A monoclonal antibody recognizing the chlorohydrin derivatives of oleic acid for probing hypochlorous acid involvement in tissue injury

Summary

A monoclonal antibody against hypochlorous acid—modified oleic acid has been raised to investigate involvement of HOCI in tissue injury. Mice were immunized with an isomeric mixture of chlorohydrin derivatives of oleic acid (18:0-chlorohydrin) conjugated to keyhole limpet haemocyanin (CH-KLH). The chlorohydrin was formed by the treatment of oleic acid with hypochlorous acid. Monoclonal antibodies were raised and the fusion was screened with 18:0-chlorohydrin-bovine serum albumin (CH-BSA) conjugate. A number of antibody-secreting clones were identified and the supernatants were characterized by binding studies and dose-response curves. In ELISA, mAb CH-1 had an equivalent titre when either the chlorohydrin or bromohydrin derivative of oleic acid, complexed to bovine serum albumin, was used as screening antigen. The mAb CH-1 recognition of CH-BSA was competed with chlorohydrin and bromohydrin conjugates of BSA and KLH. Similarly, free 18:0-chlorohydrin and the 18:0-chlorohydrin-phosphatidyl choline treated with hypochlorous acid competed with mAb CH-1 binding. The mAb CH-1 also recognised the chlorohydrin derivative of linoleic acid and chlorohydrin formed from palmitoyl, oleyl phosphatidyl choline but with a decreased avidity. Weak cross-reactivity was observed with hydroxy-linoleic acid and linoleic acid hdroperoxide, either as free fatty acid or in phosphatidyl choline. There was minimal competitive binding of mAb CH-1 to free oleic acid, 16:0/18:1 phosphatidylcholine, cholesterol, or cholesterol chlorohydrin.

The mAb CH-1 described here may be a useful probe for assessing the involvement of hypochlorous acid in tissue injury.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.