Theranostics

In the cerebral cortex, the vasculature plays important homeostatic functions, especially at the highly connected complex capillary networks. The association of focal capillary ischemia with the neurodegenerative disease as well as the laminar vascular dynamics have prompted studies of vascular micro-occlusion via photothrombosis. However, technical challenges of this approach remain, including increased temporal precision of occlusion, increasing the depth of vascular occlusion, understanding how such micro-occlusion impacts local blood flow, and ultimately the neuronal effects of such changes. Here, we have developed a novel approach that employs ultra-fast multiphoton light to induce focal Rose Bengal-induced photothrombosis. We demonstrated induction of highly precise and fast occlusion of microvessels at various types and depths. The change of the microvascular architecture and hemodynamics after occlusion revealed the autoregulation and significant difference between upstream vs downstream in layer 2/3. Further, we found that micro-occlusion at two different layers within the same vascular arbor results in distinct effects on the acute flow redistribution mechanism. To examine neuronal effects of such micro-occlusion, we produced infarct of capillaries surrounding a labeled target neuron and found this induces dramatic and rapid lamina-specific degeneration in neuronal dendritic architecture. In sum, our technique enhanced the precision and power of the photothrombotic study of microvascular function. The current results pointed to the importance of laminar scale regulation within the microvascular network, a finding which may be relevant for models of neurovascular disease.

Glycoproteins lining the luminal endothelial surface form the glycocalyx, composing the tripartite blood brain barrier. We explored the glycocalyx as a source of danger signals for complement lectin pathway after ischemic stroke. Our data indicate that hypoxic microvascular cells increased -D-mannosyl and N-acetylglucosaminyl exposure after re-oxygenation, favoring mannose binding lectin (MBL) pathogenic deposition, and overexpression of inflammatory genes (ICAM-1 and MMP-2). The hypoxia-conditioned medium induced neuronal damage (reduced MAP-2), microglia and astrocytic reactivity (increased/thickened ramifications) when applied to induced pluripotent stem cell-derived neurons, astrocytes and microglia co-cultures. All these effects were counteracted by mannose-capped gold nanoparticles (Man-GNPs), shown to bind and sequester MBL from the medium. We then tested the Man-GNPs in vivo, in an ischemic stroke model using humanized mice, knocked-in for human MBL. The ischemic mice (males:females 1:1) treated with Man-GNPs (3h after the ischemic onset) exhibited less anxiety at the elevated plus maze and reduced neuronal loss at 8d after ischemia compared to vehicle-treated. Thus, multivalent Man-GNPs represent a promising approach to take MBL away from its glycoproteic targets on the ischemic endothelium, hence preventing downstream pathogenesis. Moreover, these data support circulating MBL as a druggable pharmacological target to prevent the thrombo-inflammatory events following acute brain injury.

BACKGROUNDCurcumin has emerged as a promising candidate capable of polarization of macrophages, which promote the stability of atherosclerotic plaque. Nevertheless, a notable limitation lies in the non-specific nature of curcumins targeting. The present study endeavors to harness multifunctional lipid nanoparticles (MLNPs), which could both facilitate imaging and achieve targeted delivery of curcumin specifically to inflammatory macrophages, to effectively counteract vulnerable plaque and mitigate the risk of ischemic events. METHODSThe term "MLNPs", for targeted delivery of curcumin using multimodal imaging techniques including single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI), refers to a new type of nanoparticle designed to specifically target and modulate macrophages with phagocytic function. These nanoparticles are cholesteryl-9-carboxynonanoate-(125I-iron oxide nanoparticle/Cur)-lipid-coated nanoparticles [9-CCN-(125I-ION/Cur)-LNPs], which carry hybrid imaging agents. These agents are combinations of 125I-ION and lipids that contain phagocytic "eat-me" signals, which induces macrophages to swallow MLNPs. RESULTSThe accumulation of the devised 9-CCN-(125I-ION/Cur)-LNPs on the unstable plaque of animal models in vivo was accurately reflected and lesions were highlighted by both SPECT and MRI. The intense radioactive signals on SPECT images facilitated the identification and quantification of the target lesion, while MRI based on ION particles facilitated the visualization of the focal localization and volumetry of atherosclerotic plaque. The targeted distribution of the unstable plaque in the rabbit aorta was further confirmed by ex vivo planar images of nuclide and Prussian blue staining for ION. Additionally, 9-CCN-(125I-ION/Cur)-LNPs were found to specifically and effectively bind to the pro-inflammatory M1 macrophages present in the unstable plaque, resulting in the accumulation of radionuclide and hypointensity on T2W images. CONCLUSIONSThe 9-CCN-(125I-ION/Cur)-LNPs demonstrated remarkable capability in the delivery of both 125I-ION and curcumin to macrophages, ultimately resulting in M1-M2 macrophage polarization, which may offer valuable insights into addressing unstable atherosclerotic plaque.

