Biology subjects

Madaro, L.

Publications and source records attributed to Madaro, L..

6 recordsLinked to original sources

bioRxiv2026

Reelin engages non-canonical signaling pathways to drive endothelial remodeling and plasticity

BACKGROUNDThe vascular endothelium is a dynamic tissue central to vascular homeostasis and disease, with endothelial cells (ECs) exhibiting plasticity that drives adaptive remodeling. Reelin, a secreted extracellular matrix glycoprotein critical for neuronal migration via ApoER2/VLDLR-DAB1 signaling, may also modulate vascular function and inflammation. However, its direct role in EC biology remains unclear. We investigated Reelin as a context-dependent signaling modulator in ECs, assessing its engagement of non-canonical pathways and regulation of endothelial plasticity relevant to cardiovascular pathology. METHODSHuman endothelial cells were stimulated with recombinant Reelin and analyzed by immunoblotting, immunofluorescence, and functional assays. Time-course studies assessed signaling, including phosphorylation of FAK, AKT, and DAB1 by Western blotting, while wound-healing assays quantified endothelial migratory capacity in vitro systems. RESULTSReelin rapidly robustly activated noncanonical signaling in endothelial cells, increasing FAK and AKT phosphorylation in a time-dependent manner consistent with cytoskeletal remodeling. Canonical DAB1 activation was limited. Functionally, Reelin enhanced migration, upregulated Endoglin/CD105, and induced a remodeling-associated phenotype. Reelin silencing altered endothelial phenotype, clearly indicating a role in homeostasis. Signaling was independent of VEGFR2 interaction. Overall, Reelin preferentially engages FAK/AKT pathways to drive partial phenotypic modulation without full endothelial-to-mesenchymal transition. CONCLUSIONWe show that Reelin is a previously unrecognized regulator of endothelial signaling and plasticity, acting via non-canonical FAK- and AKT-dependent pathways. By partially and dynamically modulating endothelial phenotype, Reelin promotes a remodeling-permissive state without triggering full mesenchymal transition. These findings identify Reelin as a novel modulator of endothelial function with potential implications for vascular remodeling and cardiovascular disease. What Are the Clinical Implications?Our findings identify Reelin as a modulator of endothelial signaling with a clear bias toward non-canonical FAK- and AKT-dependent pathways that regulate endothelial plasticity and remodeling. This signaling profile is highly relevant to vascular diseases in which endothelial dysfunction is driven by maladaptive cytoskeletal reorganization, altered migration, and persistent activation rather than complete loss of endothelial identity. The ability of Reelin to promote partial and dynamically regulated phenotypic modulation suggests that it may operate at early and potentially reversible stages of vascular pathology. In this context, dysregulated Reelin signaling could contribute to pathological vascular remodeling, including processes underlying atherosclerosis, fibrosis, and microvascular dysfunction. These results also raise the possibility that circulating or locally produced Reelin may serve as an indicator of endothelial activation state, providing a novel biomarker for vascular disease progression. Importantly, the identification of a signaling bias toward FAK- and AKT-dependent pathways highlights potential therapeutic targets downstream of Reelin that could be selectively modulated to limit maladaptive endothelial remodeling while preserving essential endothelial functions. Collectively, this study positions Reelin signaling as a previously unrecognized and potentially actionable pathway in the regulation of endothelial behavior, with direct implications for the development of targeted strategies aimed at preventing or attenuating cardiovascular disease progression

cell biology↗

bioRxiv2026

A Translational Preclinical Strategy for Chronic Spinal Cord Injury: Neuroprotective and Regenerative Potential of Botulinum Neurotoxin Type A combined with Muscle Atrophy Prevention via Electrostimulation

BackgroundSpinal cord injury (SCI) triggers persistent neuroinflammation, gliosis, neuronal loss, and demyelination, leading to motor deficits and neuropathic pain. Botulinum neurotoxin type A (BoNT/A) has shown anti-inflammatory and neuroprotective effects in acute SCI, but its potential in the chronic phase remains unclear. This study investigates whether combining BoNT/A with electrical muscle stimulation (EMS) enhances recovery in chronic SCI. MethodsAdult mice with severe thoracic SCI (paraplegic) underwent EMS (30 min/day for 10 non-consecutive days starting 3 days post-injury) or no stimulation. Fifteen days after SCI, animals received a single intrathecal injection of BoNT/A (15 pg/5 L) or saline. Functional recovery was assessed up to 60 days as well as in moderate and mild SCI mice, neuropathic pain onset and maintenance were evaluated. Spinal cord tissue was analysed for astrocytic and microglial morphology, neuronal and oligodendroglia survival, myelin protein expression, and in vitro effects on oligodendrocyte precursor cells (OPCs). The phenotype of hindlimb muscles was evaluated through morphological and gene expression analyses. ResultsEMS was able to counteract muscle atrophy and fibrosis, and when combined with BoNT/A, also denervation. Moreover, the combination restored hindlimb motor function in chronic SCI, whereas BoNT/A or EMS alone were ineffective. Neuropathic pain, a common comorbidity associated with SCI, was mitigated by BoNT/A treatment even when administered in the chronic phase. BoNT/A reduced astrocytic hypertrophy and excitatory synapse association and was associated with a morphology-based redistribution of microglial profiles toward a resting-like classification, decreased apoptosis, and increased neuronal and oligodendroglia survival. Myelin basic protein expression was significantly elevated in vivo. In vitro, BoNT/A promoted OPC differentiation into myelinating oligodendrocytes, increased process complexity, and upregulated Myelin basic protein, galactocerebroside C, proteolipid protein, and myelin oligodendrocyte glycoprotein under both proliferative and differentiating conditions. Cleaved SNAP25 colocalization with OPC confirmed direct BoNT/A internalization and activity. ConclusionsBoNT/A exerts multi-cellular neuroprotective actions in chronic SCI, supporting neuronal and oligodendroglia survival, reducing neuroinflammation, enhancing remyelination and the combination with EMS promotes substantial recovery of muscle homeostasis within a permissive microenvironment shaped by early stimulation. Its efficacy depends on a permissive microenvironment achieved through EMS. These results provide strong rationale for the clinical evaluation of BoNT/A as a therapeutic strategy for chronic SCI.

