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.
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.
Chemically modified mRNA is a novel, highly efficient, biocompatible modality for therapeutic protein expression that may overcome the challenges and safety concerns with current gene therapy ...strategies. We explored the efficiency of intradermally injected modified VEGF-A165 mRNA (VEGF-A mRNA) formulated in a biocompatible citrate/saline buffer to locally produce human VEGF-A165 protein. Rabbits (n=4) and minipigs (n=3) were implanted with subcutaneous microdialysis probes close to the injection sites and interstitial-fluid samples and skin biopsies were analysed for production of VEGF-A protein over time for up to 8 hours. Three to 4 hours after the intradermal injection of VEGF-A mRNA, detectable levels of human VEGF-A protein were seen in the microdialysis eluates in both species. In the pig, the VEGF-A concentrations increased dose-dependently reaching a maximum 6 hours after dosing (62.7±28.4, 357.6±240.6, and 746.3±210.2 pg/mL following injection of 24, 120, and 600 μg VEGF-A mRNA, respectively). Likewise, in tissue biopsies harvested at study end (8 hours after VEGF-A mRNA injection), the content of VEGF-A protein increased dose-dependently. In contrast, VEGF-A protein was not detected in eluates originating from sites injected with citrate/saline vehicle. It is concluded that intradermal injection of VEGF-A mRNA is associated with a rapid and local production of VEGF-A protein. Considering the pro-angiogenic effect of VEGF-A, VEGF-A mRNA may hold promise for regenerative treatment of patients with diabetic wounds and ischemic cardiovascular disease.
Intradermal delivery of AZD8601, an mRNA designed to produce vascular endothelial growth factor A (VEGF‐A), has previously been shown to accelerate cutaneous wound healing in a murine diabetic model. ...Here, we develop population pharmacokinetic and pharmacodynamic models aiming to quantify the effect of AZD8601 injections on the dynamics of wound healing. A dataset of 584 open wound area measurements from 131 mice was integrated from 3 independent studies encompassing different doses, dosing timepoints, and number of doses. Evaluation of several candidate models showed that wound healing acceleration is not likely driven directly by time‐dependent VEGF‐A concentration. Instead, we found that administration of AZD8601 induced a sustained acceleration of wound healing depending on the accumulated dose, with a dose producing 50% of the maximal effect of 92 µg. Simulations with this model showed that a single dose of 200 µg AZD8601 can reduce the time to reach 50% wound healing by up to 5 days.
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca
ATPase and its reversible inhibitor phospholamban, induces heart failure by ...inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
Preferential utilization of fatty acids for ATP production represents an advanced metabolic phenotype in developing cardiomyocytes. We investigated whether this phenotype could be attained in human ...induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and assessed its influence on mitochondrial morphology, bioenergetics, respiratory capacity and ultra-structural architecture.
Whole-cell proteome analysis of day 14 and day 30-CMs maintained in glucose media revealed a positive influence of extended culture on mitochondria-related processes that primed the day 30-CMs for fatty acid metabolism. Supplementing the day 30-CMs with palmitate/oleate (fatty acids) significantly enhanced mitochondrial remodeling, oxygen consumption rates and ATP production. Metabolomic analysis upon fatty acid supplementation revealed a β-oxidation fueled ATP elevation that coincided with presence of junctional complexes, intercalated discs, t-tubule-like structures and adult isoform of cardiac troponin T. In contrast, glucose-maintained day 30-CMs continued to harbor underdeveloped ultra-structural architecture and more subdued bioenergetics, constrained by suboptimal mitochondria development.
The advanced metabolic phenotype of preferential fatty acid utilization was attained in hiPSC-CMs, whereby fatty acid driven β-oxidation sustained cardiac bioenergetics and respiratory capacity resulting in ultra-structural and functional characteristics similar to those of developmentally advanced cardiomyocytes. Better understanding of mitochondrial bioenergetics and ultra-structural adaptation associated with fatty acid metabolism has important implications in the study of cardiac physiology that are associated with late-onset mitochondrial and metabolic adaptations.
•metabolic shift to preferential fatty acid (FA) oxidation is developmentally attained in day 30 hiPSC-CMs•morphological remodeling of mitochondria facilitates competency of metabolic machinery to utilize FA in hiPSC-CMs•FA oxidation supports augmented mitochondrial bioenergetics in promoting developmentally advanced phenotypes in hiPSC-CMs
Bone has a remarkable potential for self-healing and repair, yet several injury types are non-healing even after surgical or non-surgical treatment. Regenerative therapies that induce bone repair or ...improve the rate of recovery are being intensely investigated. Here, we probed the potential of bone marrow stem cells (BMSCs) engineered with chemically modified mRNAs (modRNA) encoding the hBMP-2 and VEGF-A gene to therapeutically heal bone. Induction of osteogenesis from modRNA-treated BMSCs was confirmed by expression profiles of osteogenic related markers and the presence of mineralization deposits. To test for therapeutic efficacy, a collagen scaffold inoculated with modRNA-treated BMSCs was explored in an in vivo skull defect model. We show that hBMP-2 and VEGF-A modRNAs synergistically drive osteogenic and angiogenic programs resulting in superior healing properties. This study exploits chemically modified mRNAs, together with biomaterials, as a potential approach for the clinical treatment of bone injury and defects.
Myocardial ischemia is recognized as an important mechanism increasing the risk for cardiovascular events in both symptomatic and asymptomatic patients. In addition to obstructive coronary diseases, ...systemic inflammation, macro- and microvascular function are additional important mechanisms contributing to the ischemic myocardium. Accumulating evidence indicates that coronary flow reserve (CFR) is a quantitative measurement of ischemia including integrated information on structure and function of the coronary artery at all levels. Not surprisingly, CFR has been shown to confer strong prognostic value for hard cardiovascular (CV) events in a number of relevant patient cohorts. Using high-resolution imaging, it is now possible to study coronary arteries from mouse to man. Therefore, CFR may be an important translational tool to risk-stratify patients and to perform both preclinical and clinical proof-of-concept studies before investing in large-scale outcome trials, thus improving the translational value for novel CV targets.
Kidney disease is a complex disease with several different etiologies and underlying associated pathophysiology. This is reflected by the lack of effective treatment therapies in chronic kidney ...disease (CKD) that stop disease progression. However, novel strategies, recent scientific breakthroughs, and technological advances have revealed new possibilities for finding novel disease drivers in CKD. This review describes some of the latest advances in the field and brings them together in a more holistic framework as applied to identification and validation of disease drivers in CKD. It uses high-resolution ‘patient-centric’ omics data sets, advanced
in silico
tools (systems biology, connectivity mapping, and machine learning) and ‘state-of-the-art‘ experimental systems (complex 3D systems
in vitro
, CRISPR gene editing, and various model biological systems
in vivo
). Application of such a framework is expected to increase the likelihood of successful identification of novel drug candidates based on strong human target validation and a better scientific understanding of underlying mechanisms.
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.
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