Intraventricular hemorrhage (IVH) is the most fatal form of brain injury, yet a therapy directed at ameliorating intraventricular clot is very limited. There is accumulating evidence that an augmentation of the meningeal lymphatic (MLVs) functions might be a promising therapeutic target for IVH. In particular, the photostimulation (PS) of MLVs could be promising for non-invasive therapy of IVH via PS of clearance of red blood cells (RBCs) from the brain via MLVs. Indeed, we uncover that PS has therapeutic effects on IVH in mice reducing the mortality, improving the emotional status, accelerating the RBCs evacuation from the ventricles and increasing the ICP recovery. Our findings strongly suggest that the PS-mediated stimulation of drainage and clearing functions of MLVs can be a novel bedside, readily applicable and commercially viable technologies for treatment of IVH. These pilot results open new horizons in a non-invasive therapy of IVH via PS stimulation of regenerative lymphatic mechanisms.

RationaleNatural killer (NK) cells provide protective anti-cancer immunity. However, the cancer therapy induced activation gene signatures and pathways in NK cells remain unclear. MethodsWe applied a novel localized ablative immunotherapy (LAIT) by synergizing photothermal therapy (PTT) with intra-tumor delivering of the immunostimulant N-dihydrogalactochitosan (GC), to treat breast cancer using a mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) mouse model. We performed single-cell RNA sequencing (scRNAseq) analysis to unveil the cellular heterogeneity and compare the transcriptional alterations induced by PTT, GC, and LAIT in NK cells within the tumor microenvironment (TME). ResultsScRNAseq showed that NK subtypes, including cycling, activated, interferon-stimulated, and cytotoxic NK cells. Trajectory analysis revealed a route toward activation and cytotoxicity following pseudotime progression. Both GC and LAIT elevated gene expression associated with NK cell activation, cytolytic effectors, activating receptors, IFN pathway components, and cytokines/chemokines in NK subtypes. Single-cell transcriptomics analysis using immune checkpoint inhibitor (ICI)-treated animal and human samples revealed that ICI-induced NK activation and cytotoxicity across several cancer types. Furthermore, ICI-induced NK gene signatures were also induced by LAIT treatment. We also discovered that several types of cancer patients had significantly longer overall survival when they had higher expression of genes in NK cells that were also specifically upregulated by LAIT. ConclusionOur findings show for the first time that LAIT activates cytotoxicity in NK cells and the upregulated genes positively correlate with beneficial clinical outcomes for cancer patients. More importantly, our results further establish the correlation between the effects of LAIT and ICI on NK cells, hence expanding our understanding of mechanism of LAIT in remodeling TME and shedding light on the potentials of NK cell activation and anti-tumor cytotoxic functions in clinical applications.

Here, positronium imaging is presented to determine cardiac myxoma (CM) extracted from patients undergoing urgent cardiac surgery due to unexpected atrial masses. Positronium is an atom build from an electron and a positron, produced copiously in intra-molecular voids during the PET imaging. CM, the most common cardiac tumor in adults, accounts for 50-75% of benign cardiac tumors. We aimed to assess if positronium serves as a biomarker for diagnosing CM. Perioperative examinations and histopathology staining in six patients confirmed the primary diagnosis of CM. We observed significant differences in the mean positronium lifetime between tumor and normal tissues, with an average value of 1.92(02) ns and 2.72(05) ns for CM and the adipose tissue, respectively. Our findings, combined with positronium lifetime imaging, reveals the novel emerging positronium biomarker for cardiovascular imaging. One-Sentence SummaryPositronium may serve as an imaging biomarker for cancer diagnostics.

