Stiff Substrates Enhance Endothelial Oxidative Stress in Response to Protein Kinase C Activation

Abstract

Arterial stiffness, which increases with aging and hypertension, is an independent cardiovascular risk factor. While stiffer substrates are known to affect single endothelial cell morphology and migration, the effect of substrate stiffness on endothelial monolayer function is less understood. The objective of this study was to determine if substrate stiffness increased endothelial monolayer reactive oxygen species (ROS) in response to protein kinase C (PKC) activation and if this oxidative stress then impacted adherens junction integrity. Porcine aortic endothelial cells were cultured on varied stiffness polyacrylamide gels and treated with phorbol 12-myristate 13-acetate (PMA), which stimulates PKC and ROS without increasing actinomyosin contractility. PMA-treated endothelial cells on stiffer substrates increased ROS and adherens junction loss without increased contractility. ROS scavengers abrogated PMA effects on cell-cell junctions, with a more profound effect in cells on stiffer substrates. Finally, endothelial cells in aortae from elastin haploinsufficient mice (Eln+/-), which were stiffer than aortae from wild-type mice, showed decreased VE-cadherin colocalization with peripheral actin following PMA treatment. These data suggest that oxidative stress may be enhanced in endothelial cells in stiffer vessels, which could contribute to the association between arterial stiffness and cardiovascular disease.

Extracellular Matrix in Aging Aorta

“…As a result, ECs sense the substrate stiffness ( Collins et al, 2014a ), which in turn, further aggravates endothelial dysfunction ( Fels et al, 2012 ). For example, stiffening of the subendothelial space due to aberrant ECM remodeling sensitizes ECs to oxidative stress, accelerates EC senescence ( Urbano et al, 2019 ), and disrupts EC barrier function to further promote infiltration of activated immune cells ( Huynh et al, 2011 ). Aberrant cell-ECM interactions also reduce NO bioavailability and impair endothelial-dependent vasodilation ( Paar et al, 2014 ).…”

Macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by linear stiffness of the growth surface

“…Shear stress and altered flow dynamics also stimulate ROS production by endothelial cells [ 68 , 69 ]. For example, porcine aortic endothelial cells activated by phorbol 12-myristate 13-acetate (PMA) generated more ROS on stiff versus soft polyacrylamide gels [ 70 ]. These data raise the idea that increased arterial wall stiffness that precedes the development of the plaque alters mechanotransduction pathways in macrophages and other cells to favor the production of pro-inflammatory ROS that contribute to the oxidation of intimal LDL, thus propelling the progression of atherosclerosis [ 63 , 64 ].…”

Acute mitochondrial antioxidant intake improves endothelial function, antioxidant enzyme activity, and exercise tolerance in patients with peripheral artery disease

“…Furthermore, arterial stiffness is associated with atherosclerotic lesion formation (57), and patients with PAD tend to have elevated levels of arterial stiffness (18). Although mechanisms underlying the manifestation of arterial stiffness have not been fully elucidated, a potential mechanism underlying this process may be increased mitochondrial-derived ROS production and the resultant oxidative stress in both endothelial and smooth muscle cells (5,56). The role(s) of mitochondrial-derived ROS and oxidative stress in arterial stiffening and atherosclerotic development have gained significant attention over the past several years (1,5).…”