the eluted ODN was transfected into HeLa cells. Cell growth was significantly inhibited by eluted AS-ODN, indicating its full bioactivity. These results demonstrate that PC multilayered polymer films are capable of delivering a prescribed amount of anti-sense ODN with a controllable kinetic profile and that the multilayer process is more efficient and reliable than most other existing coating approaches largely based on single-layer fabrication.

Notch3 activation modulates cell growth behaviour and cross-talk to Wnt/TCF signalling pathway.

Notch3 is one of the four Notch receptors identified in mammal and expressed mainly in the arterial smooth muscle cells of human adult. Signalling via Notch3 is thought to be important in maintaining the phenotypic stability of the cells, but the nature of the signalling and its regulation to other signalling pathways are largely unknown. To understand further of the cellular function of Notch3 signalling, we generated cell lines stably expressing a constitutively active form of human Notch3 comprising of its soluble intracellular domain (N3IC). The N3IC expressing cells showed accelerated proliferation, decreased migration, increased cell surface N-cadherin, and growth in a colonised fashion that was reversible by N-cadherin blockade. N3IC expressing cells were also protected significantly against staurosporine-induced apoptosis and exhibited lower caspase 3/7 activity, accompanied by up-regulation of pAKT compared to control cells. We also found a complex cross-talk between Notch3 signalling and the Wnt pathway. N3IC stimulated Wnt-independent T-cell factor (TCF, the target transcription factor in the Wnt pathway) activation which was associated with increased Tyr-142 phosphorylation of beta-catenin. In contrast N3IC suppressed TCF activation in response to LiCl, which mimics the Wnt-dependent TCF activation mechanism. We conclude that Notch3 promotes cell growth and survival by activating PI3-kinase/AKT pathway; N-cadherin participates in the change of cell growth caused by Notch3 activation; and Notch3 signalling has dual-effects on the Wnt/TCF pathway suggesting a buffering role that Notch3 signalling may play in balancing these two important signalling pathways in regulating cell function.

Controlled delivery of antisense oligodeoxynucleotide from cationically modified phosphorylcholine polymer films.

Antisense strategy is a promising approach for the prevention of in-stent restenosis if therapeutic agents such as antisense oligodeoxynucleotides (AS-ODNs) can be successfully delivered to the implant site. Optimizing the routes and conditions for controlled loading and release of therapeutic agents from a biocompatible polymer coating is still required. In this study, phosphorylcholine (PC) polymer films bearing different cationic charge densities were deposited onto smooth silicon substrates. The thickness of these films was determined by spectroscopic ellipsometry (SE). Human c-myc AS-ODNs were incorporated into the PC polymer films by immersion in concentrated AS-ODN solution and eluted into PBS under physiological conditions. The elution profile was monitored by UV spectrometry and gel electrophoresis. Cellular uptake of the eluted AS-ODN into vascular smooth muscle cells (VSMCs) was evaluated by fluorescence microscopy. The results showed that ODN loading capacities increased with film thickness and were also strongly dependent on the cationic charge density. AS-ODN release was characterized by a slight initial burst in the first half hour followed by a period of sustained release up to 8 days. Gel electrophoresis demonstrated DNA integrity, and different transfection efficiencies were observed when the eluted ODNs were transfected into VSMCs. These results demonstrated that cationically modified PC polymers are capable of delivery of antisense ODNs in a controlled manner and that they are well suited for specific biomedical devices such as DNA-eluting stents.

VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection.

BACKGROUND: The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection. MATERIALS AND METHODS: Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis. RESULTS: The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22-transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 +/- 3.6 and 3.2 +/- 1.0 fold higher than in pMybEn-transfected cultures; pVP22MybEn-transfected cultures showed no difference in these parameters. pTK-transfected cultures underwent 60-70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions. CONCLUSIONS: The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The "intrinsic" bystander activity of TK was 4-fold greater than was "engineered" by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method.

Mechanical stretch induces phosphorylation of p38-MAPK and apoptosis in human saphenous vein.

OBJECTIVE: Failure of saphenous vein grafts remains a major limitation of coronary bypass surgery. The aims of the present study were to determine whether pressure distension of human saphenous vein induces the activation of p38-MAPK and to determine its role in apoptosis. METHODS AND RESULTS: Phosphorylated p38 was detected at basal levels in human saphenous vein obtained immediately after harvesting. Distended saphenous vein showed significantly higher levels of phosphorylated p38 compared with control vein (P<0.01) and nondistended saphenous vein maintained for 3 and 6 hours after harvesting (both P<0.01). Apoptosis in distended and nondistended vein was significantly higher at 24 hours compared with control vein, with distended vein showing increased apoptosis compared with nondistended saphenous vein at all time points investigated (P<0.001). Immunolocalization showed co-localization of phosphorylated p38 and apoptosis. Inhibition of p38 activity reduced the apoptotic index of cultured vascular smooth muscle cells by 72.1%+/-1.2% and cultured distended saphenous vein segments by 72.7%+/-0.9%. CONCLUSIONS: Pressure distension of intact human saphenous vein induces activation of p38, and this is associated with apoptosis. Inhibition of p38 kinase activity in saphenous vein smooth muscle cells and intact vein reduces apoptosis. These findings contribute to our understanding of the mechanisms of saphenous vein graft failure.

Angiopeptin-eluting stents: observations in human vessels and pig coronary arteries.

Local drug delivery from polymer-coated coronary stents may reduce the incidence of in-stent restenosis. Angiopeptin, an inhibitor of smooth muscle cell proliferation, may reduce the clinical impact of restenosis. The objectives of this study were to characterize the release kinetics and distribution of angiopeptin-loaded phosphorylcholine (PC)-coated drug delivery (DD) BiodivYsio stents and assess their safety and efficacy at reducing neointima formation. I125-angiopeptin-loaded DD-PC-coated stents were implanted into human saphenous vein segments ex vivo, and I125 angiopeptin was detected in the medial layer at 1 hour. When implanted in pig coronary arteries, I125 angiopeptin was found adjacent to the stent at intervals up to 28 days. No significant amounts were found elsewhere. To assess efficacy, twelve angiopeptin-loaded DD-PC-coated stents, twelve non-loaded DD-PC stents, ten standard PC-coated stents and 8 uncoated stents were implanted into normal porcine coronary arteries. Stents were harvested at 28 days and neointima formation was assessed by computerized morphometry. No adverse tissue reactions were seen with any of the PC-coated stents. No significant differences were seen in neointimal or luminal cross-sectional areas between study groups. Local delivery of I125 angiopeptin into the vascular wall can be achieved using a PC-coated stent. Delivery of angiopeptin from drug delivery PC-coated stents is safe, but does not lead to a significant reduction in neointimal growth at 28 days within the parameters of this study.