While a subset of patients with metastatic melanoma achieves durable responses to immune checkpoint blockade (ICB) therapies, the majority ultimately exhibit either innate or acquired resistance to these treatments. However, the molecular mechanisms underlying resistance to ICB therapies remain elusive and are warranted to elucidate. Here, we comprehensively investigated the tumor and tumor immune microenvironment (TIME) of paired pre- and post-treatment tumor specimens from metastatic melanoma patients who were primary or secondary resistance to anti-CTLA-4 and/or anti-PD-1/PD-L1 therapies. Differentially expressed gene (DEG) analysis and single-sample gene set enrichment analysis (ssGSEA) with transcriptomic data identified cell cycle and c-MYC signaling as pathway-based resistance signatures. And weighted gene co-expression network analysis (WGCNA) revealed the activation of a cross-resistance meta-program involving key signaling pathways related to tumor progression in ICB resistant melanoma. Moreover, spatially-resolved, image-based immune monitoring analysis by using NanoStrings digital spatial profiling (DSP) and Cyclic Immunofluorescence (CyCIF) showed infiltration of suppressive immune cells in the tumor microenvironment of melanoma with resistance to ICB therapies. Our study reveals the molecular mechanisms underlying resistance to ICB therapies in patients with metastatic melanoma by conducting such integrated analyses of multi-dimensional data, and provides rationale for salvage therapies that will potentially overcome resistance to ICB therapies. Statement of translational relevanceThis study paves the way for the creation of innovative therapeutic strategies, aimed at subverting resistance to immune checkpoint blockade (ICB) therapies in metastatic melanoma patients. By unraveling the specific molecular mechanisms underlying resistance, scientists can design effective alternative treatments that target pathways such as pathways associated with cell cycle dysregulation and c-MYC signaling. Furthermore, through the application of advanced immune monitoring techniques such as NanoString Digital Spatial Profiling (DSP) and Cyclic Immunofluorescence (CyCIF), this study has significantly enriched our understanding of the tumor microenvironment. This enhanced characterization facilitates the discovery of potential biomarkers that may forecast a patients response to ICB treatment. Ultimately, these advancements could potentially refine patient outcomes and foster the development of more personalized cancer treatments in the future.

Cranial irradiation is used for prophylactic brain radiotherapy as well as treatment of primary brain tumors. Despite its high efficiency, it often induces unexpected side effects, including cognitive dysfunction. Herein, we observed that mice exposed to cranial irradiation exhibited cognitive dysfunction, including altered spontaneous behavior, decreased spatial memory, and reduced novel object recognition. Analysis of actin cytoskeleton revealed that ionizing radiation (IR) disrupted the filamentous/globular actin (F/G-actin) ratio and downregulated the actin turnover signaling pathway p21-activated kinase 3 (PAK3)-LIM kinase 1 (LIMK1)-cofilin. Furthermore, we found that IR could upregulate microRNA-206-3p (miR-206-3p) targeting PAK3. As the inhibition of miR-206-3p through antagonist (antagomiR), IR-induced disruption of PAK3 signaling is restored. In addition, intranasal administration of antagomiR-206-3p recovered IR-induced cognitive impairment in mice. Our results suggest that cranial irradiation-induced cognitive impairment could be ameliorated by regulating PAK3 through antagomiR-206-3p, thereby affording a promising strategy for protecting cognitive function during cranial irradiation, and promoting quality of life in patients with radiation therapy.

Neurovasculoglial crosstalk is critical in establishing and maintaining a functional neurovascular unit. Breakdown in the unit is central to many neurodegenerative disorders of the CNS of which the retina is a component. A growing literature indicated that primary fatty acid amides (PFAMs) can regulate this crosstalk between vasculature and neuronal tissues. In this study we describe a central role for erucamide, a 22:1 mono-unsaturated omega-9 fatty acid amide, in degenerating retinal tissues. Using high-resolution global mass spectrometry-based metabolomics, we cataloged metabolites in murine models of retinal degeneration and show that while PFAMs, in general, are highly dysregulated, erucamide is the one most significantly diminished during photoreceptor atrophy. Using rodent models of retinal degeneration and novel organosilane-modified porous silicon nanoparticles (pSiNPs) for the in vivo delivery of erucamide, we demonstrate that erucamide activates CD11b+ myeloid cells, leading to the upregulation of angiogenic and neurotrophic cytokines that stabilize retinal degeneration. We identified TMEM19 as a novel binding protein for erucamide that is crucial for human iPSC-derived macrophage precursor cells activation and subsequent neurotrophic and angiogenic factor production. These findings reveal a previously unknown PFAM pathway that is modulated during retinal degenerative diseases, demonstrating that erucamide or functional analogues and their action through TMEM19 may be useful as a therapeutic alternative to neuroprotective and stem cell-based approaches for the treatment of retinal degenerative diseases.