neuroscience↗

bioRxiv2025

Spatial and Multiomic profiling of muscle regeneration dynamics in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a pediatric degenerative myopathy caused by the absence of functional dystrophin. As a result, DMD muscles exhibit compromised myofiber integrity and increased susceptibility to mechanical damage. In early disease stages, muscles undergo repeated cycles of degeneration and regeneration; over time, however, this regenerative capacity declines, leading to the gradual replacement of muscle tissue with fat and fibrosis. While several signaling pathways have been identified as deregulated in dystrophic muscle, the cellular and molecular mechanisms underlying this regenerative exhaustion remain to be fully elucidated. To address this, we constructed a comprehensive cellular atlas of human dystrophic muscle using high-resolution spatial transcriptomics (Visium HD), capturing the cellular crosstalk within regenerative regions. Cell-to-cell communication analysis revealed activation of Notch signaling mediated by NOTCH3 in activated satellite cells. Immunostaining confirmed elevated NOTCH3 expression in both DMD patient samples and in the mdx mouse model at late disease stages. Silencing of NOTCH3 in primary myoblasts improved myogenic differentiation, pinpointing NOTCH3-mediated signaling as a contributor to regeneration impairment. To further dissect the dynamics of regeneration and infer the gene regulatory networks governing myogenic differentiation, we integrated paired snRNA-seq and snATAC-seq data from young, adult, and aged mdx mice. This analysis identified GLIS3 upregulation as an additional barrier to effective myogenesis. GLIS3 displayed an overall increase in dystrophic muscles, while silencing experiments enhanced differentiation in myoblasts. Together, our work reveals intrinsic defects in the dystrophic stem cell compartment that emerge during disease progression and hinder the execution of the myogenic program. These findings suggest NOTCH3 and GLIS3 as potential therapeutic targets to enhance regeneration and maintain muscle integrity in DMD. This study provides a high-resolution map of the dystrophic regenerative landscape and offers a valuable resource for future translational research.

cell biology↗

bioRxiv2022

Spatially resolved transcriptomics reveals innervation-responsive functional clusters in skeletal muscle

Striated muscle is a highly organized structure composed by well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we followed the changes in the transcriptomics profile of specific domains in muscle in a model of denervation. Furthermore, our approach allowed us to identify the spatial distribution and nerve dependence of atrophic signalling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate a pronounced alteration of polyamine homeostasis upon denervation. Our dataset will serve as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/486563v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@6ee767org.highwire.dtl.DTLVardef@11a4eb4org.highwire.dtl.DTLVardef@25228eorg.highwire.dtl.DTLVardef@1ffa60a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

scRNA-seq-based analysis of skeletal muscle response to denervation reveals selective activation of muscle-resident glial cells and fibroblasts

Developmental synaptogenesis toward formation of neuromuscular junctions (NMJs) is regulated by the reciprocal exchange of signals derived from nerve or muscle ends, respectively. These signals are re-deployed in adult life to repair NMJ lesions. The emerging heterogeneity of skeletal muscle cellular composition and the functional interplay between different muscle-resident cell types activated in response to homeostatic perturbations challenge the traditional notion that muscle-derived signals uniquely derive from myofibers. We have used single cell RNA sequencing (scRNA-seq) for a longitudinal analysis of gene expression profiles in cells isolated from skeletal muscles subjected to denervation by complete sciatic nerve transection. Our data show that, unlike muscle injury, which massively activates multiple muscle-resident cell types, denervation selectively induced the expansion of two cell types - muscle glial cells and activated fibroblasts. These cells were also identified as putative sources of muscle-derived signals implicated in NMJ repair and extracellular matrix (ECM) remodelling. Pseudo-time analysis of gene expression in muscle glial-derived cells at sequential timepoints post-denervation revealed an initial bifurcation into distinct processes related to either cellular de-differentiation and commitment to specialized cell types, such as Schwann cells, or ECM remodeling. However, at later time points muscle glial-derived cells appear to adopt a more uniform pattern of gene expression, dominated by a reduction of neurogenic signals. Consensual activation of pro-fibrotic and pro-atrophic genes from fibroblasts and other muscle-resident cell types suggests a global conversion of denervated muscles into an environment hostile for NMJ repair, while conductive for progressive development of fibrosis and myofiber atrophy.

cell biology↗

bioRxiv2020

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetes patients

Aging and type 2 diabetes mellitus (T2DM) are associated with impaired skeletal muscle function and degeneration of the skeletal muscle microenvironment. However, the origin and mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscles of T2DM patients exhibit pathological degenerative remodeling of the extracellular matrix that was associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90) - the FAPCD90+. We identified Platelet-derived growth factor (PDGF) signaling as key regulator of human FAP biology, as it promotes proliferation and collagen production at the expense of adipogenesis, an effect accompanied with a metabolic shift towards glycolytic lactate fermentation. FAPsCD90+ showed a PDGF-mimetic phenotype, with high proliferative activity and clonogenicity, increased production of extracellular matrix production and enhanced glycolysis. Importantly, the pathogenic phenotype of T2DM FAPCD90+ was reduced by treatment with the anti-diabetic drug Metformin. These data identify PDGF-driven conversion of a sub-population of FAPs as a key event in the pathogenic accumulation of extracellular matrix in T2DM muscles.

cell biology↗