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First published online 27 February 2007
doi: 10.1242/jcs.002212

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The ADMA/DDAH pathway is a critical regulator of endothelial cell motility

¹ BHF Laboratories, Department of Medicine, University College London, 5 University Street, London, WC1 E6JJ, UK
² Institut of Cardiovascular Physiology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany

View larger version (81K): [in this window] [in a new window]   Fig. 1. ADMA induces formation of stress fibres and focal adhesions in PAECs and inhibits cell motility. (A-D) Control untreated cells; (E-H) cells treated with 100 µM SDMA; (I-L) cells treated with 100 µM ADMA for 24 hours. (A,E,I) Phase contrast microscopy images showing sparsely growing PAECs. (B,C,F,G,J,K) Fluorescent images showing staining for F-actin and a focal adhesions component vinculin. (D,H,L) Representative images of cell trajectories. Bar, 100 µm (A,E,I), 10 µm (B,C,F,G,J,K) and 130 µm (D,H,L). (M) Changes in the mean speed of cell movement in untreated cells, cells treated with SDMA (100 µM), ADMA (100 µM) or the cells grown in arginine-free medium with or without ADMA. (N) Total cell translocation defined as a distance between starting point and an end point of each trajectory. *P<0.05; **P<0.01 (comparison with untreated controls).

View larger version (60K): [in this window] [in a new window]   Fig. 2. Overexpression of DDAHI and DDAHII prevents ADMA-induced increase in stress fibre and focal adhesion formation and restores normal motility in ADMA-treated cells. (A-E) Cells incubated with ADMA and infected with control adenoviruses to express AdGFP. (F-O) Cells treated with ADMA and overexpressed AdDDAHI (F-J) and AdDDAHII (K-O). (P-T) Cells overexpressing AdDDAHI without the addition of ADMA. In A-T the columns from the left to right show F-actin distribution, vinculin distribution, the cells overexpressing recombinant proteins, composite images and cell trajectories, as indicated. In the composite images F-actin is red, vinculin is green and the cells overexpressing GFP are blue (pseudocolour). Bar, 10 µm (180 µm in E, J, O, T). (U) Changes in the cell speed; (W) changes in total cell translocation in non-treated cells and cells overexpressing AdGFP, AdDDAHI, AdDDAHII. The cells were left untreated or were treated with 100 µM of ADMA or SDMA, as indicated. *P<0.05; **P<0.01, comparison with untreated controls. (Bottom left) The two images are an example of fluorescently labelled cells expressing recombinant DDAHII that were used for cell tracking. Only overexpressing cells (96% of total cell population) were tracked.

View larger version (38K): [in this window] [in a new window]   Fig. 3. ADMA but not SDMA activates RhoA and inhibits Rac1 and Cdc42 in PAECs. The three graphs at the top show changes in the activity of RhoA, Rac1 and Cdc42 following 4 or 24 hours incubation with the drugs. Representative examples of western blots illustrating changes in Rho GTPases activity upon treatment with 100 µM ADMA or SDMA are shown below. The two graphs at the bottom show concentration-dependent changes in RhoA, Rac1 and Cdc42 activities in cells treated with SDMA or ADMA for 24 hours. *P<0.05; **P<0.01 (comparison with untreated controls).

View larger version (97K): [in this window] [in a new window]   Fig. 4. Inhibition of RhoA and Rho kinase prevents the effects of ADMA on the actin cytoskeleton and focal adhesions, and restores cell speed and translocation to control values. Adenoviral gene transfer was used to induce expression of GFP (A-C), inhibitory mutant of RhoA, N19RhoA (D-F), activated Rac1, V12Rac1 (J-L) GFP-tagged activated Cdc42, L61Cdc42 was introduced by transfection (M-O). The cells in G-I were treated with the Rho kinase inhibitor, Y-27632 (10 µM) for 2 hours. The cells were stained for F-actin (left column), vinculin (middle column) and Myc (right column) to identify overexpressing cells. Bar, 15 µm. (P) Cell speed in ADMA-treated PAECs. (Q) The effect of mutant Rho GTPases on cell translocation. Cell cytoskeleton and motility were studied 24 hours after adenoviral infection. **P<0.01 (comparison with untreated controls); &&P<0.01 and &P<0.05 (comparison with ADMA-treated cells).

View larger version (120K): [in this window] [in a new window]   Fig. 5. The effects SNAP and L-NAME on the actin cytoskeleton, focal adhesion formation and cell movement in PAECs. (A-C) Cells incubated with 10 µM SNAP for 4 hours; (D-F) cells incubated with ADMA (100 µM) and SNAP (10 µM) for 24 hours; (G-I) cells incubated with L-NAME for 24 hours. The columns from left to right show F-actin distribution in cells, vinculin distribution and cell trajectories, as indicated. Bars, 15 µm (A,B,D,E,G,H) and 170 µm (C,F,I).

