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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 30, 2002 as doi:10.1096/fj.01-0563fje. |
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2,3
,3
* Max Planck Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Bad Nauheim, Germany;
University of Amsterdam, Academic Medical Center, Department of Cardiology, Amsterdam, the Netherlands;
University of Freiburg, Department of Cardiology and Angiology, Research Group for Experimental and Clinical Arteriogenesis, Freiburg, Germany; and
|| Clinic for Tumor Biology, Angiogenesis Research Center, Freiburg, Germany
2Correspondence: Department of Cardiology, Room B2–250, Academic Medical Center, University of Amsterdam, Meibergdreef 9 1105 AZ, Amsterdam, the Netherlands. E-mail: n.vanroyen{at}amc.uva.nl
SPECIFIC AIMS
The role of monocytes/macrophages during arteriogenesis became evident in recent years and transforming growth factor beta 1 (TGF-ß1) was reported to be chemoattractive for circulating monocytes and to induce the expression of various growth factors by these cells. However, the in vivo effects of exogenously applied TGF-ß1 on arteriogenesis as well as the influence of TGF-ß1 on monocyte adhesion and their subsequent migration (decisive steps in the process of arteriogenesis) are unclear. Our aim was to study 1) the expression of TGF-ß1 in a model of arteriogenesis in the rabbit hind limb, 2) the effects of exogenously applied TGF-ß1 on the process of arteriogenesis, and 3) the effects of TGF-ß1 on monocyte adhesion and trans-endothelial migration.
PRINCIPAL FINDINGS
1. Expression of TGF-ß1 is increased around growing collateral arteries in a specific model of arteriogenesis
Using Western blotting, three different bands were recognized by the TGF-ß1 antibody in collateral vessels of experimental and sham-operated animals. The 55 kDa band, which represents the monomer of pro-TGF-ß1, did not change after occlusion. The 25 kDa band, corresponding to the mature form of TGF-ß1, was significantly up-regulated around growing collateral arteries vs. vessels from sham-operated animals. (25 kDa: 131.04±20.48 vs. 166.56±12.70, P<0.05; 50 kDa: 166.72±58.23 vs. 205.08±44.98, P=ns; 55kDa: 173.60±15.89 vs. 188.34±4.68, P=ns; arbitrary units).
2. TGF-ß1 stimulates arteriogenesis when applied exogenously
In vivo effects of exogenously applied TGF-ß1 on collateral artery growth were determined in a model of arteriogenesis in rabbits.
Three days after femoral artery ligation, nuclei of arterial wall cells stained positive for the proliferation marker Ki-67 in control and TGF-ß1-treated animals. The proliferation index was significantly higher in TGF-ß1-treated animals vs. control animals. Double staining with Ki-67 and alpha-smooth muscle antibody showed a significantly higher proliferation of smooth muscle cells in TGF-ß1-treated animals. In control sections from nonligated limbs, no Ki-67 positive nuclei were found in the vessel wall. Angiograms performed 1 wk after ligation of the femoral artery showed several typically corkscrewed, collateral arteries spanning from the arteria profunda femoris and the arteria circumflexa femoris to the arteria genualis and the arteria saphena parva. TGF-ß1 infusion for a 1 wk significantly increased the number of visible collateral arteries vs. the PBS control group (15.2±3.4, TGF-ß1; 24.6±4.1, P<0.05).
Collateral conductance as measured with fluorescent microspheres was 4.1 ± 0.5 ml/min/100 mmHg in the control group. TGF-ß1 significantly increased collateral conductance to > sevenfold compared with the PBS-treated group (28.9±3.7 ml/min/100 mmHg) (Fig. 1
). A conductance value of 161.5 ± 10.8 ml/min/100 mmHg was measured in the nonoccluded control group.
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3. TGF-ß1 up-regulates monocytic MAC-1 expression and stimulates monocyte adhesion and transmigration
Strongly increased adhesion to a HUVEC layer was observed after stimulation of monocytes with TGF-ß1. Adhesion of monocytes was linearly related to TGF-ß1 dose. Using FACS analysis, we found that the expression of the MAC-1 receptor increased significantly (dose dependently) after stimulation of human monocytes with TGF-ß1. A similar dose-dependent response of MAC-1 expression (CD11b/CD18) upon TGF-ß1 stimulation was found in isolated rabbit monocytes.
TGF-ß1 showed no chemoattractive potency toward monocytes in the trans-endothelial migration assays. When TGF-ß1 was diluted at concentrations ranging from 0.001 to 100 ng/ml into the lower chamber of the assay, the migration of monocytes did not differ significantly from the control assay and was significantly lower than MCP-1.
When the HUVEC-layer was stimulated with TGF-ß1, no increase in the number of transmigrated cells was observed. However, when monocytes were prestimulated with TGF-ß1, an increased trans-endothelial migration of monocytes was seen compared with the control group. When monocytes and endothelium were stimulated simultaneously with TGF-ß1, the transmigration rate was similar to that after monocyte stimulation alone. Maximum migration of monocytes was achieved when MCP-1 was added to the lower chamber of the transmigration assay along with TGF-ß1 stimulation of monocytes.
