Oxidized Low‐Density Lipoprotein Alters the Effect of Matrix Stiffness on the Formation of Endothelial Networks and Capillary Lumens

Abstract

Formation of new blood vessels is essential for vascular repair and remodeling, and it is known that biomechanical properties of extracellular matrix play a major role in this process. Our earlier studies have also shown that exposing endothelial cells to oxidized modification of low-density lipoproteins (oxLDL) increases endothelial stiffness and facilitates their ability to form cellular networks, suggesting that it facilitates endothelial angiogenic potential. The goal of this study, therefore, was to test the interrelationship between matrix stiffness and oxLDL in the regulation of angiogenesis. Our results show that, as expected, an increase in matrix stiffness inhibited endothelial network formation and that exposure to oxLDL significantly facilitated this process. We also show, however, that oxLDL-induced facilitation of endothelial networks was observed only in stiff (3 mg/mL) but not in soft (1 mg/mL) collagen gels, resulting in blunting the effect of matrix stiffness. Also unexpectedly, we show that an increase in matrix stiffness results in a significant increase in the number of capillary lumens that are formed by single cells or pairs of cells, suggesting that while endothelial connectivity is impaired, formation of single-cell lumens is facilitated. oxLDL facilitates lumen formation, but this effect is also matrix dependent and is observed only in soft gels and not in stiff gels. Finally, an increase in both matrix stiffness and oxLDL exposure results in changes in capillary morphology, with the formation of larger capillary lumens. Overall, our study suggests that oxLDL plays an important role in formation of new capillaries and their morphology and that this effect is critically dependent on the extracellular environment's compliance, thereby underlining the importance of the interdependence of these parameters.

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation

“…Earlier studies suggested that oxLDL inhibits endothelial proliferation (14), migration (15), and in vitro tube formation (16), as well as angiogenesis in the hind limb ischemia model (17). Our studies, however, showed that oxLDL may have a positive effect on the formation of endothelial networks in a 3D in vitro model of angiogenesis (3,18). Consistent with these findings, a similar effect was shown in a 2D system (19).…”

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading

“…Our studies demonstrated that oxLDL also has a major impact on endothelial biomechanical properties, resulting in increased en-dothelial stiffness and contractility (9,54). Furthermore, we showed that this effect is accompanied by enhanced formation of endothelial networks and capillary lumens in threedimensional cell cultures (9,19,54). Recently, we showed that preexposure to oxLDL facilitates formation of functional capillaries in matrigels in vivo via activation of the RhoA/Rho kinase (ROCK) signaling pathway (45).…”

“…Eighty to 90 percent confluent cells were transfected in Opti-MEM (Thermo Fisher Scientific) media using Lipofectamine 3000 Reagent (Invitrogen) and a siRNA mix for 4 h. HAEC-enriched cell culture media (Lonza) was then used to replace the Opti-MEM solution, and cells were allowed to grow for 48 h before experiments were performed. GFP-tagged-RhoGDI-1 was generated by PCR amplification (PFU; Stratagene) of the cDNA clone pOTB7-GDI-1 into p-EGFP-C3 vector using specific primer pairs [p-EGFP-FL-GDI-1 (AA1-204)] as described (19): forward primer 5=-AGGAATTCGAATGGCTGAG-CAGGAGCCCAC-3= and reverse primer 5=-CGGGATCCTCAT-CAGTCCTTCCAGTCCTTC-3=. We used a mutant containing the p115-RGS domain (RGS mutant) to address the role of p115RhoGEF in regulating RhoA activity as described (24) LDL isolation and oxidation.…”

Paradoxical impact of cholesterol on lipid packing and cell stiffness

“…More specifically, an increase in endothelial stiffness was shown to impair generation of nitric oxide in response to shear stress forces generated by blood flow (85), but also to facilitate flow-induced realignment of endothelial cells, a hallmark of their sensitivity to flow (77) and enhance transmigration of neutrophils through the endothelial layer (76). A series of studies also implicate endothelial stiffness as an important factor in angiogenesis (49,73,86,87). Furthermore, an increase in smooth muscle stiffness has been proposed to underlie the stiffening of blood vessels in hypertension and aging (88,89).…”