CD109-regulated mechanical properties of endothelial cells

CD109 antigen on the endothelial cell surface plays an important role in vascular pathology. The aim of the work was to investigate the effect of the immobilization of CD109 antigen with specific antibodies on nanomechanical properties of human umbilical endothelial cells (HUVECs) using atomic force microscopy in quantitative nanomechanical property mapping mode (PeakForce QNM). Anti-CD109 antibodies induced significant stiffening of the cell surface Me(LQ; UQ): in 1.45(1.07;2.29) times with respect to control cells for fixed cells and in 4.9(3.6;5.9) times with respect to control cells for living cells, and changes in the spatial distribution of cell surface mechanical properties. The changes in the HUVEC's mechanical properties were accompanied by the activation of the TGF-/Smad2/3 signaling pathway and reorganization of the vimentin and actin cytoskeletal elements. Our findings show that blocking CD109 antigen using anti-CD109 antibodies leads in HUVECs to the processes similar to that occur after cell TGF- β -signaling activation. Therefore, we sug-gest that CD109 antigen may be involved in regulating the mechanical behavior of endothelial cells.

Among others, TGF-β has been shown to induce cardiac fibrosis, which leads to increased myocardial stiffness and, ultimately, to cardiac dysfunction and heart failure (Souders, Borg, Banerjee, & Baudino, 2012;Sullivan & Black, 2013). For example, the expression of TGF-β1 increases with the development of cardiac hypertrophy (Ruwhof, 2000), dilated cardiomyopathy (Khan & Sheppard, 2006) and aortic stenosis (Beaumont et al., 2014), and crucially, myocardial infarction (de Sousa Lopes et al., 2003;Frantz et al., 2008). In atherosclerosis, the fibrous and inflammatory components of the lesion in the vessel wall is mostly likely modulated by TGF-β. For instance, an increased expression of TGF-β1 in atherosclerotic plaques has been shown to correlate with progressive atherosclerosis patients (Herder et al., 2012;Panutsopulos et al., 2005).
Endothelial cells lining the interior of blood and lymph vessels can undergo a transition to mesenchymal cells, a process known as endothelial-to-mesenchymal transition (Kovacic, Mercader, Torres, Boehm, & Fuster, 2012). Concomitant loss of functional endothelial cells could also lead to a decrease in capillary density, thus causing tissue ischemia, which is a potent factor in the fibrous process (Kruithof, Duim, Moerkamp, & Goumans, 2012).
In the present work, to test the hypothesis that a CD109 antigen can be involved into the regulation of the mechanical behavior of endothelial cells we use specific antibodies (ABs) against CD109 antigen for immobilization of the antigen and blocking its ability to downregulate TGF-β signaling in endothelial cells using the human umbilical vein endothelial cells (HUVECs) as a cell model. We examine CD109-dependent changes of the nanomechanical properties of cell surface and cytoskeleton remodeling in HUVECs.

| Effect of anti-CD109 antibodies on the morphology and cytoskeleton of HUVECs
CD109 is known to be a negative regulator of the TGF-β pathways that activate the endothelial-to-mesenchymal transition with cytoskeleton rearrangement and cell morphology change. Anti-CD109 ABs have, in addition to specific CD109 binding sites on Fab fragments, Fc fragments that bind to Fc receptors on the cell surface, which can blur or even mask the effects of specific interactions of endothelial cells with anti-CD109 ABs. Fc receptors are known to be expressed by endothelial cells in various organs (Roopenian & Akilesh, 2007). FcRn (the neonatal Fc receptor) participates in the transport of IgG produced by HUVECs into the umbilical vein and arteries (Zhao, Liu, Chen, Korteweg, & Gu, 2011). Therefore, to eliminate the specific CD109-associated effect, firstly, it was necessary to block Fc receptors with IgG. To block the nonspecific interaction of ABs with cellular Fc receptors of the HUVEC surface, we used IgG following a protocol from R&D systems (https://www.rndsystems.com/resources/ protocols). The study of the expression of SMAD proteins of the classical mechanism of TGF-β signaling showed an overexpression of SMAD2 and SMAD3 proteins in HUVECs after exposure of IgGpretreated cells to anti-CD109 ABs ( Figure A in Appendix S1), which indicated the activation of SMAD2/3 signaling pathways in HUVECs using anti-CD109 ABs.
The changes in the cell morphology induced by treatment with IgG and anti-CD109 ABs were studied by both light microscopy

| EFFECT OF ANTI-CD109 ANTIBODIES ON HUVEC'S MECHANICAL PROPERTIES
The reorganization of the cytoskeleton leads to a change in HUVEC's mechanical properties. The heterogeneity in the nanomechanical parameters of HUVECs has been recently demonstrated by the AFM technique (Starodubtseva et al., 2021). Three different zones of HUVECs, the nuclear, perinuclear, and peripheral zones had relatively different mechanical properties (Starodubtseva et al., 2021). Figure 3 presents the AFM images recorded on three channels (topography, DMT modulus, and adhesion) of the microscale areas of the fixed HUVEC's surface for three experimental samples (control cells, cells after IgG treatment, and cells after IgG + anti-CD109 AB treatment).
The visual analysis of the images in Figure 3  F I G U R E 3 Typical AFM images of the microscale surface areas of control HUVECs, HUVECs after IgG treatment, and HUVECs after IgG + anti-CD109 AB treatment. The figure represents AFM images for two cell zones: nuclear (a) and perinuclear (b) zones. Peripheral cell zone is a thin layer consisting mainly the developed actin cytoskeleton network and the plasmalemma. Because the structural changes within the peripheral zone were insignificant, the AFM images for the peripheral zone were not presented. The scan sizes are 1 μm Â 1 μm, resolution is 256 Â 256 pixels. The adhesion maps clearly show spatially confined nanoscale regions with varying coloration grades for cell samples after IgG + anti-CD109 AB treatment (areas with lower adhesion are marked with number 1, and areas with higher adhesion are marked with number 2). modulus in 1.45(1.07;2.29) times stiffer than one for control cells and    since changes in cell stiffness assessed using AFM are mainly associated with rearrangements of the cortical actin cytoskeleton in cells (Starodubtseva, 2022;Starodubtseva et al., 2021).

Recently, the effect of neutralization of CD109 with anti-CD109
ABs has been studied in human fibroblast-like synoviocytes. In that study, cells were stimulated with TNF-α and IL-1β for 12 h, and then subsequently treated with anti-CD109 ABs or IgG for another 24 h.
Anti-CD109 AB treatment dose-dependently reduced the levels of IL-6, IL-8, MMP-1, and MMP-3 and the levels of phosphorylated Akt, NF-κB, Stat3, and p38 MAPK irrespective of TNF-α or IL-1β stimulation, as well as reduced cellular migration and invasion (Song