Dataset Information

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

ABSTRACT:

Purpose

To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.

Methods

CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot.

Results

The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate.

Conclusions

Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

SUBMITTER: Liu S

PROVIDER: S-EPMC10929747 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Publications

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Liu Shuting S Chen Hua H Xie Huatao H Liu Xin X Zhang Mingchang M

Investigative ophthalmology & visual science 20240301 3

<h4>Purpose</h4>To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.<h4>Methods</h4>CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscop ...[more]

PMID: 38466286

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

Dataset Information

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Purpose

Methods

Results

Conclusions

Publications

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Similar Datasets

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