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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 18, 2001 as doi:10.1096/fj.00-0651fje. |
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3 integrin/tetraspanin complexes in the angiogenic response induced by angiotensin II1
Servicio de Inmunología y
* Nefrología, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, Spain
3Correspondence: Servicio de Inmunología, Hospital Universitario de la Princesa, C/Diego de León no. 62, 28006 Madrid, Spain. E-mail: fsanchez{at}hlpr.insalud.es
SPECIFIC AIMS
The effect of angiotensin II (Ang II) on endothelial cell (EC) function has important potential implications under physiological conditions and in the pathogenesis of different cardiovascular diseases. In this study, we analyzed the effect of Ang II on the cellular distribution of representative molecules from different EC junctions and their possible consequences on angiogenesis and EC permeability.
PRINCIPAL FINDINGS
1. Ang II affects the subcellular localization of 
3ß1
integrin/tetraspanin complexes at EC lateral junctions through
interaction with AT1 receptor
We first studied the effect of Ang II on the cellular distribution
of representative molecules from different endothelial junctions: ZO-1
(tight junctions), VE-cadherin/ß-catenin (adherens junctions), CD31,
the tetraspanins CD151, CD9, and CD81, and the 3ß1 integrin. In
untreated EC, all these molecules were concentrated at intercellular
junctions. Ang II induced a selective decrease in fluorescence staining
intensity of 3ß1 integrin, CD151, and CD9 at lateral junctions
(Fig. 1A
) whereas ZO-1, VE-cadherin/ß-catenin, CD31, and CD81
remained unchanged. Likewise, focal adhesion structures were not
affected by Ang II, as shown by staining of v and activated ß1
integrins. Furthermore, the cellular morphology and actin cytoskeleton
of EC were not affected by Ang II (Fig. 1A
). Flow cytometry
analysis showed that the expression levels of the molecules studied
were not significantly modified by Ang II, indicating that the changes
observed were the result of molecular redistribution on the EC surface.
Moreover, biochemical analysis showed that the 3ß1/CD151 molecular
association was unaffected by Ang II treatment (Fig. 1B
).
Pretreatment of HUVEC with the selective antagonist Losartan abolished
the Ang II effect on 3ß1, CD9, and CD151 redistribution (Fig. 1A
), indicating this effect was induced through the AT1
receptor.
|
Since intercellular junctions play a critical role in endothelial permeability and leukocyte transmigration, we studied the effect of Ang II on these EC functions. Ang II treatment had no effect on HUVEC monolayer permeability or PMN transmigration levels. These results indicate that the barrier functions of HUVEC monolayers are not affected by Ang II.
2. Angiotensin II increases in vitro angiogenesis
The remarkable and selective effect of Ang II on 3
integrin/tetraspanin complexes suggested to us that it could be related
to an important EC function, such as angiogenesis. We found that in an
in vitro angiogenesis model on Matrigel, Ang II induced a significant
increase in the number of tubes formed by HUVEC (P<0.001;
Fig. 2A
, B
). Similar results were found at 8 and 16 h (Fig. 2B
) and at concentrations ranging from
10-5 to at least 10-8 M
(Fig. 2C
).
|
The expression of the two known Ang II receptors in HUVEC preparations
was assessed by Western blot analysis. As shown in Fig. 2D
,
HUVEC expressed both AT1 and AT2 at least up to the fourth passage. To
determine which receptor mediated the effect of Ang II on angiotube
formation, we used specific pharmacological antagonists. The Ang II
effect was selectively abolished by Losartan, an antagonist of AT1
receptor, but not by PD123,319, the inhibitor of AT2 receptor (Fig. 2D
). Neither antagonist affected the number of tubes formed
when used alone (Fig. 2D
).
3. The 3 integrin has a critical role in the tubulogenesis
induced by Ang II
To assess the involvement of 3ß1/tetraspanin complexes in the
angiogenesis induced by Ang II, Matrigel assays in the presence of
blocking mAbs were performed. As shown in Fig. 2E
, the
VJ1/18 anti-3 integrin mAb abolished the effect of Ang II. This mAb
did not alter the number of tubes formed in basal conditions,
suggesting it is specifically blocking Ang II-mediated effect (Fig. 2E
). Unexpectedly, blocking anti-tetraspanins CD9, CD81, and
CD151 mAb did not exert any significant effect on the angiotube
formation induced by Ang II. Moreover, an anti- 2 integrin mAb had
no effect (Fig. 2E
) regardless of the high expression of
this integrin in HUVECs. No significant effect was exerted by Ang II on
EC adhesion to collagen I, laminin 5, and
fibronectin.;F3>
CONCLUSIONS AND SIGNIFICANCE
Angiotensin II has a key role as a physiological modulator of
vascular tone and is involved in the pathogenesis of different
important conditions, including hypertension and myocardial
hypertrophy. It has also been reported that Ang II promotes
angiogenesis in vivo and is involved in the pathogenesis of diseases
characterized by abnormal angiogenic activity such as diabetic
retinopathy and solid tumor growth. The angiogenic properties of Ang II
have been previously reported in different in vivo models, although
these studies did not analyze the direct effect of this agent on EC.
