Synthesis and expression of C1 inhibitor by human umbilical vein endothelial cells

A H Schmaier,S C Murray,G D Heda,A Farber,A Kuo,K Mccrae,D B Cines

doi:10.1016/s0021-9258(19)84693-8

A H Schmaier, S C Murray + Show 5 more

Open Access

https://doi.org/10.1016/s0021-9258(19)84693-8

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Abstract

The biologic activity of C1 esterase, activated forms of factor XII and kallikrein at sites of vascular inflammation may be regulated by C1 inhibitor (C1 INH) elaborated by endothelial cells. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) in culture produce C1 INH. Passaged HUVEC contain 1.6 +/- 0.8 micrograms of C1 INH/10(8) cells (mean +/- S.D.; n = 7) which was immunochemically similar to plasma C1 INH measured by a competitive enzyme-linked immunosorbent assay. Methylamine-treated lysates of HUVEC contained a functional inhibitor of purified kallikrein (2.7 +/- 0.8 micrograms activity/10(8) cells, mean +/- S.D.; n = 4). The HUVEC-derived kallikrein inhibitory activity was mostly C1 INH because it was reversed by chemically treating the lysate with chloroform and was neutralized by anti-C1 INH antibody. A lysate of HUVEC derived from an umbilical cord from a patient with Type I hereditary angioedema contained less than 30% of the normal levels of C1 INH antigen and activity. Immunohistochemical staining of HUVEC demonstrated a diffuse pattern of staining for C1 INH. HUVEC C1 INH was also expressed on the endothelial cell surface as detected by binding of anti-C1 INH antibody to intact monolayers and was elaborated progressively into the overlying media over the first 24 h in culture. HUVEC incubated with [35S] methionine secreted a metabolically labeled protein having a molecular mass of 92 kDa immunoisolated using polyclonal or monoclonal antibodies to human C1 INH. A mRNA transcript encoding for C1 INH was detected by slot blot hybridization. Incubation of HUVEC with gamma-interferon stimulated the expression of the 2.1 kilobase mRNA for C1 INH and increased the level of C1 INH produced by these cells. Production and expression of C1 INH by endothelial cells may help modulate the complement system and the contact system of plasma proteolysis on the vascular surface in vivo.

Highlights

S.D.; n = 7) which was immunochemically similar to other cells within the vasculatureof inhibitors of proteolytic enzymes
A lysate of Humuamnbilical vein endothelial cells (HUVEC) derived from an umbilical cord forms of factor XI1 to liberate thevasoactive peptide bradyfromapatientwithType
Further studies were performed to determine if the kallikrein neutralizing activity of the HUVEC lysates was due to the presence of C1 INH (Table I)

Summary

EXPERIMENTA LP ROCEDURES

2.1 kilobasemRNA for C1INH and increased thleevel purchased from Atlantic Antibodies (Scarborough, ME). Incubation of C1 INH produced by these cells. Mouse IgG in ascites (NS-1) was obtained from Organon-Teknika (Malvern, PA). A rabbitanti-human von Willebrandfactorantibody was purchased from Dako Corp. 12sII-Labeledgoat anti-mouse IgG was purchased from Du Pont-. B. C.), and Grant 2381 from The Tobacco Research Council Iodogen (chloroamide, 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril) was obtained from Pierce Chemical Co. Nitrocellulose (0.45-pm poresize), was purchased from Schleicher & Schuell. All reagent and molecular weight standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)were purchased from Bio-Rad. Pansorbin was purchased from Behring Diagnostics. §Recipient of Research Career DevelopmentAward HL01615 from thelial cells; C1 INH, C1 inhibitor;CELISA,competitiveenzymethe National Institutes of Health. To whom reprint requests should linkedimmunosorbent assay; SDS-PAGE, sodium dodecyl sulfatebe addressed Hematology/Oncology SectionT, empleUniversity polyacrylamide gel electrophoresis; IFN-y, y-interferon; SSC, stand-.

RESULTS

Additional studies were performed to determine if HUVEC

ACllNH CN

Findings

DISCUSSION