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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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MMP-9 and HSP70 are closely associated in the membrane fraction of HUVECs. After the incubation time of 20 hrs, cell cultures in both absence (control) and presence of peaks 1 and 2 from the PG column (10 ng/ml) were lysed to obtain cytosol and membrane fractions, as specified in Materials and Methods. In the figure, representative Western blot analyses with anti-MMP-9 (reacting with both inactive and active species of the protease), anti-HSP70 and anti-VEGF mono-clonal Abs on both the membrane fraction of cell lysates and corresponding conditioned media are presented. The same quantity of proteins was loaded in each lane in SDS-PAGE in non-reducing conditions of samples. Upper panel: the arrow on left marks the band of MMP-9 at 40 kD visible in HUVECs treated with peaks 1 and 2 but not in the control. Arrows in the lower panel mark the bands of interest (see text).
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fig08: MMP-9 and HSP70 are closely associated in the membrane fraction of HUVECs. After the incubation time of 20 hrs, cell cultures in both absence (control) and presence of peaks 1 and 2 from the PG column (10 ng/ml) were lysed to obtain cytosol and membrane fractions, as specified in Materials and Methods. In the figure, representative Western blot analyses with anti-MMP-9 (reacting with both inactive and active species of the protease), anti-HSP70 and anti-VEGF mono-clonal Abs on both the membrane fraction of cell lysates and corresponding conditioned media are presented. The same quantity of proteins was loaded in each lane in SDS-PAGE in non-reducing conditions of samples. Upper panel: the arrow on left marks the band of MMP-9 at 40 kD visible in HUVECs treated with peaks 1 and 2 but not in the control. Arrows in the lower panel mark the bands of interest (see text).

Mentions: Since MMPs are specifically involved in the development and the finest modulation of angiogenesis [13], we investigated whether effects on HUVECs were mediated by an increase in activity and/or expression of MMP-9, the secreted MMP capable of degrading the extra-cellular matrix in various physiological and pathological conditions [13–15]. Proteolytic activity of conditioned media, measured on gel of gelatin, unexpectedly showed that both peaks caused a significant reduction in the secretion of inactive MMP-9 (the 92 kD form that is activated artificially by zymography) at 6 and 9 hrs (Fig. 7). Reductions were significantly different at 6 hrs (unpaired t-test: P= 0.005 and P= 0.0001 for peak 1 and 2, respectively, with respect to control), being still significantly different at 9 hrs for peak 2 only (P= 0.044, n= 4). The secretion of inactive MMP-9 started to increase after 9 hrs of incubation, reaching values similar to those of control at 20 hrs (Fig. 7). Also the secretion of the 90-kD active form of MMP-9, only slightly detectable at 6 and 9 hrs, increased significantly after 20 hrs independently of the treatment with peaks 1 and 2 (two-way ANOVA, P= 0.0001 for both control and treated HUVECs compared to respective values at 6 and 9 hrs, n= 4). Apparently, thus, the mechanisms leading to complete angiogenic transformation did not involve an increased activity of MMP-9. However, since the measurement of proteolytic activity does not give precise information about the protein expression, we also measured in Western blotting the level of MMP-9 in both conditioned media and cell lysates after 20 hrs of incubation (Fig. 8). The expression of both active and inactive MMP-9 in the media of control and treated HUVECs did not differ at all, nor there was any difference in MMP-9 species of 50 and 40 kD, mostly representing inactive products of MMP-9, not visible in zymography (Fig. 8, lower panels, arrows). Instead, in the membrane fraction of cell lysates, the bands of MMP-9 were more numerous and more intensely stained in treated HUVECs than in the control (Fig. 8, upper panels). This pattern strongly resembled that described by others who showed that a series of inactive and active species of MMP-9 with various molecular weight, form intra-cellularly in response to various growth factors [16, 17]. Consistent with the result that membrane-bound species of MMP-9 were expressed at a higher level in HUVECs treated with both peaks than in the control, in the corresponding cytosol fraction of cell lysates after treatment, MMP-9 was only weakly detected in two bands at 67 and 60 kD, whereas in control HUVECs the pattern of MMP-9 bands was similar to that seen in the membrane fraction (data not shown). Thus, after challenging HUVECs with Ab-containing peaks, MMP-9 was almost exclusively confined to cell membranes, whereas an insignificant part of it was in soluble form in the cytosol.