Our results indicate that the redistribution of 3ß1/tetraspanin
complexes on EC is associated with induction of angiogenesis by Ang II.
Endothelial intercellular adhesion includes tight, adherens, and gap
junctions as well as integrin/tetraspanin complexes. Endothelial cell
junctions are dynamic structures whose composition can be modulated by
different stimuli such as tumor necrosis factor and interferon 
or shear stress. Our data demonstrate for the first time a selective
change in the subcellular distribution of 3ß1 integrin/tetraspanin
molecular complexes induced by Ang II. This effect is exerted without
altering the level of expression or the association of 3ß1 with
tetraspanins. The 3ß1 and CD151 redistribution induced by Ang II
is more evident compared to CD9 and CD81. These results agree with the
association affinities of 3ß1 with different tetraspanins (high
for CD151, mild for CD9, and weak for CD81) and indicate that the
effect of Ang II is primarily exerted on 3ß1.
Blood vessel neoformation requires substantial changes in cell-to-cell
endothelial interactions as well as cell proliferation, migration, and
vasodilatation. It is very likely that the redistribution of
3ß1/tetraspanins induced by Ang II is causally related to these
phenomena. We had previously found that these molecular complexes are
involved in EC migration and wound healing. However, the lack of effect
of anti-tetraspanin mAbs on angiotube formation suggests that these
molecules are not directly involved in Ang II-mediated angiotube
formation in Matrigel.
Angiogenesis plays a central role in different physiological processes and pathological conditions. It has been postulated that Ang II plays additional functions in blood vessels distinct from its classical role as a vasoconstrictor peptide. Thus, Ang II has different effects on EC that are involved in atherogenesis, aneurysm formation, thrombosis, and inflammation. Here we provide evidence that the direct interaction of Ang II with the AT1 receptor of EC increases the number of capillary-like structures formed in an in vitro model of angiogenesis. The effect of Ang II on isolated EC is quantitatively comparable to those observed in in vivo models.
Induction of the synthesis of VEGF by Ang-II on EC has been described.
In other reports, however, no production of VEGF was detected, but a
potentiation of the VEGF-induced angiogenic activity through an
increase of the VEGF receptor KDR/Flk-1 by Ang II. Under our
experimental conditions, no significant levels of VEGF were induced by
Ang II at the times of angiotube formation assay. Likewise, the Ang II
induced redistribution of 3ß1 integrin/tetraspanins is a rapid
event that does not seem to be related to VEGF induction. Finally, the
addition of exogenous VEGF did not increase the angiotube formation
rate in our short-term assay with HUVEC. These data suggest that the
effect of Ang II on tube formation is not mediated through VEGF
up-regulation.
Cell adhesion molecules are key in the maintenance of EC monolayer
integrity and play a fundamental role in angiogenesis. The v
integrins participate in angiogenesis by providing survival signals to
EC. Furthermore, inhibition of 2ß1 and 1ß1 integrins prevents
the VEGF-promoted blood vessels neoformation within a collagen-rich
matrix. Last, it has been reported that both fibronectin and its
receptor integrin 5ß1 directly regulate angiogenesis. Our results
indicate that the 3ß1 integrin has a critical role in the
angiogenic effect of Ang II. However, it has been reported that Ang II
does not have a significant effect on angiotube formation in collagen
matrices. This apparent discrepancy could be explained by differences
in the angiogenesis assays used. Our data agree with the defective
blood vessel formation and perinatal lethality observed in
3-deficient mice. The effect of Ang II was exerted through an
adhesion receptor that shows a discrete expression on EC, a phenomenon
that further underscores the specificity of the observed effect. Hence,
the regulation of angiogenesis by Ang II involves subtle cellular
changes that lead to an overall enhancement in the formation of tubular
structures. In this regard, it has been reported that the 3ß1
integrin modulates the function of other integrins.
The clinical implications of our findings must be elucidated through future studies. However, it is evident that the pharmacologic modulation of Ang II effects on EC may have a great therapeutic potential to enhance or block them in conditions characterized by ischemia and pathological angiogenesis, respectively.
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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.00-0651fje ; to cite this
article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0651fje 
2 C.D.-J. and M.Y.-M contributed equally to this article.
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