Unraveling the Metabolic Derangements Occurring in Non-infarcted Areas of Pig Hearts With Chronic Heart Failure.

Objective: After myocardial infarction (MI), the non-infarcted left ventricle (LV) ensures appropriate contractile function of the heart. Metabolic disturbance in this region greatly exacerbates post-MI heart failure (HF) pathology. This study aimed to provide a comprehensive understanding of the metabolic derangements occurring in the non-infarcted LV that could trigger cardiovascular deterioration. Methods and Results: We used a pig model that progressed into chronic HF over 3 months following MI induction. Integrated gene and metabolite signatures revealed region-specific perturbations in amino acid- and lipid metabolism, insulin signaling and, oxidative stress response. Remote LV, in particular, showed impaired glutamine and arginine metabolism, altered synthesis of lipids, glucose metabolism disorder, and increased insulin resistance. LPIN1, PPP1R3C, PTPN1, CREM, and NR0B2 were identified as the main effectors in metabolism dysregulation in the remote zone and were found differentially expressed also in the myocardium of patients with ischemic and/or dilated cardiomyopathy. In addition, a simultaneous significant decrease in arginine levels and altered PRCP, PTPN1, and ARF6 expression suggest alterations in vascular function in remote area. Conclusions: This study unravels an array of dysregulated genes and metabolites putatively involved in maladaptive metabolic and vascular remodeling in the non-infarcted myocardium and may contribute to the development of more precise therapies to mitigate progression of chronic HF post-MI.

In vivo imaging of hemodynamic redistribution and arteriogenesis across microvascular network.

OBJECTIVE: Arteriogenesis is an important mechanism that contributes to restoration of oxygen supply in chronically ischemic tissues, but remains incompletely understood due to technical limitations. This study presents a novel approach for comprehensive assessment of the remodeling pattern in a complex microvascular network containing multiple collateral microvessels. METHODS: We have developed a hardware-software integrated platform for quantitative, longitudinal, and label-free imaging of network-wide hemodynamic changes and arteriogenesis at the single-vessel level. By ligating feeding arteries in the mouse ear, we induced network-wide hemodynamic redistribution and localized arteriogenesis. The utility of this technology was demonstrated by studying the influence of obesity on microvascular arteriogenesis. RESULTS: Simultaneously monitoring the remodeling of competing collateral arterioles revealed a new, inverse relationship between initial vascular resistance and extent of arteriogenesis. Obese mice exhibited similar remodeling responses to lean mice through the first week, including diameter increase and flow upregulation in collateral arterioles. However, these gains were subsequently lost in obese mice. CONCLUSIONS: Capable of label-free, comprehensive, and dynamic quantification of structural and functional changes in the microvascular network in vivo, this platform opens up new opportunities to study the mechanisms of microvascular arteriogenesis, its implications in diseases, and approaches to pharmacologically rectify microvascular dysfunction.

Oral Delivery of the P2Y12 Receptor Antagonist Ticagrelor Prevents Loss of Photoreceptors in an ABCA4-/- Mouse Model of Retinal Degeneration.

Purpose: Accumulation of lysosomal waste is linked to neurodegeneration in multiple diseases, and pharmacologic enhancement of lysosomal activity is hypothesized to reduce pathology. An excessive accumulation of lysosomal-associated lipofuscin waste and an elevated lysosomal pH occur in retinal pigment epithelial cells of the ABCA4-/- mouse model of Stargardt's retinal degeneration. As treatment with the P2Y12 receptor antagonist ticagrelor was previously shown to lower lysosomal pH and lipofuscin-like autofluorescence in these cells, we asked whether oral delivery of ticagrelor also prevented photoreceptor loss. Methods: Moderate light exposure was used to accelerate photoreceptor loss in albino ABCA4-/- mice as compared to BALB/c controls. Ticagrelor (0.1%-0.15%) was added to mouse chow for between 1 and 10 months. Photoreceptor function was determined with electroretinograms, while cell survival was determined using optical coherence tomography and histology. Results: Protection by ticagrelor was demonstrated functionally by using the electroretinogram, as ticagrelor-treated ABCA4-/- mice had increased a- and b-waves compared to untreated mice. Mice receiving ticagrelor treatment had a thicker outer nuclear layer, as measured with both optical coherence tomography and histologic sections. Ticagrelor decreased expression of LAMP1, implicating enhanced lysosomal function. No signs of retinal bleeding were observed after prolonged treatment with ticagrelor. Conclusions: Oral treatment with ticagrelor protected photoreceptors in the ABCA4-/- mouse, which is consistent with enhanced lysosomal function. As mouse ticagrelor exposure levels were clinically relevant, the drug may be of benefit in preventing the loss of photoreceptors in Stargardt's disease and other neurodegenerations associated with lysosomal dysfunction.

Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes.

Chemically modified mRNA is an efficient, biocompatible modality for therapeutic protein expression. We report a first-time-in-human study of this modality, aiming to evaluate safety and potential therapeutic effects. Men with type 2 diabetes mellitus (T2DM) received intradermal injections of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) or buffered saline placebo (ethical obligations precluded use of a non-translatable mRNA control) at randomized sites on the forearm. The only causally treatment-related adverse events were mild injection-site reactions. Skin microdialysis revealed elevated VEGF-A protein levels at mRNA-treated sites versus placebo-treated sites from about 4-24 hours post-administration. Enhancements in basal skin blood flow at 4 hours and 7 days post-administration were detected using laser Doppler fluximetry and imaging. Intradermal VEGF-A mRNA was well tolerated and led to local functional VEGF-A protein expression and transient skin blood flow enhancement in men with T2DM. VEGF-A mRNA may have therapeutic potential for regenerative angiogenesis.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration.

The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt's disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

Cardiac safety assays.

Cardiac safety, including the risk of drug-induced 'torsades de pointes' (TdP) arrhythmia, is a major concern in the development, approval and prescription of new drugs. Assessment of surrogate markers of TdP-risk, such as QT-interval prolongation or inhibition of the rapid delayed-rectifier K(+)-current (IKr) encoded by the human ether-a-go-go-related gene (hERG), is therefore required before drug approval. Here, we review some methodologies employed to assess proarrhythmia liability of drugs, discuss the challenges involved in this process, and highlight promising novel cardiac-safety assays.

Reduced ventricular proarrhythmic potential of the novel combined ion-channel blocker AZD1305 versus dofetilide in dogs with remodeled hearts.

BACKGROUND: AZD1305 is an investigational antiarrhythmic agent for management of atrial fibrillation. It blocks various cardiac ion currents at different potencies and has atrial-predominant electrophysiological effects. We investigated the electrophysiological and proarrhythmic effects of AZD1305 versus dofetilide in dogs with chronic complete atrioventricular block and myocardial hypertrophic remodeling. METHODS AND RESULTS: AZD1305 was administered to anesthetized mongrel dogs before and >2 weeks after the induction of atrioventricular block and ventricular and atrial electrophysiological parameters were assessed. In all dogs, the selective I(Kr) blocker dofetilide was used to examine susceptibility to acquired torsades de pointes in chronic atrioventricular block and for comparison. At normal sinus rhythm, AZD1305 increased QT and RR intervals from 290±7 to 397±15 ms (+37%, P<0.0001) and from 603±22 to 778±32 ms (+29%, P=0.002), respectively. In the same animals at chronic atrioventricular block, AZD1305 increased the QT interval from 535±28 to 747±36 ms (+40%, P<0.0001), similar to the QT prolongation by dofetilide (511±22 to 703±45 ms [+38%, P<0.0001]). AZD1305 slightly slowed the idioventricular rhythm. Whereas all (n=14) chronic atrioventricular block animals exhibited torsades de pointes on dofetilide, the arrhythmia was induced in only 4 of 11 dogs after AZD1305. Beat-to-beat variability of left-ventricular monophasic-action-potential duration increased after dofetilide (2.3±0.2 to 6.3±0.7 ms; P<0.0001) but not after AZD1305 (2.8±0.3 to 3.7±0.3 ms; P=0.20) despite similar left-ventricular monophasic-action-potential duration prolongations. CONCLUSIONS: Despite causing similar degrees of repolarization delay as the selective I(Kr) blocker dofetilide, the combined ion-channel blocker AZD1305 induces less repolarization instability and has a lower ventricular proarrhythmic potential in the remodeled dog heart.