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)

MMP-9 and HSP70 are closely associated in the membrane fraction of HUVECs. After the incubation time of 20 hrs, cell cultures in both absence (control) and presence of peaks 1 and 2 from the PG column (10 ng/ml) were lysed to obtain cytosol and membrane fractions, as specified in Materials and Methods. In the figure, representative Western blot analyses with anti-MMP-9 (reacting with both inactive and active species of the protease), anti-HSP70 and anti-VEGF mono-clonal Abs on both the membrane fraction of cell lysates and corresponding conditioned media are presented. The same quantity of proteins was loaded in each lane in SDS-PAGE in non-reducing conditions of samples. Upper panel: the arrow on left marks the band of MMP-9 at 40 kD visible in HUVECs treated with peaks 1 and 2 but not in the control. Arrows in the lower panel mark the bands of interest (see text).
© Copyright Policy
Related In: Results  -  Collection

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

fig08: MMP-9 and HSP70 are closely associated in the membrane fraction of HUVECs. After the incubation time of 20 hrs, cell cultures in both absence (control) and presence of peaks 1 and 2 from the PG column (10 ng/ml) were lysed to obtain cytosol and membrane fractions, as specified in Materials and Methods. In the figure, representative Western blot analyses with anti-MMP-9 (reacting with both inactive and active species of the protease), anti-HSP70 and anti-VEGF mono-clonal Abs on both the membrane fraction of cell lysates and corresponding conditioned media are presented. The same quantity of proteins was loaded in each lane in SDS-PAGE in non-reducing conditions of samples. Upper panel: the arrow on left marks the band of MMP-9 at 40 kD visible in HUVECs treated with peaks 1 and 2 but not in the control. Arrows in the lower panel mark the bands of interest (see text).
Mentions: Since MMPs are specifically involved in the development and the finest modulation of angiogenesis [13], we investigated whether effects on HUVECs were mediated by an increase in activity and/or expression of MMP-9, the secreted MMP capable of degrading the extra-cellular matrix in various physiological and pathological conditions [13–15]. Proteolytic activity of conditioned media, measured on gel of gelatin, unexpectedly showed that both peaks caused a significant reduction in the secretion of inactive MMP-9 (the 92 kD form that is activated artificially by zymography) at 6 and 9 hrs (Fig. 7). Reductions were significantly different at 6 hrs (unpaired t-test: P= 0.005 and P= 0.0001 for peak 1 and 2, respectively, with respect to control), being still significantly different at 9 hrs for peak 2 only (P= 0.044, n= 4). The secretion of inactive MMP-9 started to increase after 9 hrs of incubation, reaching values similar to those of control at 20 hrs (Fig. 7). Also the secretion of the 90-kD active form of MMP-9, only slightly detectable at 6 and 9 hrs, increased significantly after 20 hrs independently of the treatment with peaks 1 and 2 (two-way ANOVA, P= 0.0001 for both control and treated HUVECs compared to respective values at 6 and 9 hrs, n= 4). Apparently, thus, the mechanisms leading to complete angiogenic transformation did not involve an increased activity of MMP-9. However, since the measurement of proteolytic activity does not give precise information about the protein expression, we also measured in Western blotting the level of MMP-9 in both conditioned media and cell lysates after 20 hrs of incubation (Fig. 8). The expression of both active and inactive MMP-9 in the media of control and treated HUVECs did not differ at all, nor there was any difference in MMP-9 species of 50 and 40 kD, mostly representing inactive products of MMP-9, not visible in zymography (Fig. 8, lower panels, arrows). Instead, in the membrane fraction of cell lysates, the bands of MMP-9 were more numerous and more intensely stained in treated HUVECs than in the control (Fig. 8, upper panels). This pattern strongly resembled that described by others who showed that a series of inactive and active species of MMP-9 with various molecular weight, form intra-cellularly in response to various growth factors [16, 17]. Consistent with the result that membrane-bound species of MMP-9 were expressed at a higher level in HUVECs treated with both peaks than in the control, in the corresponding cytosol fraction of cell lysates after treatment, MMP-9 was only weakly detected in two bands at 67 and 60 kD, whereas in control HUVECs the pattern of MMP-9 bands was similar to that seen in the membrane fraction (data not shown). Thus, after challenging HUVECs with Ab-containing peaks, MMP-9 was almost exclusively confined to cell membranes, whereas an insignificant part of it was in soluble form in the cytosol.

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