Changes in expression of VE-cadherin and MMPs in endothelial cells: Implications for angiogenesis - PubMed (original) (raw)
Changes in expression of VE-cadherin and MMPs in endothelial cells: Implications for angiogenesis
Manikantan S Kiran et al. Vasc Cell. 2011.
Abstract
The mechanism of cell-cell contact dependent regulation of pericellular proteolysis in angiogenesis was examined by studying the expression of MMPs using isolated HUVECs in culture. Zymography, Immunoblot and RT-PCR analysis showed that the production and secretion of matrixmetalloproteinase-2 and matrixmetalloproteinase-9 by HUVECs in culture were high when they remain as individual cells and significantly decreased during later stages of culture when cells developed cell-cell contact and tubular network-like structure. As MMPs decreased there was significant upregulation of VE-cadherin in cells undergoing angiogenic transition. Investigations to understand the signaling pathways downstream of VE-cadherin showed a relatively high level of β-catenin in the nucleus of endothelial cells in culture during initial stages and decrease in its levels in the nucleus, associated with an increase in the cytosol during later stages of culture. The distribution of β-catenin was found to be regulated by Tyr/Ser phosphorylation status of this protein. Cell-cell contact dependent downregulation of MMPs during angiogenesis was also observed in experiments using proangiogenic substances which caused a rapid rate of downregulation of MMP-2 and MMP-9 and absence of downregulation of MMPs when treated with anti-angiogenic agents.
Figures
Figure 1
Production of MMP-2 and MMP-9 by HUVECs: Equivalent aliquots of the medium normalized to the total cell protein from the cultures of HUVECs maintained in MCDB 131 medium collected at intervals of 24 hrs were subjected to zymography (A) and ELISA (C) as described in text. Lanes 1-5 1st-5th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm2/mg protein (B). Values given are the average of intensity measurement of 6 experiments ± SEM. The protein level expression quantitated for MMP-9 and MMP-2 by ELISA using specific antibody (C). Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day p < 0.05.
Figure 2
Expression of MMP-2 and MMP-9 in HUVECs - RT-PCR: Cells were maintained in MCDB 131 medium for different time intervals. Medium was removed and the cell layer harvested, total RNA was isolated and subjected to RT-PCR as described in the text. Samples were resolved on 1.75% agarose gel MMP-2 (A) and MMP-9 (B). Intensity of bands were quantitated and normalized with the intensity of band for internal control GAPDH and expressed as intensity/mm2 (C). * Statistically significant when compared to their production on first day p < 0.05.
Figure 3
Effect of modulators of angiogenesis on the activity and production of MMPs: Equivalent aliquots of the medium normalized to the total cell protein from the culture of control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells on days 1 to 4 were subjected to zymography (A) as described in the text.(a) Lanes 1-4, 50 μM ursolic acid treated cells 1st -4th day, Lanes 5-8, control 1st-4th day. (b) Lanes 1-4, 10 μM curcumin treated cells 1st -4th day, Lanes 5-8, aspirin treated cells 1st-4th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm2/mg protein (B). Values given are the average of intensity measurement of 6 experiments ± SEM. The culture medium collected from control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells at intervals of 24 hrs (normalized to the total cell protein) were quantitated for MMP-9 and MMP-2 protein expression by ELISA using specific antibody (C). Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day (p < 0.05) # Statistically significant compared to controls (p < 0.05).
Figure 4
Effect of supplementation of curcumin in presence of serum on the production and activities of MMP-2 and MMP-9: (A) Equivalent aliquots of the medium appropriately diluted (1:10)from the culture of FCS control and curcumin +FCS treated cells on days 1 to 4 were normalized to the total cell protein were subjected to zymography as described in the text. (a) Lane1-4 curcumin + FCS treated cells 1st -4th day, Lane 5-9 control 1st-4th day. The activity of MMP-9(b) and MMP-2(c) expressed in arbitrary units of intensity/mm2/mg protein. Values given are the average of intensity measurement of 6 experiments ± SEM. (B) ELISA The culture medium collected from controls and curcumin treated cells in presence of serum at intervals of 24 hrs which were normalized to the total cell protein were quantitated for MMP-9(d) and MMP-2(e) protein expression by ELISA using specific antibody. Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant when compared to their production on first day p < 0.05. # Statistically significant when compared to control p < 0.05.
