Abstract
Injury to the central nervous system (CNS) usually leads to the activation of astrocytes, followed by glial scar formation. The formation of glial scars from active astrocytes in vivo has been found to be dependent on the cell microenvironment. However, how astrocytes respond to different microenvironmental cues during scar formation, such as changes in matrix stiffness, remains elusive. In this work, we established an in vitro model to assess the responses of astrocytes to matrix stiffness changes that may be related to pathophysiology. The investigated hydrogel backbones are composed of collagen type I and alginate. The stiffness of these hybrid hydrogels can be dynamically changed by association or dissociation of alginate chains through adding crosslinkers of calcium chloride or a decrosslinker of sodium citrate, respectively. We found that astrocytes obtain different phenotypes when cultured in hydrogels of different stiffnesses. The obtained phenotypes can be switched in situ when changing matrix stiffness in the presence of cells. Specifically, matrix stiffening reverts astrogliosis, whereas matrix softening initiates astrocytic activation in 3D. Moreover, the effect of matrix stiffness on astrocytic activation is mediated by Yes-associated protein (YAP), where YAP inhibition enhances the upregulation of GFAP and contributes to astrogliosis. To investigate the underlying mechanism of matrix stiffness-dependent GFAP expression, we also developed a mathematical model to describe the time-dependent dynamics of biomolecules involved in the matrix stiffness mechanotransduction process of astrocytes. The modeling results further indicate that the effect of matrix stiffness on cell fate and behavior may be related to changes in the cytoskeleton and subsequent activity of YAP. The results from this study will guide researchers to re-examine the role of matrix stiffness in reactive astrogliosis in vivo and inspire the development of a novel therapeutic approach for controlling glial scar formation following injury, enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.
Highlights
Injury to the central nervous system (CNS) usually induces astrocytic reactivity, followed by glial scar formation[1]
We developed a mathematical model to study the underlying mechanism of matrix stiffness-dependent glial fibrillary acidic protein (GFAP) expression, and the modeling results were consistent with the experimental results
Matrix stiffness regulates astrocyte activation in 3D Recent studies have proven that the stiffness of glial scars is significantly lower than that of healthy neural tissue, which is associated with increased concentrations of softer extracellular matrix (ECM) components[6] (e.g., CSPGs22 and GAGs23)
Summary
Injury to the central nervous system (CNS) usually induces astrocytic reactivity, followed by glial scar formation[1]. As the key effector cells in glial scar formation, astrocytes have been found to change their phenotype from naive astrocytes to reactive astrocytes and. Page 2 of 15 35 gradually into scar-forming astrocytes, upregulating glial fibrillary acidic protein (GFAP)[9,10,11] and vimentin[12] and inflammatory proteins such as IL-1β13 Such astrocytic phenotype transformation has been found to depend on the cell microenvironment in vivo[14]. The phenotypic changes of astrocytes are affected by multiple microenvironmental cues, one of which is matrix stiffness[15,16,17,18] Glial scars in both the rat cortex and spinal cord are significantly softer than those in healthy CNS tissues[6]. It is still of great interest to study the effects of dynamic changes in matrix stiffness on astrocyte phenotypic switching in 3D culture
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