EXTRACELLULAR-MATRIX INCORPORATION OF NORMAL AND NEM-ALKYLATED FIBRONECTIN - LIVER AND SPLEEN DEPOSITION

The incorporation of plasma fibronectin (pFn) into the extracellular m atrix (ECM) is believed to influence tissue integrity, wound repair, a nd vascular permeability. In vitro, matrix assembly of Fn requires the binding of soluble Fn to cell-associated matrix assembly sites. Alkyl ation of human pFn (HFn) with N-ethylmaleimide (NEM) prevents the init ial binding of Fn to matrix assembly sites as well as its in vitro inc orporation into the ECM as reflected by detergent-insoluble I-125-labe led Fn (pool II Fn). We determined the kinetics of Fn matrix incorpora tion in tissue and whether NEM treatment of rat pFn (NEM-RFn) would li mit its in vivo incorporation into ECM by analysis of pool I [deoxycho late (DOG) soluble] and pool II (DOG insoluble) I-125-Fn in tissues af ter 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 l ocalization of normal I-125-RFn in liver, lung, spleen, heart, and int estine was greater (P < 0.05) than I-125-NEM-RFn at 4 h. Normal HFn, b ut not NEM-HFn, was incorporated into tissues and colocalized with end ogenous 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 cell s. 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 contin ued 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-HF n in pool II was observed over 24 h. Kupffer cells were not able to in corporate significant amounts of I-125-HFn into matrix. NEM-HFn displa yed limited incorporation into ECM by both fibroblast and hepatocyte c ultures. These novel observations suggest that the interaction of solu ble pFn with matrix assembly sites is necessary to its in vivo incorpo ration into the ECM.