Figure 5
Production of VE-cadherin in HUVECs (A) RT-PCR: Cells were maintained in culture in MCDB 131 medium for different time intervals. Total RNA was isolated and subjected to RT-PCR as described in the text. Intensity of bands were quantitated and normalized with the intensity of band for internal control GAPDH and expressed in intensity units/mm2 (B). * Statistically significant when compared to their production on first day p < 0.05. C. Western blotting HUVECs were maintained in culture in MCDB 131 medium for different time intervals. Medium was removed, the cells were harvested and lysed in Laemmeli buffer and protein equivalent amount of the cell lysates were subjected to SDS PAGE (bottom) and western blot (top) analysis with anti VE-cadherin antibody as described in the text. Inset, Lane1-4 represents 1st -4th day. The intensity of the immunoblotted bands were measured and expressed in intensity units/mm2/mg protein. * Statistically significant when compared to the production on first day p < 0.05.
Figure 6
Changes in nuclear and cytosolic distribution of β-catenin in HUVECs: HUVECs were maintained in culture for different time intervals as described in the text. Medium was removed, the cells were harvested, fractionated and nuclear and cytosol were isolated. Protein equivalent amount of the cytosolic and nuclear fractions were subjected to SDS PAGE and western blot analysis with anti β-catenin antibody as described in the text. Lane1-4, 1st -4th day (A). The intensity of the immunoblotted bands were measured and expressed in intensity units/mm2 (B). Percentage distribution of β-catenin in the nucleus and cytosol (C) * Statistically significant when compared to their relative levels in nucleus and cytosol on first two days p < 0.05.
Figure 7
Changes in the production and distribution of VE Cadherin and β-catenin in HUVECs: Effect of curcumin. (A) Production of VE Cadherin-Western blotting: HUVECs were maintained in culture in MCDB 131 medium in presence of curcumin (10 μM) for different time intervals. Medium was removed, the cells were harvested and lysed in Laemmeli buffer and protein equivalent amount of the cell lysates normalized to the total cell protein were subjected to SDS PAGE (bottom) and western blot (top) analysis with anti VE-cadherin antibody as described in the text. Lanes 1-4 represents 1st -4th day (A). The intensity of the immunoblotted bands were measured and expressed in intensity units/mm2/mg protein. Values for control in intensity units/mm2 (Day 1: 1256 ± 145, Day 2: 2678 ± 156, Day 3: 2287 ± 189, Day 4: 4018 ± 178) * Statistically significant when compared to the production on first day p < 0.05. (B) Changes in the distribution of β-catenin in HUVECs HUVECs were maintained in culture in presence of curcumin for (10 μM) for different time intervals as above. The cells were harvested and nuclear and cytosol were isolated. Protein equivalent amount of the cytosolic and nuclear fractions were subjected to SDS PAGE and western blot analysis with anti β-catenin antibody as described in the text. Values for control in intensity units/mm2/mg protein (Nucleus Day 1: 1877 ±, 145 Day 2: 1687 ± 102, Day 3: 987 ± 89, Day 4: 417 ± 96 and Cytosol Day 1: 0, Day 2: 0, Day 3: 887 ± 69, Day 4: 1567 ± 58). Lane1-4 1st -4th day (inset). The intensity of the immunoblotted bands were measured and expressed in intensity units/mm2/mg protein (a). Percentage distribution of β-catenin in the nucleus and cytosol was calculated and expressed in the percentage bar diagram (b). Values for control (Nucleus Day 1: 100, Day 2: 100, Day 3: 52, Day 4: 21 and Cytosol Day 1: 0, Day 2: 0, Day 3: 48, Day 4: 79). * Statistically significant when compared to their relative levels in nucleus and cytosol on first two days p < 0.05.
Figure 8
Phosphorylation of β-catenin. (A) Cell lysates equivalent to same amount of β-catenin, from cells treated with curcumin (10 μM) in the absence and presence of serum for different time intervals were treated with anti-β-catenin antibody. The immunoprecipitated β-catenin was electrophoresed and immunoblotted with anti-Phospho-Tyr and Phospho-Ser antibody (C control, Cu curcumin, FCS (FCS control), CF (Curcumin + FCS). (B) The intensity of bands were quantitated and the extent of phosphorylation was expressed as percentage bar diagram. Values given are average of quarduplicate experiments. * Statistically significant when compared to the relative ratio in FCS control p < 0.05.
Figure 9
Effect of lithium chloride on the activity of MMP-2 and MMP-9. HUVECs were maintained in culture with 10 μM curcumin for 48, 72 and 96 hrs as described in text with and without GSK-3 inhibitor lithium chloride 1 mM. Medium was removed and the activities of MMP-2 and MMP-9 were determined by gelatin zymography. The intensity of MMP-9 and MMP-2 were measured and expressed in intensity units/mm2/mg protein. * Statistically significant when compared to untreated control p < 0.05.
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