Fibronectin Fragments Promote Human Retinal Endothelial Cell Adhesion and Proliferation and ERK Activation through α5β1Integrin and PI 3-Kinase
Investigative Ophthalmology and Visual Science
PURPOSE. Extracellular matrix degradation is associated with neovascularization in diabetic retinas. Fibronectin fragments (Fn-fs) are generated during vascular remodeling. The effects of cellular fibronectin (Fn) and selected Fn-fs on adhesion, proliferation, and signal transduction in human retinal endothelial cells (HRECs) were characterized. METHODS. Relative quantitative RT-PCR, flow cytometry, and immunocytochemistry determined integrin expression on HRECs. Adhesion was evaluated by
... evaluated by coating plastic with Fn or Fn-fs of 45, 70, 110, or 120 kDa, and MTT conversion was used to measure proliferation and survival. Peptide inhibitors and blocking antibodies determined adhesive sites and integrins used for adhesion. Pharmacologic inhibitors and Western analyses were used to evaluate intracellular signaling. RESULTS. HRECs produced significant levels of ␣ 2 , ␣ 3 , ␣ 5 , ␣ v , ␤ 1 , ␤ 3 , and ␤ 5 integrin subunit mRNA. Flow cytometry of surface integrin expression revealed high levels of ␣ 3 , ␣ 5 , and ␤ 1 and lower levels of ␣ 1 , ␣ v , ␤ 3 , and ␤ 5 . These results were confirmed by immunocytochemistry. For adhesion to Fn and Fn-fs. the ␣ 5 ␤ 1 integrin was essential. Pharmacologic inhibitors of PI 3-kinase blocked adhesion to Fn and Fn-fs, whereas the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 blocked phosphorylation. The 110-and 120-kDa Fn-fs showed a concentration-dependent increase in proliferation, whereas 500 ng of the 70 kDa Fn-f-induced proliferation. Addition of III1-C, a matrix assembly domain, increased the proliferative effect of these Fn-fs. CONCLUSIONS. Fn and its Fn-fs modulate HREC adhesion and proliferation through signal-transduction pathways involving coupling of the ␣ 5 ␤ 1 integrin through PI 3-kinase. Mitogenic signals for endothelial cells from degraded extracellular matrix may contribute to the development of diabetic retinopathy.