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

Tramentozzi E, Pagetta A, Frasson M, Brunati AM, Montopoli M, Finotti P - J. Cell. Mol. Med. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy.

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.

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Cell-growth stimulation and angiogenic transformation of HUVECs. Cells were grown as specified in Materials and Methods and after starvation they were challenged with serum-free medium, in the absence (control) and presence of 10 ng/ml of the indicated plasma-purified peaks, control human IgG and native Grp94. After incubation for 20 hrs, cells from duplicate wells were counted. (A) Histograms are means (±S.D.) of at least three separate experiments. The mean (±S.D.) cell number in the control and after incubation with peaks 1 and 2 from the PG column was 180 (±51) × 103, 207 (±38) × 103 and 224 (±52.4) × 103, respectively. No significant differences were observed in multiple comparisons between control and treatments. (B) Microscopic evaluation of the morphogenic response of HUVECs to the indicated treatments (10 ng/ml each). Representative pictures (of several other made with any plasma) are shown. Original magnification of 10×.
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fig04: Cell-growth stimulation and angiogenic transformation of HUVECs. Cells were grown as specified in Materials and Methods and after starvation they were challenged with serum-free medium, in the absence (control) and presence of 10 ng/ml of the indicated plasma-purified peaks, control human IgG and native Grp94. After incubation for 20 hrs, cells from duplicate wells were counted. (A) Histograms are means (±S.D.) of at least three separate experiments. The mean (±S.D.) cell number in the control and after incubation with peaks 1 and 2 from the PG column was 180 (±51) × 103, 207 (±38) × 103 and 224 (±52.4) × 103, respectively. No significant differences were observed in multiple comparisons between control and treatments. (B) Microscopic evaluation of the morphogenic response of HUVECs to the indicated treatments (10 ng/ml each). Representative pictures (of several other made with any plasma) are shown. Original magnification of 10×.

Mentions: To test the effects on HUVECs of Ab-containing plasma-purified fractions, we first incubated cells for 20 hrs and noted that after this time peak 2 from the mono-Q column did not stimulate the cell growth at all, whereas peaks 1 and 2 from the PG column caused average stimulations of 15% and 24.4%, respectively (Fig. 4A). To rule out that also non-immune Abs contributed to this effect, IgG purified from normal plasma were used as additional control in cell cultures. In this case, the cell count overlapped that of control in absence of FBS (Fig. 4A). In both absence and presence of IgG from normal plasma, control HUVECs showed typical cobblestone morphology, whereas after the addition of Ab-containing peaks – especially peak 2 from the PG column – cells underwent morphologic changes consistent with angiogenic transformation. To mimic the condition found in Ab-containing fractions from diabetic plasma, we also used native Grp94 as further control in cell culture experiments. Native Grp94 (10 ng/ml) caused an average cell growth stimulation of 32% also displaying strikingly similar angiogenic effects on HUVECs after 20 hrs of incubation (Fig. 4B).


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

Tramentozzi E, Pagetta A, Frasson M, Brunati AM, Montopoli M, Finotti P - J. Cell. Mol. Med. (2008)

Cell-growth stimulation and angiogenic transformation of HUVECs. Cells were grown as specified in Materials and Methods and after starvation they were challenged with serum-free medium, in the absence (control) and presence of 10 ng/ml of the indicated plasma-purified peaks, control human IgG and native Grp94. After incubation for 20 hrs, cells from duplicate wells were counted. (A) Histograms are means (±S.D.) of at least three separate experiments. The mean (±S.D.) cell number in the control and after incubation with peaks 1 and 2 from the PG column was 180 (±51) × 103, 207 (±38) × 103 and 224 (±52.4) × 103, respectively. No significant differences were observed in multiple comparisons between control and treatments. (B) Microscopic evaluation of the morphogenic response of HUVECs to the indicated treatments (10 ng/ml each). Representative pictures (of several other made with any plasma) are shown. Original magnification of 10×.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496147&req=5

fig04: Cell-growth stimulation and angiogenic transformation of HUVECs. Cells were grown as specified in Materials and Methods and after starvation they were challenged with serum-free medium, in the absence (control) and presence of 10 ng/ml of the indicated plasma-purified peaks, control human IgG and native Grp94. After incubation for 20 hrs, cells from duplicate wells were counted. (A) Histograms are means (±S.D.) of at least three separate experiments. The mean (±S.D.) cell number in the control and after incubation with peaks 1 and 2 from the PG column was 180 (±51) × 103, 207 (±38) × 103 and 224 (±52.4) × 103, respectively. No significant differences were observed in multiple comparisons between control and treatments. (B) Microscopic evaluation of the morphogenic response of HUVECs to the indicated treatments (10 ng/ml each). Representative pictures (of several other made with any plasma) are shown. Original magnification of 10×.
Mentions: To test the effects on HUVECs of Ab-containing plasma-purified fractions, we first incubated cells for 20 hrs and noted that after this time peak 2 from the mono-Q column did not stimulate the cell growth at all, whereas peaks 1 and 2 from the PG column caused average stimulations of 15% and 24.4%, respectively (Fig. 4A). To rule out that also non-immune Abs contributed to this effect, IgG purified from normal plasma were used as additional control in cell cultures. In this case, the cell count overlapped that of control in absence of FBS (Fig. 4A). In both absence and presence of IgG from normal plasma, control HUVECs showed typical cobblestone morphology, whereas after the addition of Ab-containing peaks – especially peak 2 from the PG column – cells underwent morphologic changes consistent with angiogenic transformation. To mimic the condition found in Ab-containing fractions from diabetic plasma, we also used native Grp94 as further control in cell culture experiments. Native Grp94 (10 ng/ml) caused an average cell growth stimulation of 32% also displaying strikingly similar angiogenic effects on HUVECs after 20 hrs of incubation (Fig. 4B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy.

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.

Show MeSH
Related in: MedlinePlus