Limits...
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.

Show MeSH

Related in: MedlinePlus

Time-dependent stimulation of P-ERK1/2 in HUVECs treated with peaks eluting from the PG column. Whole lysates of cells grown in serum-free medium and treated with peaks from the PG column were analysed in Western blotting with anti-total and anti-P-ERK1/2 polyclonal Abs, as specified in Materials and Methods. The same membranes were then stripped and tested with anti-β actin Abs for normalization of the protein content. In the figure are shown: a representative blotting of two separate experiments (above), and the corresponding graphical representation of the intensity (means ± S.D., n= 2) of the P-ERK1/2 bands measured by the densitometric analysis (below).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4496147&req=5

fig06: Time-dependent stimulation of P-ERK1/2 in HUVECs treated with peaks eluting from the PG column. Whole lysates of cells grown in serum-free medium and treated with peaks from the PG column were analysed in Western blotting with anti-total and anti-P-ERK1/2 polyclonal Abs, as specified in Materials and Methods. The same membranes were then stripped and tested with anti-β actin Abs for normalization of the protein content. In the figure are shown: a representative blotting of two separate experiments (above), and the corresponding graphical representation of the intensity (means ± S.D., n= 2) of the P-ERK1/2 bands measured by the densitometric analysis (below).

Mentions: To see whether the ERK1/2 pathway was involved in the cell growth stimulation and angiogenic differentiation induced by peaks 1 and 2 from the PG column, total and P-ERK1/2 were measured in whole cell lysates. In control, serum-free HUVECs, a slight stimulation of P-ERK1/2 was detected only in first hours, disappearing completely after 20 hrs (Fig. 6). With both peaks (mostly peak 2) there was an intense activation, especially of P-ERK2 at 6 and 9 hrs, which decreased in time, remaining however still significantly elevated with respect to the control even after 20 hrs.


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)

Time-dependent stimulation of P-ERK1/2 in HUVECs treated with peaks eluting from the PG column. Whole lysates of cells grown in serum-free medium and treated with peaks from the PG column were analysed in Western blotting with anti-total and anti-P-ERK1/2 polyclonal Abs, as specified in Materials and Methods. The same membranes were then stripped and tested with anti-β actin Abs for normalization of the protein content. In the figure are shown: a representative blotting of two separate experiments (above), and the corresponding graphical representation of the intensity (means ± S.D., n= 2) of the P-ERK1/2 bands measured by the densitometric analysis (below).
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Time-dependent stimulation of P-ERK1/2 in HUVECs treated with peaks eluting from the PG column. Whole lysates of cells grown in serum-free medium and treated with peaks from the PG column were analysed in Western blotting with anti-total and anti-P-ERK1/2 polyclonal Abs, as specified in Materials and Methods. The same membranes were then stripped and tested with anti-β actin Abs for normalization of the protein content. In the figure are shown: a representative blotting of two separate experiments (above), and the corresponding graphical representation of the intensity (means ± S.D., n= 2) of the P-ERK1/2 bands measured by the densitometric analysis (below).
Mentions: To see whether the ERK1/2 pathway was involved in the cell growth stimulation and angiogenic differentiation induced by peaks 1 and 2 from the PG column, total and P-ERK1/2 were measured in whole cell lysates. In control, serum-free HUVECs, a slight stimulation of P-ERK1/2 was detected only in first hours, disappearing completely after 20 hrs (Fig. 6). With both peaks (mostly peak 2) there was an intense activation, especially of P-ERK2 at 6 and 9 hrs, which decreased in time, remaining however still significantly elevated with respect to the control even after 20 hrs.

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