Difluoroboron β–diketonate poly(lactic acid) materials exhibit both fluorescence (F) and oxygen sensitive room-temperature phosphorescence (RTP). Introduction of halide heavy atoms (Br and I) is an effective strategy to control the oxygen sensitivity in these materials. A series of naphthyl-phenyl (nbm) dye derivatives with hydrogen, bromide and iodide substituents were prepared for comparison. As nanoparticles, the hydrogen derivative was hypersensitive to oxygen (0–0.3%), while the bromide analogue was suited for hypoxia detection (0–3% O2). The iodo derivative, BF2nbm(I)PLA, showed excellent F to RTP peak separation and an 0–100% oxygen sensitivity range unprecedented for metal-free RTP emitting materials. Due to the dual emission and unconventionally long RTP lifetimes of these O2 sensing materials, a portable, cost-effective camera was used to quantify oxygen levels via lifetime and red/green/blue (RGB) ratiometry. The hypersensitive H dye was well matched to lifetime detection, simultaneous lifetime and ratiometric imaging was possible for the bromide analogue, whereas the iodide material, with intense RTP emission and a shorter lifetime, was suited for RGB ratiometry. To demonstrate the prospects of this camera/material design combination for bioimaging, iodide boron dye-PLA nanoparticles were applied to a murine wound model to detect oxygen levels. Surprisingly, wound oxygen imaging was achieved without covering (i.e. without isolating from ambient conditions, air). Additionally, would healing was monitored via wound size reduction and associated oxygen recovery, from hypoxic to normoxic. These single-component materials provide a simple tunable platform for biological oxygen sensing that can be deployed to spatially resolve oxygen in a variety of environments.

Mitochondria are fundamental for metabolic homeostasis in all multicellular eukaryotes. In the nervous system, mitochondria-generated adenosine triphosphate (ATP) is required to establish appropriate electrochemical gradients and reliable synaptic transmission. Notably, several mitochondrial defects have been identified in central nervous system disorders. Membrane leakage and electrolyte imbalances, pro-apoptotic pathway activation, and mitophagy are among the mechanisms implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s disease, as well as ischemic stroke. In this review, we summarize mitochondrial pathways that contribute to disease progression. Further, we discuss pathological states that damaged mitochondria impose on normal nervous system processes and explore new therapeutic approaches to mitochondrial diseases.

Treatment of many pathologies of the brain could be improved markedly by the development of noninvasive therapeutic approaches that elicit robust, endothelial cell-selective gene expression in specific brain regions that are targeted under MR image guidance. While focused ultrasound (FUS) in conjunction with gasfilled microbubbles (MBs) has emerged as a noninvasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce an MR image-guided FUS method that elicits endothelialselective transfection of the cerebral vasculature (i.e., "sonoselective" transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing MBs with pulsed low-pressure (0.1 MPa) 1.1-MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e., 0.3-MPa and 0.4-MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low-pressure regimen does not up-regulate inflammatory or immune responses. Single-cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cellsecreted factors to stimulate nerve regrowth, or recruit neural stem cells.focused ultrasound | microbubbles | endothelium | gene delivery

BackgroundArteriogenesis is initiated by increased shear stress and is thought to continue until shear stress is returned to its original “set point.” However, the molecular mechanism(s) through which shear stress set point is established by endothelial cells (ECs) are largely unstudied. Here, we tested the hypothesis that DNA methyltransferase 1 (DNMT1)–dependent EC DNA methylation affects arteriogenic capacity via adjustments to shear stress set point.Methods and ResultsIn femoral artery ligation–operated C57BL/6 mice, collateral artery segments exposed to increased shear stress without a change in flow direction (ie, nonreversed flow) exhibited global DNA hypermethylation (increased 5‐methylcytosine staining intensity) and constrained arteriogenesis (30% less diameter growth) when compared with segments exposed to both an increase in shear stress and reversed‐flow direction. In vitro, ECs exposed to a flow waveform biomimetic of nonreversed collateral segments in vivo exhibited a 40% increase in DNMT1 expression, genome‐wide hypermethylation of gene promoters, and a DNMT1‐dependent 60% reduction in proarteriogenic monocyte adhesion compared with ECs exposed to a biomimetic reversed‐flow waveform. These results led us to test whether DNMT1 regulates arteriogenic capacity in vivo. In femoral artery ligation–operated mice, DNMT1 inhibition rescued arteriogenic capacity and returned shear stress back to its original set point in nonreversed collateral segments.ConclusionsIncreased shear stress without a change in flow direction initiates arteriogenic growth; however, it also elicits DNMT1‐dependent EC DNA hypermethylation. In turn, this diminishes mechanosensing, augments shear stress set point, and constrains the ultimate arteriogenic capacity of the vessel. This epigenetic effect could impact both endogenous collateralization and treatment of arterial occlusive diseases.