4. TGF-ß1 has no direct effect on sprouting angiogenesis and inhibits proangiogenic activity of VEGF
In the in vivo model, no increase in capillary number was found after TGF-ß1 treatment compared with control animals. EC spheroids have a low baseline sprouting activity that can be strongly stimulated by exogenous VEGF (50 ng/ml). There was no significant sprouting originating from TGF-ß1 pretreated spheroids (24 h, 5 ng/ml) or after stimulation with TGF-ß1 (0.1–10 ng/ml). TGF-ß1 pretreated spheroids show significant inhibition of VEGF-mediated sprouting (Fig. 2
).
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CONCLUSIONS
With Western blot analysis we show that theexpression of the mature form of TGF-ß1 is increased around growing collateral arteries. This TGF-ß1 expression during arteriogenesis is not dependent on the presence of ischemia (growing collaterals are surrounded by normoxic tissue and are perfused by arterial blood) but may be dependent on increased shear stress.
Upon exogenous intravascular application, TGF-ß1 was taken up and stored in the perivascular space, especially near zones of high proliferation of vascular smooth muscle cells as well as other endoluminal and perivascular cells. The labeling index of smooth muscle cells was significantly higher in collateral vessels of treated than in untreated animals. X-ray angiograms of TGF-ß1-treated animals showed a higher number of visible collateral vessels (i.e., vessels with a diameter >50 µm). TGF-ß1 increased the conductance of the collateral circulation by 
sevenfold 1 wk after femoral artery ligation. Conductance represents the maximal capacity of the collateral circulation and is the most direct quantitative measurement of collateral artery growth.
Wiseman reported maximal chemoattractive activity at concentrations ranging from 0.1 to 1 pg/ml and monocyte activation at concentrations of 10–100 pg/ml. These data are confirmed in part by our own experiments, which showed monocyte activation (increased adhesion) at higher doses. However, the reported chemotactic activity of TGF-ß1 toward monocytes was not reproducible over a layer of endothelial cells in a wide range of dosages. The chemoattractive activity of TGF-ß1 was reported from experiments with regular chemotaxis chambers without an endothelial layer. This suggests that factors other than chemoattractive activity alone are decisive for the number of transmigrated cells in the trans-endothelial migration assay.
A strong increase in adhesion to endothelial cells was observed for TGF-ß1-stimulated monocytes, which led to an increase in the number of trans-endothelial migrated monocytes. FACS analysis showed that TGF-ß1 led to an increase in monocytic adhesion receptor MAC-1 expression, which may have caused the increased adhesion and transmigration. Proof for MAC-1 involvement in arteriogenesis will be provided by studies using specific MAC-1 antibodies and MAC-1 knockout mice. Activation of monocytes with TGF-ß1 leads not only to increased adhesion and transmigration of monocytes, but also to enhanced expression of growth factors and cytokines like b-FGF, platelet-derived growth factor (PDGF), TNF-
, IL-1, and IL-6.
Several studies reported anti-angiogenic properties of TGF-ß1 and overexpression of TGF-ß1 after direct arterial gene transfer did not show an increase in angiogenesis. This was confirmed by our own data showing no stimulation of angiogenesis by TGF-ß1 in either the in vivo or the spheroid model and an inhibition of the proangiogenic effects of VEGF. This inhibitory effect of TGF-ß1 on the angiogenic potency of VEGF was described earlier by Pepper et al.
In our in vivo setting of arteriogenesis, inhibition of endothelial cell proliferation is probably compensated for by the mechanisms of increased monocyte activation, adhesion, and transmigration and the subsequent release of vascular growth factors. A second explanation for the seemingly paradoxical arteriogenic effect of TGF-ß1 is the fact that the process of arteriogenesis, in contrast to angiogenesis, consists mainly of proliferation of smooth muscle cells. Therefore, it is of notice that TGF-ß1 induces vascular smooth muscle cell proliferation via PDGF. This was also confirmed by our histological data, showing an increased proliferation index for smooth muscle cells in TGF-ß1-treated animals.
To our knowledge, the present report describes for the first time TGF-ß1 as a specific proarteriogenic substance. The number of collateral arteries on the X-ray angiograms as well as conductance of the collateral vessels showed a significant increase upon TGF-ß1 treatment. Others have reported that TGF-ß1 induces the expression of several growth factors by monocytes/macrophages. This explains the observed arteriogenic effects of TGF-ß1. As another mechanistic explanation for the arteriogenic effects of TGF-ß1, we show that TGF-ß1 induces MAC-1 receptor expression and increases monocytic adhesion and trans-endothelial migration, necessary steps during arteriogenesis. Involvement of the MAC-1 receptor in arteriogenesis needs to be further clarified in studies using MAC-1 antibodies and MAC-1 knockout mice.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0563fje; to cite this article, use FASEB J. (January 30, 2002) 10.1096/fj.01-0563fje 
3 Both authors contributed equally to the study.
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