Extracellular matrix distribution and hillock formation in human mesangial cells in culture without serum

Smooth muscle cell and mesangial cell hillock formation have been proposed as in vitro models of vascular sclerosis and glomerular sclerosis. This growth pattern is characterized by multilayered ridges and nodules, termed hills or hillocks, separated by less populated areas termed valleys. In this study, it was discovered that an extracellular matrix rich in pericellular fibronectin-fibrils was key to hillock formation. Human mesangial cells were plated onto serum-coated or noncoated substrata in serum-free medium. Subconfluent cells on serum-coated substrata migrated together, forming aggregates, but cells on noncoated substrata remained evenly dispersed. When plated at confluent densities, cells in serum-coated dishes formed hillocks, but cells in noncoated dishes did not. In serum-coated dishes, the substratum underlying subconfluent cells was vitronectin-rich but fibronectin-poor, whereas the pericellular matrix contained abundant fibronectin fibrils. In contrast, the substratum of subconfluent cells plated in noncoated dishes lacked vitronectin but was fibronectin-rich, whereas the pericellular matrix contained few fibronectin fibrils. The distributions of integrin receptors for fibronectin (rabbit anti-alpha 5 beta 1) and vitronectin (rabbit anti-alpha V, beta 3, and beta 5) followed the distributions of their ligands, fibronectin and vitronectin, respectively. Antibodies to fibronectin blocked hillock formation by cells on serum-coated substrata and prevented spreading of cells on noncoated substrata. In summary, key steps in hillock formation are: (1) migration, (2) secretion of fibronectin and assembly of pericellular fibrils, (3) fibronectin fibril-mediated cell-cell adhesion, and (4) aggregation of cells with further migration to form multiple layers. A similar mechanism may play a role in vascular and glomerular sclerosis.