Non-invasive drug and gene delivery to the brain to treat central nervous system pathologies has long been inhibited by the blood-brain barrier. The activation of microbubbles with focused ultrasound has emerged as a promising non-invasive approach to circumvent this obstacle, by transiently disrupting the blood-brain barrier and permitting passage of systemically administered therapeutics into the tissue. Clinical trials are underway to evaluate the safety of this technique; however, concerns remain regarding the potential for the treatment to induce sterile inflammation or petechiae. In this issue of Theranostics, Jones et al.[1] address these concerns through the development of an advanced three-dimensional imaging system for monitoring acoustic emissions from oscillating microbubbles. When subharmonic emissions are detected with this system, focused ultrasound pressure is reduced by 50% for the remainder of the treatment. This serves to transiently open the blood-brain barrier without generating adverse effects. While the ideal configuration of the transducer array for treatment and monitoring still presents an area for further optimization, the approach indicates that the acoustic signature of microbubble behavior within the skull can be used to ensure safe and effective blood-brain barrier opening using focused ultrasound.

Arteriogenesis, the growth of endogenous collateral arteries bypassing arterial occlusion(s), is a fundamental shear stress-induced adaptation with implications for treating peripheral arterial disease (PAD). Nonetheless, endothelial mechano-signaling during arteriogenesis is incompletely understood. Here we tested the hypothesis that a mechanosensitive microRNA, miR-199a-5p, regulates perfusion recovery and collateral arteriogenesis following femoral arterial ligation (FAL) via control of monocyte recruitment and pro-arteriogenic gene expression. We have previously shown that collateral artery segments exhibit distinctly amplified arteriogenesis if they are exposed to reversed flow following FAL in the mouse. We performed a genome-wide analysis of endothelial cells exposed to a biomimetic reversed flow waveform. From this analysis, we identified mechanosensitive miR-199a-5p as a novel candidate regulator of collateral arteriogenesis. In vitro, miR-199a-5p inhibited pro-arteriogenic gene expression (IKKβ, Cav1) and monocyte adhesion to endothelium. In vivo, following FAL in mice, miR-199a-5p overexpression impaired foot perfusion and arteriogenesis. In contrast, a single intramuscular anti-miR-199a-5p injection elicited a robust therapeutic response, including complete foot perfusion recovery, markedly augmented arteriogenesis (>3.4-fold increase in segment conductance), and improved gastrocnemius tissue composition. Finally, we found plasma miR-199a-5p to be elevated in human PAD patients with intermittent claudication compared to a risk factor control population. Through our transformative analysis of endothelial mechano-signaling in response to a biomimetic amplified arteriogenesis flow waveform, we have identified miR-199a-5p as both a potent regulator of arteriogenesis and a putative target for treating PAD.

Background: Focused ultrasound (FUS) blood brain barrier disruption (BBBD) permits the noninvasive, targeted, and repeatable delivery of drugs to the brain. FUS BBBD also elicits secondary responses capable of augmenting immunotherapies, clearing amyloid-β and hyperphosphorylated tau, and driving neurogenesis. Leveraging these secondary effects will benefit from an understanding of how they correlate to the magnitude of FUS BBBD and are differentially affected by the mechanical and biochemical stimuli imparted during FUS BBBD. Methods: We aggregated 75 murine transcriptomes in a multiple regression framework to identify genes expressed in proportion to biochemical (i.e. contrast MR image enhancement (CE)) or mechanical (i.e. harmonic acoustic emissions from MB-activation (MBA)) stimuli associated with FUS BBBD. Models were constructed to control for potential confounders, such as sex, anesthesia, and sequencing batch. Results: MBA and CE differentially predicted expression of 1,124 genes 6 h or 24 h later. While there existed overlap in the transcripts correlated with MBA vs CE, MBA was principally predictive of expression of genes associated with endothelial reactivity while CE chiefly predicted sterile inflammation gene sets. Over-representation analysis identified transcripts not previously linked to BBBD, including actin filament organization, which is likely important for BBB recovery. Transcripts and pathways associated with neurogenesis, microglial activation, and amyloid-β clearance were significantly correlated to BBBD metrics. Conclusions: The secondary effects of BBBD may have the potential to be tuned by modulating FUS parameters during BBBD, and MBA and CE may serve as independent predictors of transcriptional reactions in the brain.

Gene delivery via focused ultrasound (FUS) mediated blood-brain barrier (BBB) opening is a disruptive therapeutic modality. Unlocking its full potential will require an understanding of how FUS parameters (e.g., peak-negative pressure (PNP)) affect transfected cell populations. Following plasmid (mRuby) delivery across the BBB with 1 MHz FUS, we used single-cell RNA-sequencing to ascertain that distributions of transfected cell types were highly dependent on PNP. Cells of the BBB (i.e., endothelial cells, pericytes, and astrocytes) were enriched at 0.2 MPa PNP, while transfection of cells distal to the BBB (i.e., neurons, oligodendrocytes, and microglia) was augmented at 0.4 MPa PNP. PNP-dependent differential gene expression was observed for multiple cell types. Cell stress genes were upregulated proportional to PNP, independent of cell type. Our results underscore how FUS may be tuned to bias transfection toward specific brain cell types in vivo and predict how those cells will respond to transfection.