Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: Liver and spleen deposition

The incorporation of plasma fibronectin (pFn) into the extracellular matrix (ECM) is believed to influence tissue integrity, wound repair, and vascular permeability. In vitro, matrix assembly of Fn requires the binding of soluble Fn to cell-associated matrix assembly sites. Alkylation of human pFn (HFn) with N-ethylmaleimide (NEM) prevents the initial binding of Fn to matrix assembly sites as well as its in vitro incorporation into the ECM as reflected by detergent-insoluble I-125-labeled Fn (pool II Fn). We determined the kinetics of Fn matrix incorporation in tissue and whether NEM treatment of rat pFn (NEM-RFn) would limit its in vivo incorporation into ECM by analysis of pool I [deoxycholate (DOG) soluble] and pool II (DOG insoluble) I-125-Fn in tissues after its intravenous injection into rats. After intravenous injection, tissue incorporation of normal rat I-125-pFn was especially intense in liver and spleen, in agreement with the large amount of endogenous Fn detected in the matrices of these organs. Tissue deposition of plasma-derived I-125-RFn in liver and spleen peaked by 4 h, with significant (P < 0.01) loss over 24 h, indicating turnover of matrix Fn. Tissue localization of normal I-125-RFn in liver, lung, spleen, heart, and intestine was greater (P < 0.05) than I-125-NEM-RFn at 4 h. Normal HFn, but not NEM-HFn, was incorporated into tissues and colocalized with endogenous Fn in the matrix. To identify the cells mediating the intense incorporation of pFn into liver ECM, we compared matrix assembly of I-125-HFn by cultured fibroblasts, hepatocytes, and hepatic Kupffer cells. With fibroblasts, I-125-HFn in pool I reached steady state by 3 h, whereas I-125-HFn in pool II exceeded that in pool I by 6 h and continued to increase over 24 h. With hepatocytes, pool I I-125-HFn reached steady state by 1 h, and a progressive increase (P < 0.05) of I-125-HFn in pool II was observed over 24 h. Kupffer cells were not able to incorporate significant amounts of I-125-HFn into matrix. NEM-HFn displayed limited incorporation into ECM by both fibroblast and hepatocyte cultures. These novel observations suggest that the interaction of soluble pFn with matrix assembly sites is necessary to its in vivo incorporation into the ECM.