Substrate stiffness induces aged like-phenotypes in endothelial cells

Cardiovascular diseases (CVD) are the leading cause of death globally and ageing is a major risk factor. Cardiac ageing is characterized by fibrosis, microcirculatory dysfunction, inflammation and the secretion of senescence-associated molecules. Histological analysis of the aged heart revealed increased capillary diameter and endothelial cell size. However, the molecular mechanisms by which endothelial cells respond to age associated mechanical alterations like increasing tissue stiffness remains unclear.

To address this question, we have cultured endothelial cells on substrates of different stiffness. We have generated matrices that mimic embryonic (12 kPa), healthy (30 kPa) and fibrotic (123 kPa) myocardium using Sylgard 184, a non-reactive polymer. Endothelial cells cultured on stiffer matrices resembled aged cardiac endothelial cells. Specifically, they presented increased size, showed shorter telomeres and increased expression of pro-inflammatory cytokines like IL1B and IL6. Furthermore, increased stiffness reduced endothelial cell adhesion. Moreover, Bulk-RNA sequencing revealed the upregulation of genes associated with extracellular matrix (ECM) deposition, such as ADAMTS1 and ITGA11, and the endothelial to mesenchymal transition related FoxO signaling pathway, including FBXO32 and IL7R.

Endothelial cells regulate the local microenvironment by secreting angiocrine signals. The supernatant of endothelial cells cultured on stiff matrix, impaired endothelial barrier, increased the size of endothelial cells and reduced the uptake of fatty acids and glucose, in a paracrine manner. Mass spectrometry analysis of the supernatants detected 1600 proteins, 102 enriched in the supernatant of endothelial cells cultured on stiff 123 kPa matrix and 27 enriched on the soft 12 kPa. Analysis of aged single-nucleus RNA sequencing revelated that 30% of the stiff-enriched molecules were also upregulated in aged endothelial cells. Gene ontology analysis of the differentially enriched proteins revealed regulation across several biological processes. Notably, these included chromatin organization (SMARCC2, NPM1, HMGB2), cellular senescence (RSL1D1, B2M), and focal adhesion and cell-substrate junctions (PDIA3, MCAM, PPIA). Interestingly, we observe an upregulation of TIMP2 and a downregulation of MMP1. Additionally, growth factors associated with fibrosis like MDK or arterial stiffness like BMP4 were also enriched in the stiff 123 kPa condition. These observations suggest that stiffness may induce a pro-fibrotic response of endothelial cells and impair their capacity to remodel extracellular matrix

In conclusion, our findings suggest that substrate stiffness induces age like features in endothelial cells and drive the secretion of an 'aged secretome', potentially contributing to endothelial dysfunction, one of the hallmarks of cardiac ageing.