Angiogenic transforming capacity of IgG purified from plasma of type 1 diabetic patients

Abstract

We previously demonstrated that plasma of type 1 diabetic patients contains antibodies complexed irreversibly with Grp94 that also display proteolytic activity. In this work, we wanted to test whether antibodies obtained from diabetic plasma may convey an inflammatory risk on vascular cells. To this aim, IgG were purified on the Protein-G column from individual plasma of eight type 1 diabetic patients, and then tested on HUVECs to measure effects on cell growth and morphologic changes at different incubation times. The purified fractions of IgG contained a significant amount of Fab/(Fab)2, both free and in big aggregates, and anti-Grp94 antibodies, mostly irreversibly linked with, but also free of Grp94. The purified fractions of both Fab/(Fab)2 and whole IgG stimulated the proliferation and sustained the angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) at sub-nanomolar concentrations. IgG from normal plasma neither stimulated the cell growth nor induced any differentiation of HUVECs. The maximum cell growth stimulation occurred at 6–9 hrs and associated with the strong activation of the ERK1/2 pathway, whereas angiogenic transformation was completed later when the ERK1/2 activation was silenced and cell growth stimulation significantly reduced. Neither proteolytic activity of MMP-9 nor VEGF were apparently involved in mediating the angiogenic differentiation of HUVECs that mostly correlated with an increased expression of HSP70 closely coupled with cell membrane-bound inactive species of MMP-9. Results indicate that effects displayed on HUVECs by antibodies purified from diabetic plasma are likely sustained by immune complexes with Grp94 that may thus predict an increased risk of angiogenic transformation in vivo.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

“…In this work we wanted to address the unsolved question concerning the nature and structure of Grp94-IgG complexes that circulate in plasma of T1 diabetic subjects with still uncertain pathogenetic significance. Previous observations suggested that these complexes could be immune in nature, based on the finding that anti-Grp94 Abs were present in diabetic plasma in response to extracellular exposure of Grp94 [ 8 ]. However, the discovery that Grp94 was able to form rapidly stable complexes also with nonimmune IgG in vitro [ 9 ] raised the possibility that the mechanism leading to the formation of Grp94-IgG complexes in vivo could also exclude an antigenic recognition.…”

“…This was also supported by the finding that anti-Grp94 antibodies are present in diabetic plasma in response to the immune stimulation driven by extracellular Grp94 [ 2 ]. Other observations indicated that Grp94-IgG complexes in diabetic plasma can induce an intense activation of inflammatory cell signaling pathways with angiogenic-like transformation of vascular cells [ 8 ]. This has led to the proposal that Grp94-IgG complexes might be a marker of increased risk of vascular complications in T1 diabetes [ 2 ].…”

Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

“…However, the possibility that Grp94 can bind to IgG in a manner different from that predicted from its chaperoning function has been raised in a previous work in which complexes have been observed to form between Grp94 and human non-immune IgG in vitro [25]. Such complexes in addition turned out to be functionally similar to those identified in the plasma of diabetic subjects in whom they drive a high risk of vascular damage [21], [24].…”

“…In ex vivo experiments on plasma of type 1 diabetic subjects we observed that Grp94, besides being present at a higher-than-normal concentration [23], circulated only linked to plasma proteins, mostly IgG, forming complexes of various masses prevalently immune in nature [21], [24]. We further demonstrated that Grp94 could also bind to IgG irrespective of their immune nature, forming non-immune complexes (NICs) in which binding occurs at sites other than the antigen-binding site [25].…”

Grp94 in complexes with IgG is a soluble diagnostic marker of gastrointestinal tumors and displays immune-stimulating activity on peripheral blood immune cells

“…Previous works had stably demonstrated that when liberated in the extracellular milieu - as it also occurs in autoimmune diseases [27, 31] - Grp94 is never detected as a single protein, but is always found linked in big, stable complexes with IgG [26, 28]. To explore the possibility that Grp94-IgG complexes could also circulate in cancer patients, we first tested any single plasma sample with anti-Grp94 Abs in Western blotting (WB) (Figure 2A).…”