Granulation Tissue

Fibrotic scarring remains a critic obstacle to axonal regeneration after spinal cord injury (SCI). Current strategies primarily concentrating on eliminating extracellular matrix (ECM) components neglect their dispensable roles in maintaining tissue integrity. Here, it is reported that the mechanical strength of an integrated hydrogel composed of hyaluronic acid-graft-dopamine and HRR peptide directs fibroblast migration, determining ECM deposition. The mechanical strength matching that of spinal cord induces fibroblast alignment, reshaping fibrotic scars into a parallel matrix, while the mechanical strength deviating from that of spinal cord fails to do so. Mechanical investigation identifies a previously unknown Il11ra1+/Itga11+ fibroblast subset that is specially associated with aligned infiltration and parallel ECM via mechanotransduction signaling cascade LRP6/β-Catenin/MMP7, promoting axonal regeneration and boosting neural reconnections across the lesion. The study uncovers the mechanotransduction mechanism that remodels fibrosis progression through manipulating cellular components of fibrotic scars, providing novel insights into discovering potential therapeutic targets to resolve fibrosis after SCI.

Scarring is an insurmountable obstacle for axonal regeneration in recovery from spinal cord injury (SCI). It impedes the repair effects of therapeutic targets in cortical neurons, such as PTEN-/- and hyper-IL-6, which cannot break through dense scar barriers to reconstruct neural circuits. However, methods for eliminating this process remain elusive. Here, we conducted a multiomics analysis of SCI and identified FBXL12 as an effective target for inhibiting scarring, further promoting spontaneous crossing of axons at the epicenter. We identified N6-Methyladenosine (m6A) modification as the predominant mRNA modification in SCI, with Fbxl12 being a major modification target. Furthermore, m6A modification specifically promoted FBXL12 synthesis in activated microglia. The overexpression of FBXL12 in microglia contributed to its homogeneous distribution and maintained a "scar-less healing" phenotype. Remarkably, FBXL12 therapy effectively reduced extracellular matrix deposition and decreased the scar area by ~70%. Importantly, axons grew through the epicenter and reached a length of more than 2.4 mm 56 days post-SCI, significantly improving motor function and reconstructing the neural circuit. Mechanistically, FBXL12 promoted cytoskeletal reorganization and migration in microglia by catalyzing the K63-linked ubiquitylation of Myosin heavy chain 14 (MYH14). Together, our results identify m6A-FBXL12-MYH14 axis as a novel cytoskeletal reorganization pathway in activated microglia and suggest FBXL12 as an effective target for a novel microglia-based approach to facilitate scarless functional recovery in SCI.

Activation of fibroblasts and formation of myofibroblasts are essential for granulation tissue formation following injury. In fibrotic reactions, excessive deposition of ECM by the activated fibroblasts determines scar formation and functional failure. Although these events critically depend on the activity of a plethora of growth factors and cytokines, TGFβ1 is a unique player controlling the immune response and proliferation of many cell types. Different cell types contribute to its release and activation, which is also regulated by the interaction with the ECM and by mechanical forces. The aim of this study was to elaborate whether fibroblast-derived TGFβ1 plays a critical role during these processes. The data demonstrate a dynamic expression of TGFβ1 during tissue repair. Cell-specific ablation of Tgfb1 in fibroblasts revealed that deletion of TGFβ1 attenuates bleomycin-induced skin fibrosis and perturbs maturation of granulation tissue in skin wounds. Absence of fibroblast-derived TGFβ1 induced vascular alterations (less vascular density and branching, haemorrhage) in early wound healing. This was associated with alterations in the formation of stable ECM structure. This can be explained by paracrine regulation of endothelial cells or pericytes by fibroblast-released TGFβ1 and by impaired expression of pro-angiogenic factors in TGFβ1-deficient fibroblasts. Our findings provide novel mechanistic insights into the central role of fibroblast-derived TGFβ1 for early stages of tissue repair and fibrosis in the skin.

Background: Reactive astrocytes form a chemical and mechanical glial scar that inhibits neuro-regeneration after stroke. Astrocyte heterogeneity is accompanied by changes in morphology and mechanical properties altering during scar formation after injury. This work aimed to elucidate the relationship between glial scar stiffness and astrocyte subtype transformation. Methods: Astrocyte-specific archaerhodopsin-3 and channelrhodopsin-2 knock-in C57BL/6J mice underwent distal MCAO. Atomic force microscopy, ultrasound elastography and synchrotron radiation were used to determine changes in glial scar stiffness. A proteomic analysis of astrocyte subtypes was performed ex vitro using single-cell laser capture microdissection-MS. Furthermore, optogenetics was employed in vivo to reduce the glial scar stiffness, thereby facilitating neural regeneration following brain injury. Results: Glial scar stiffness systematically increases following stroke and correlates with an increased number of Wnt7b+ fibrotic astrocytes. Furthermore, these results indicate that Piezo1 is the key regulator of astrocytic stiffness and anisotropy, which contributes to the glial scar stiffness in the peri-infarct area. The downregulation of Piezo1 expression promotes activation of the Wnt7b-Ca2+ nonclassical signaling pathway to modulate cytoskeletal reorganization. Finally, the specific optogenetic inhibition of Ca2+ signaling in astrocytes can effectively reduce glial scar stiffness by decreasing the proportion of Wn7b+ astrocytes, which further promotes neuro-regeneration and improves the recovery of motor function after ischemic stroke. Conclusions: This study successfully revealed astrocyte subtype transformation as a key determinant of glial scar physical barrier formation after stroke and highlighted Piezo1 as a potential therapeutic target for modulating the mechanical microenvironment post-injury.