TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC–mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.

Endothelial cells: source, barrier, and target of defensive mediators

“…Very few reports describe the possibility of EC to secrete components of the classical and lectin pathways. While C2 was detected on mRNA and protein level, C4 was detected only at mRNA level . C1s but not C1r were detected also in HUVEC medium but stimulation with IFNγ was reported to increase the mRNA level and protein production, reaching equimolar C1r to C1s ratio .…”

“…Following cell activation or under inflammatory conditions, endothelium switches from a complement‐regulatory surface to a complement activating surface. This is true in particular for the alternative pathway, since EC activation is associated with a local increase in C3 and FB production . Sartain et al.…”

“…For example, classical pathway genes are highly expressed in coronary compared to iliac artery endothelium in a swine model . Thrombomodulin (TM) is overexpressed in GEC compared HUVEC in resting conditions, and decreased stronger in GECs upon cells activation by TNFα …”

Thrombomodulin and Endothelial Dysfunction: A Disease-Modifier Shared between Malignant Hypertension and Atypical Hemolytic Uremic Syndrome

“…Simultaneously, conditions that cause endothelial stress such as DIC and sepsis, or more specifically exposure to cytokines, endotoxin, human leukocyte antigen antibodies, fibroblast growth factor 23, or Shiga toxin temporarily decrease the endothelial synthesis and ex-DOI: 10.1159/000490201 pression of membrane-bound TM (Fig. 3, Table 2) [21][22][23][24][25]. A low endothelial concentration of TM contributes to the procoagulant and proinflammatory vascular state of these conditions.…”

A new therapeutic strategy for atypical HUS

“…In vitro, the powerful inflammatory cytokine TNF stimulates endothelial cell secretion/anchorage of ULVWF strings plus associated AP activation. 9 Subsequent clinical studies are likely to clarify whether or not discontinuation of eculizumab in patients with aHUS in remission is safe in the absence of underlying inflammation or infection. Conflict-of-interest disclosure: The author declares no competing financial interests.…”