View larger version (53K): [in this window] [in a new window]   Fig. 6. The effects of SNAP and L-NAME on cell speed (A), translocation (B) and nitrite levels (C). SNAP (10 µM) and L-NAME (1 mM) were added to untreated cells or cells treated with ADMA and incubated for 24 hours. Nitrite levels in culture medium were measured using a standard Griess assay. In (D) two sets of corresponding images show localisation of endogenous DDAHI in normal PAECs. The cells were stained for F-actin (left) and DDAHI (right). White arrows point to colocalisation of F-actin and DDAHI in membrane ruffles and lamellipodium. Bar, 8 µm. (E) shows expression of DDAHI in untreated PAECs (western blot on the left). Differences in expression levels between DDAHI and DDAHII in PAECs are also shown in a graph and a corresponding western blot on the right.

View larger version (35K): [in this window] [in a new window]   Fig. 7. The effect of L-NAME and SNAP on the activity of RhoA. (A,B) Graphs show RhoA activity in cells treated with (A) L-NAME (0.5 mM and 1 mM) for 24 hours, or (B) treated with L-NAME (1 mM) for 4 and 24 hours. (C) Graph shows time-dependent changes in RhoA activity in cells treated with SNAP (10 µM). (D) Graph shows changes in Ser188 phosphorylation of RhoA in cells treated with ADMA (100 µM), SDMA (100 µM), SNAP (10 µM) or SNAP and ADMA. ADMA and SDMA were added to the cells for 24 hours whereas SNAP was added for 4 hours. Representative examples of western blots are shown below the graphs. Values in A and C are means of three experiments whereas values in B are means of two experiments. *P<0.05, **P<0.01 (comparison with untreated controls).

View larger version (39K): [in this window] [in a new window]   Fig. 8. The effects of overexpression of non-phosphorylatable mutant of RhoA, Ala188RhoA in PAECs. The cells were stained for F-actin (A,D), vinculin (B,E) and haemagglutinin (C,F) 24 hours after transfection. Bar, 10 µm. (G) Western blot of control cells and cells overexpressing Ala188RhoA-HA. The blot was probed for RhoA and haemagglutinin (HA), as indicated. (H,I) Cell speed and translocation in control, untreated cells, cells treated with 100 µM ADMA, cells transfected with an empty vector (transfection control) and cells transfected with Ala188RhoA. **P<0.01 (comparison with untreated controls).

View larger version (57K): [in this window] [in a new window]   Fig. 9. The effects of the PKG activator, Br-cGMP, and PKG inhibitor, Rp-cGMPS, on untreated and ADMA-treated PAECs. (A-H) The effects of Br-cGMP and Rp-cGMPS on F-actin and vinculin distribution, in untreated cells and ADMA-treated cells, as indicated. Bar, 10 µm. (I,J) The effects of the drugs on cell speed and translocation, respectively. (K) Changes in RhoA activity in cells treated with ADMA (100 µM), ADMA (100 µM) and Br-cGMP (500 µM), Br-cGMP (500 µM) alone or Rp-cGMPS (100 nM). A representative example of a western blot is shown beside the graph. In A-H and K, ADMA was added to the cells for 20 hours while Br-cGMP and the Rp-cGMPS were added for 2 hours. In motility assays (I,J) all drugs were added to the cells at the same time and incubated for 24 hours. Values in I,J are means of three experiments whereas values in K are means of two experiments. **P<0.01 (comparison with untreated controls). &P<0.05 and &&P<0.01 (comparison with ADMA-treated cells).

View larger version (44K): [in this window] [in a new window]   Fig. 10. Pulmonary microvascular endothelial cells from DDAHI heterozygous knockout mice (HT) show phenotypical and functional differences from cells taken from their wild-type (WT) littermates. (A,B) Phase contrast microscopy images; (C,D) fluorescent confocal images of F-actin in WT and HT cells, as indicated. (E,F) Cell trajectories, 24 hour recording. Bar, 50 µm (A,B), 20 µm (C,D) and 120 µm (E,F). (G) Changes in nitrite levels in culture medium of cells from wild-type (WT) mice, heterozygous DDAHI knockout (HT) mice, and cells from WT mice treated with ADMA (100 µM); (H,I) changes in speed and translocation of these cells. (J) Graphs and western blots showing activities of RhoA, Rac1 and Cdc42 in cultured PMVECs taken from WT and HT mice.**P<0.01; *P<0.05 (comparison with WT controls).

View larger version (19K): [in this window] [in a new window]   Fig. 11. Microvessel outgrowth from aortic rings from wild-type (WT) and heterozygous DDAHI knockout (HT) mice. The rings were left untreated or were treated with ADMA (100 µM) or SDMA (100 µM) in culture. Microvessel outgrowth was studied 6-7 days after plating. Bar, 500 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 12. Proposed signalling pathway of asymmetric methylarginine (ADMA). ADMA inhibits NO production in PAECs. This contributes to a decrease in RhoA phosphorylation on Ser188 which facilitates it membrane localisation and activation. Phosphorylation of RhoA on Ser188 is likely to be carried out by protein kinase G (PKG) but other kinases, such as protein kinase A (PKA) may also be involved. Activation of RhoA and its downstream effector, Rho kinase inhibits endothelial motility, possibly by increasing cell adhesion to the substratum or slowing down turnover of focal adhesions. Impairment of endothelial motility inhibits angiogenic responses of endothelial cells. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) improve persistence of cell movement and prevent the effects of ADMA on cells.