Introduction: As we age, our organ tissues become stiffer due to the increased deposition and crosslinking of collagen and other matrix proteins. These changes impact how blood vessel cells interact with the surrounding extracellular matrix (ECM) and neighboring cells. Particularly in highly vascularized organs like the brain and kidneys, such stiffening often accompanies endothelial dysfunctions including the aging of endothelial cells (ECs), increased vascular permeability, a decrease in microvascular density, and enhanced inflammation. To mitigate these pathological aging responses, it is important to understand how tissue stiffness regulates EC senescence and barrier dysfunction. However, the lack of tissue models for monitoring dynamic EC responses impedes research into the direct link between tissue stiffening and EC senescence, as well as the development of therapeutic strategies to rejuvenate ECs and restore their function.
Materials and
Methods: We have developed an on-demand stiffening hydrogel model using methacrylated collagen 1 (Col-MA) and methacrylated hyaluronic acid (HA-MA). First, we encapsulated human ECs in a soft matrix through collagen physical crosslinking to develop microvascular networks that closely mimic the properties of young, healthy tissue. Subsequently, we stiffened the microvasculature-laden hydrogel via light-mediated methacrylate polymerization 48h post-encapsulation (Fig 1A). This approach models progressive stiffness increase in tissue matrix seen with aging. Since ECs are quiescent in intact blood vessels, it is essential to form vasculatures in 3D to accurately analyze how stiffness impacts EC behaviors, rather than using single-EC suspensions or monolayers on 2D, which tend to proliferate more. We produced hydrogels with different stiffness levels under the same light exposure conditions while decoupling the potential effects of radicals on EC senescence from matrix stiffening. This setup allows us to characterize the stiffness effect on EC aging comprehensively. Our hydrogel model offers an innovative platform for time-series monitoring of human EC dynamics in a mechanically defined 3D environment. To understand how cells respond to this dynamically changing environment and to investigate the mechanisms by which ECM stiffening leads to microvascular EC senescence, we utilized techniques such as qPCR, immunofluorescence, and western blotting. Endothelial networks grown under soft conditions served as controls.
Results, Conclusions, and Discussions: Human ECs cultured in dynamic hydrogel platforms maintained high viability throughout the stiffening culture environment, as evidenced by live/dead staining (Fig 1B-C). Following the stiffening process, ECs exhibited two responses: (1) altered vascular network morphology (Fig 1B) and (2) increased expression of senescence markers. Notably, hydrogel stiffening significantly upregulated the expression of cyclin dependent kinase inhibitor 1A and 2A (CDKN1A and CDKN2A) as well as p16 and p21, indicating that increased matrix stiffness acts as a causative factor in endothelial senescence (Fig 1D-G). To determine whether matrix stiffening alone triggers p21 expression rather than changes in the vascular network in our system, we treated networks in soft hydrogels with the Tie2 inhibitor, Bay 826, which led to vascular regression, but did not alter p21 expression levels (Fig 1H). Importantly, enhanced senescence in ECs within the stiffened matrix was demonstrated by a significant increase in senescence-associated beta-galactosidase (SA-β-gal) activity (Fig 1I-J). At the gene level, expression of senescence-associated secretory phenotype (SASP) elements showed an increase in TNFα and MMP1, alongside a decrease in CTGF (Fig 1 K-M), suggesting that stiffening-mediated EC senescence modulates SASP secretion patterns. This series of in vitro experiments confirms that tissue stiffness alteration is a key regulator for endothelial senescence. To further delineate the molecular mechanisms underlying stiffness-dependent EC aging, we investigated the expression of Notch signaling components, including the Notch1 receptor, and the ligands JAG1 and JAG2, as well as HEY1, a known target gene of Notch signaling. All tested genes were significantly upregulated, implying that the mechanoreceptor function of the Notch pathway may play a role in directing EC fate and senescence (Fig 1N-Q). Our dynamic matrix model facilitated the in situ examination of EC networks in response to alterations in matrix stiffness and its underlying mechanisms. This in vitro approach enhances our understanding of the mechanotransduction role of the Notch pathway in EC senescence. The findings from this study are expected to be directly applicable to various vascularized tissues that routinely experience mechanical stresses with aging.