Patients receiving cranial radiotherapy for primary and metastatic brain tumors may experience radiation-induced brain injury (RIBI). So far there is a lack of effective preventive and therapeutic strategies for RIBI. Due to its complicated underlying pathogenic mechanisms, it is rather difficult to develop a single approach to target them simultaneously. We have recently reported that Reprimo (RPRM), a tumor suppressor gene, is a critical player in DNA damage repair, and RPRM deletion significantly confers radioresistance to mice. Here in this study, by using RPRM knockout (KO) mouse model established in our laboratory, we found that RPRM deletion alleviated RIBI in mice via targeting its multiple underlying mechanisms. Specifically, RPRM knockout significantly reduced hippocampal DNA damage and apoptosis shortly after mice were exposed to whole brain irradiation (WBI). For the late-delayed effect of WBI, RPRM knockout obviously ameliorated radiation-induced decline in neurocognitive function and dramatically diminished WBI-induced neurogenesis inhibition. Moreover, RPRM KO mice exhibited a significantly lower level of acute and chronic inflammation response and microglial activation than wild type (WT) mice did post WBI. Finally, we uncovered that RPRM knockout not only protected microglia against radiation-induced damage, thus prevented microglial activation, but also protected neurons and decreased the induction of CCL2 in neurons after irradiation, in turn attenuating the activation of microglial cells nearby through paracrine CCL2. Taken together, Our results indicate that RPRM plays a crucial role in the occurrence of RIBI, suggesting that RPRM may serve as a novel potential target for the prevention and treatment of RIBI.

We utilized positron emission tomography (PET) imaging in vivo to map the spatiotemporal biodistribution/expression (protein density) of class-IIa histone deacetylases (class-IIa HDACs) in the brain. Herein, we report an improved radiosynthesis of [18F]-NT160 using 4-hydroxy-TEMPO which led to a significant improvement in radiochemical yield and molar activity. PET imaging with [18F]-NT160, a highly potent class-IIa HDAC inhibitor with sub-nM affinity for HDAC4 and 5 isoforms, led to high-quality and high-contrast images among various brain regions. [18F]-NT160 displayed excellent pharmacokinetic and imaging characteristics: brain uptake is high in gray matter regions, leading to high-quality PET images; tissue kinetics are appropriate for an 18F tracer and specific binding for class-IIa HDACs is demonstrated by self-blockade. Higher uptake with [18F]-NT160 was observed in the hippocampus, thalamus, and cortex while there was relatively lower uptake in the cerebellum and striatum. Overall, our current studies with [18F]-NT160 will likely facilitate the development and clinical translation of class-IIa HDACs of the next generation of PET tracers for imaging and targeted therapy of cancer and the diseases of the central nervous system (CNS).

ObjectivesFutile recanalization (FR) after endovascular thrombectomy (EVT) is driven by neutrophil-mediated thromboinflammation, yet conventional neuroprotectants fail to address the spatiotemporal complexity of stroke injury. This study aimed to identify the clinical link between circSCMH1, traditionally viewed as a late-stage repair molecule, and FR, while evaluating its hyperacute anti-inflammatory potential via a neutrophil-targeted delivery system. MethodsWe analyzed thrombi and plasma from LVO-AIS patients stratified by functional outcomes. We engineered neutrophil-targeted lipid nanoparticles (circSCMH1@pepLNP) for delivery in a mouse transient middle cerebral artery occlusion model. Therapeutic outcomes were evaluated through infarct volume measurement, histological assessment of NETosis, and intravital two-photon microscopy to monitor real-time neutrophil dynamics and capillary stalling. ResultsClinical analyses showed circSCMH1 was significantly downregulated in FR patients, with its levels negatively correlated with NET burden. Furthermore, circSCMH1-negative neutrophils exhibited a higher propensity for NETosis within the thrombus microenvironment. In mice, circSCMH1@pepLNP achieved specific neutrophil delivery, significantly reduced infarct volume, and suppressed the expression of citrullinated histone H3 and neutrophil elastase. Intravital two-photon and laser speckle imaging further confirmed that this intervention attenuated neutrophil adhesion, increased migratory velocity, and alleviated capillary stalling, thereby restoring cortical microvascular perfusion. ConclusionsOur findings associate neutrophil-specific circSCMH1 downregulation with clinical FR in LVO-AIS. Targeted intracellular delivery reveals an acute anti-thromboinflammatory function that complements established reparative properties, proposing a novel adjunctive approach for EVT to address the spatiotemporal complexity of stroke and improve clinical outcomes.

Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand--N-acetylgalactosamine (GalNAc)--which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.

Therapeutic angiogenesis has been the focus of hundreds of clinical trials but approval for human treatment remains elusive. Current strategies often rely on the upregulation of a single proangiogenic factor, which fails to recapitulate the complex response needed in hypoxic tissues. Hypoxic oxygen tensions dramatically decrease the activity of hypoxia inducible factor prolyl hydroxylase 2 (PHD2), the primary oxygen sensing portion of the hypoxia inducible factor 1 alpha (HIF-1) proangiogenic master regulatory pathway. Repressing PHD2 activity increases intracellular levels of HIF-1 and impacts the expression of hundreds of downstream genes directly associated with angiogenesis, cell survival, and tissue homeostasis. This study explores activating the HIF-1 pathway through SpCas9 knockout of the PHD2 encoding gene EGLN1 as an innovative in situ therapeutic angiogenesis strategy for chronic vascular diseases. Our findings demonstrate that even low editing rates of EGLN1 lead to a strong proangiogenic response regarding proangiogenic gene transcription, protein production, and protein secretion. In addition, we show that secreted factors of EGLN1 edited cell cultures may enhance human endothelial cell neovascularization activity in the context of proliferation and motility. Altogether, this study reveals that EGLN1 gene editing shows promise as a potential therapeutic angiogenesis strategy.

Glioblastoma (GBM) is an aggressive tumor with very bad prognosis. The urgent need to find new effective therapies is challenged by the unique characteristics of GBM, including high intra and intertumoral heterogeneity. Using single-nucleus transcriptomics (snRNA-seq), we characterized the panorama of a preclinical immunocompetent murine model based in the implantation of mouse glioblastoma stem cells (GL261-GSCs) into the brain parenchyma. Additionally, we performed Visium spatial transcriptomics in the in vivo model to confirm the location of annotated cells. To understand the technical bias of this approach, we performed two scRNA-seq methods in GBM cells. We thoroughly characterized the tumor microenvironment (TME) at early and late stages of tumor development and upon treatment with temozolomide (TMZ), the standard of care for patients with GBM, and with Tat-Cx43266-283, a promising experimental treatment. We identified prominent GBM targets that can be addressed using this preclinical model, such as Grik2, Nlgn3, Gap43 or Kcnn4, which are involved in electrical and synaptic integration of GBM cells into neural circuits, as well as the expression of Nt5e, Cd274 or Irf8, which indicates the development of immune evasive properties in these GBM cells. In agreement, snRNA-seq unveiled high expression of several immunosuppressive-associated molecules in immune cells, such as Csf1r, Arg1, Mrc1 and Tgfb1, suggesting the development of an immunosuppressive microenvironment. We also show the landscape of cytokines, cytokine receptors, checkpoint ligands and receptors in tumor and TME cells, which are crucial data for a rational design of immunotherapy studies. Thus, Mrc1, PD-L1, TIM-3 or B7-H3 are among the immunotherapy targets that can be addressed in this model. Finally, the comparison of the preclinical GL261-GSC GBM model with human GBM subtypes unveiled important similarities with the recently identified TMEmed human GBM, indicating that preclinical data obtained in GL261-GSC GBM model might be applied to TMEmed human GBM, improving patient stratification in clinical trials. In conclusion, this work provides crucial information for future preclinical studies in GBM improving their clinical application.

Immigration and activation of immune cells play a significant role in damage progression after ischemic stroke. It has been shown that the nuclear receptor NR4A1 exerts a crucial role within the inflammatory response of various immune diseases via regulating immune cell activation. In this study, we investigated the role of NR4A1 on the activation and recruitment of brain resident and peripheral immune cells after cerebral ischemia. Here, we show that NR4A1 mediates an anti-inflammatory and damage limiting effect after ischemic stroke through immigrating neutrophil granulocytes. Importantly, NR4A1-activation with its ligand Cytosporone-B improves functional outcome and diminishes brain damage. Therefore, modulation of NR4A1 is a promising therapeutic target in the treatment of stroke.

COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via still elusive mechanisms. SARS-CoV-2 infects host cells by binding to angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor that recognizes the viral spike (S) protein. Brain pericytes were recently shown to express ACE2 at the neurovascular interface, outlining their possible implication in microvasculature injury in COVID-19. Yet, pericyte responses to SARS-CoV-2 is still to be fully elucidated. Using cell-based assays, we report that ACE2 expression in human brain vascular pericytes is highly dynamic and is increased upon S protein stimulation. Pericytes exposed to S protein underwent profound phenotypic changes translated by increased expression of contractile and myofibrogenic proteins, namely -smooth muscle actin (-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). These changes were associated to an altered intracellular calcium (Ca2+) dynamic. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-{kappa}B) signalling pathway, which was potentiated by hypoxia, a condition associated to vascular comorbidities, which exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely interleukin-8 (IL-8), IL-18, macrophage migration inhibitory factor (MIF), and stromal cell-derived factor-1 (SDF-1). Finally, we found that S protein could reach the mouse brain via the intranasal route and that reactive ACE2-expressing pericytes are recruited to the damaged tissue undergoing fibrotic scarring in a mouse model of cerebral multifocal micro-occlusions, a main reported vascular-mediated neurological condition associated to COVID-19. Our data demonstrate that the released S protein is sufficient to mediate pericyte immunoreactivity, which may contribute to microvasculature injury in absence of a productive viral infection. Our study provides a better understanding for the possible mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in lymphedematous arm compared to normal arm in patients. The role of APLN in LD was confirmed in APLN-knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of safe and non-integrative RNA-delivery LentiFlash(R) vector that will be evaluated for phase I/IIa clinical trial.

The development of advanced therapies for stroke, spinal cord injury or neurodegenerative diseases -main causes of death, disability and dementia- requires a profound understanding of the complex interactions established among excitotoxic neuronal death, aberrant neurotrophic-signaling, glial reactivity, and neuroinflammation. However, the master proteins coordinating these mechanisms have not been yet defined. Different evidence suggests that the truncated form of the neurotrophin tyrosine kinase receptor, TrkB-T1, might play such a key role. The levels of this TrkB isoform increase in stroke while those of the full-length pro-survival isoform (TrkB-FL) are reduced. Additionally, ischemic stroke and, specifically, excitotoxicity induce TrkB-T1 regulated intramembrane proteolysis (RIP), a process releasing a receptor ectodomain able to bind the brain-derived neurotrophic factor (BDNF) and leading to decreased BDNF-signaling. We hypothesize that the second RIP product, TrkB-T1 intracellular domain (TrkB-T1-ICD), might similarly contribute to neurotoxicity but also reactive gliosis and neuroinflammation. Herein, we first demonstrate migration of the cytoplasmic TrkB-T1-ICD to the nuclei of neurons undergoing excitotoxicity, suggesting a possible role in the transcriptional control induced by injury. Then, taking advantage of cell-penetrating peptides (CPPs), we produce a TrkB-T1-ICD mock peptide (Bio-LTT1Ct) containing the short TrkB-T1 intracellular region (23 amino acids) and test it in vitro and in vivo. Notably, this peptide migrates to the nucleus of both neurons and astrocytes cultured in vitro and provokes cell death. Additionally, Bio-LTT1Ct induces early transcriptional changes in neurons resembling those triggered by excitotoxicity such as the inhibition of the promoter activity of pro-survival transcription factors CREB and MEF2, and altered mRNA levels of their regulated genes. In vivo, Bio-LTT1Ct is accessible to the brain cortex after intranasal delivery, being efficiently distributed into cortical neurons and astrocytes of both hemispheres. Moreover, peptide administration is sufficient to promote important pathological hallmarks of stroke such as the imbalance of the TrkB isoforms, and the reactivity of astrocyte and microglia, cells that acquire proinflammatory profiles. Altogether, these results establish TrkB-T1 RIP as a central mechanism of ischemic damage and demonstrate that the receptor intracellular region is sufficient to recapitulate stroke-like effects on neurotoxicity, glial reactivity and neuroinflammation.

PurposeHuman OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. MethodsNbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. ResultsOur selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. ConclusionConsidering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.