Matrix Mechanics Induces Microvascular Senescence and Pro-inflammation

Introduction: : Extracellular matrix (ECM) stiffening is a hallmark of aging and many diseases. The resulting increase in mechanical stress alters vascular phenotype, disrupts homeostasis, and contributes to a pro-inflammatory microenvironment by dysregulating cell-matrix communication. Endothelial cells (ECs), a key component of microvasculature, play a central role in maintaining vascular integrity and regulating inflammatory responses. Their dysfunction or premature senescence can therefore impair tissue regeneration and overall vascular health. Although ECM remodeling commonly occurs during aging or disease progression, the direct contribution of ECM stiffening to EC senescence, particularly within 3D microvascular networks, remains poorly understood. This knowledge gap impedes the development of effective therapeutic strategies targeting microvascular aging and its associated pathologies. To address this, we engineered a dynamic hydrogel platform capable of modulating stiffness to enable real-time monitoring of human EC aging in a physiologically relevant 3D microenvironment.

Materials and

Methods: : We developed a photo-tunable hydrogel system, composed of methacrylated collagen I (Col-MA) and methacrylated hyaluronic acid (HA-MA), to recapitulate the dynamic changes in matrix stiffness observed in pathological tissue environments (Fig. 1A). Initially, human endothelial colony-forming cells were encapsulated via collagen physical crosslinking to form 3D microvascular networks in a soft matrix, mimicking a young, healthy tissue condition (Fig. 1B). Subsequently, the vessel networks were exposed in situ to a stiffer microenvironment through light-initiated methacrylate polymerization (Fig. 1B).
Day 4 endothelial networks were analyzed for senescence marker expression at both gene and protein levels, using qRT-PCR, Western blotting, and immunofluorescence imaging. The senescence-associated secretory phenotype (SASP) was profiled using a Luminex assay. To investigate the underlying mechanism, we examined whether integrin-mediated mechanotransduction promoted Notch signaling by enhancing receptor-ligand complex formation. Gene and protein expression levels of key Notch components were assessed, and the γ-secretase inhibitor nirogacestat was used to evaluate the impact of Notch inhibition on stiffness-induced EC senescence.

Results, Conclusions, and Discussions:: Our two-step crosslinking strategy, combining collagen physical crosslinking and methacrylate photopolymerization, allowed for precise temporal modulation of matrix stiffness. A two-fold increase in stiffness significantly elevated senescence marker expression, including p16, p21, and SA-β-Gal activity, compared to the soft matrix condition. Control experiments confirmed that exposure to photoinitiator-generated radicals alone did not induce senescence, validating matrix stiffening as the primary driver of these effects.
In addition to promoting senescence, matrix stiffening induced a distinct SASP profile. Levels of pro-inflammatory mediators such as IL-33, IL-1α, IFN-γ, CCL3, CCL26, MMP1, and MMP3 were elevated, whereas canonical SASP factors like IL-6, IL-8, and CXCL1 were reduced with increased stiffness (Fig. 1C). These findings suggest that mechanical stress triggers an early-stage inflammatory response, with the elevated SASP components acting upstream of IL-6 and IL-8 networks.
Given the role of the Notch pathway in mechanotransduction, we examined Notch activation and tested pharmacological inhibition of Notch signaling. We found reduced p16 expression, thereby rescuing network phenotype (Fig. 1D). Mechanistically, we propose that matrix stiffening enhances formation of Notch1 receptor-JAG ligand complexes, leading to Notch activation (Fig. 1E). Our 3D stiffening model successfully recapitulated Notch-JAG interactions implicated in pathological vascular remodeling and inflammation related to EC aging.
Altogether, our hydrogel model provides a physiologically relevant system for studying microvascular aging and offers mechanistic insights into how ECM stiffness drives endothelial senescence. Notably, it highlights its potential for identifying and testing therapeutic targets to mitigate stiffness-induced microvascular aging.

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