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Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel

Karina Laflamme^*, Charles J. Roberge^*, Guillaume Grenier^*, Murielle Rémy-Zolghadri^*, Stéphanie Pouliot^*, Kathleen Baker^*, Raymond Labbé^*, Pédro D’Orléans-Juste, François A. Auger^* and Lucie Germain^*,1

^* Laboratoire d’Organogénèse Expérimentale/LOEX, Hôpital Saint-Sacrement du Centre Hospitalier Affilié Universitaire de Quebec and Department of Surgery, Laval University, Québec, Canada; and

Institute of Pharmacology, Medical School, Sherbrooke University, Sherbrooke, Quebec, Canada

¹Correspondence: Laboratoire d’Organogénése Expérimentale Hôpital du Saint-Sacrament du CHA, 1050 Chemin Ste-Foy, Québec, QC G1S 4L8, Canada. E-mail: lucie.germain{at}chg.ulaval.ca

SPECIFIC AIM

It is well accepted that the media of a blood vessel is responsible for the vasomotor tone control by contracting and relaxing in response to different hormonal factors released. The adventitia, on the other hand, has long been thought to mainly serve as a structural support for the media, its main contribution to vascular compliance being controlled by autonomous perivascular innervation. Interestingly, recent studies suggest that the adventitia influences vascular function. Nonetheless, whether the adventitia can directly participate in the regulation of vasomotor tone of blood vessels still remains to be demonstrated. The lack of appropriate technical procedures to separate the adventitia tunica from the other components of a native blood vessel (stripping) have prevented direct investigations on the possible role of that layer in the regulation of vasomotor tone.

We have recently developed, using the self-assembly technique, a human tissue- engineered blood vessel (TEBV) produced in vitro and composed of the three tunicae found in a native blood vessel. In the present study, we took advantage of the self-assembly method to produce in vitro 3 independent vascular constructs from vascular smooth muscle cells (VSMCs) and vascular fibroblasts (VFs) derived from the media and adventitia, respectively, of the same human saphenous vein biopsy. The first vascular construct was composed of only an adventitia (TEVA), a second vascular construct contained only a media (TEVM), and the third contained a media and an adventitia (TEVMA).

These three vascular models (TEVA, TEVM, and TEVMA) were reconstructed in vitro from cultured cells to investigate the role of the adventitia in the modulation of vascular tone by comparing the response of each of these vascular constructs to endothelin (ET), the most powerful vasopressor agent known to date. Studies in humans have demonstrated the importance of ET in the maintenance of vascular tone and blood pressure. Three endogenous isoforms of ET have been discovered, endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). ET binds two different receptor subtypes: endothelin A (ETA) receptors, which have a higher affinity for ET-1 and ET-2 than ET-3, and endothelin B (ETB) receptors, which have equal affinity for ET-1, ET-2, and ET-3.

The specific aim of this study was to investigate the role of the adventitia in vasoconstriction using endothelin and our different vascular constructs. The ET receptors (ETA and ETB) implicated in the observed responses to the peptides were also investigated in our three different vascular constructs.

PRINCIPAL FINDINGS

The present work describes four novel findings by using well-characterized in vitro vascular tissue-engineered pharmacological models that among other phenomena unmask the contractile properties of the adventitia. In the present study, we show that: 1) the adventitia (TEVA) has the capacity of contracting to endothelin, 2) there is a heterogeneity in the response of the different vascular constructs tested to endothelin, 3) the adventitia (TEVA) expresses only the ET_A receptor, and 4) the adventitia (TEVA) has the capacity of vasodilating in response to a relaxing agent such as sodium nitroprusside (SNP).

1. The tissue-engineered vascular adventitia contracts to endothelin
We took advantage of in vitro models developed by the self-assembly approach of tissue engineering to produce three independent vascular constructs: 1) only an adventitia (TEVA), 2) only a media (TEVM), and 3) a media and an adventitia (TEVMA). These constructs were produced from vascular smooth muscle cells (VSMCs) and vascular fibroblasts (VFs) that were derived by culturing explants of the media and adventitia of the same human saphenous vein biopsy and amplified in vitro.

Classical vasoconstriction studies were performed to investigate and compare the effect of endothelins (ET-1, ET-2, and ET-3) on the various tissue constructs (TEVA, TEVM, and TEVMA). Tissue rings from the various vascular constructs were placed in separate isolated organ baths and increasing concentrations of each of the various agonists were added. TEVMAs dose dependently contracted to ET-1, ET-2, or ET-3. The maximal contractile responses were 192%, 190%, and 193%, respectively (Fig. 1 ). When taken separately, both the media (TEVM) and the adventitia (TEVA) component of the TEVMA also contracted to endothelin.

View larger version (10K): [in this window] [in a new window]   Figure 1. Vasocontractile responses to endothelin showing the presence of different receptors on the various vascular constructs. Tissue-engineered vascular media (TEVM) composed of a four-layer-rolled sheet of vascular smooth muscle cells (VSMCs), Tissue-engineered vascular adventitia (TEVA) composed of a four-layer-rolled sheet of fibroblast cells, and tissue-engineered vascular media + adventitia (TEVMA) composed of a rolled-sheet comprising two layers of VSMCs and two layers of fibroblasts cells. Cumulative concentration–effect curves of ET-1 (A), ET-2 (B) and ET-3 (C) producing contractions (% maximal response, Emax obtained with 3 mM ATP). Albeit maximal response was not reached, Emax for ET-3 was set from the responses of TEVM and TEVMA to the highest dose (10–6M) of the agonist used in the present study. Data points are the mean results of five separate rings from different TEVM, TEVA, or TEVMA constructs. Vertical bars represent SEM. *P < 0.01, significantly different from control using Student’s unpaired t test.

2. Heterogeneity in the response of vascular constructs to endothelin
Heterogeneity in the contractile response to endothelin was observed for the different constructs tested. ET-1 and ET-2 were found to induce a similar dose-dependent contractile response in TEVMs and TEVAs (Fig. 1A, B ). However, the maximal responses were different 241 and 236% for TEVMs and 108 and 132%, for TEVAs for ET-1 and ET-2, respectively. In contrast, ET-3 induced a contraction of the TEVMs but failed to affect the tone of the TEVAs (Fig. 1C ). Taken together, these results show that the reconstructed adventitia displays a vasoconstrictor response to ET-1 and ET-2, but not to ET-3.

Contraction experiments in the presence of specific ET_A or ET_B receptor antagonists were performed. The results indicated that ET-1 induced contraction occurred via the ET_A receptor in the TEVA, but via ET_A and ET_B receptors in TEVM and TEVMA. In addition, ET-3 induced contraction in the TEVM and TEVMA via interaction with the ET_B receptor (data not shown).

3. The tissue-engineered vascular adventitia presents vasodilation properties
To evaluate the relaxing potential of the TEVA, the effect of SNP, a pharmacological agent widely used in relaxing studies of blood vessels as it forms NO in solution, was tested. A dose-dependent relaxation was observed in TEVMA, TEVM, and TEVA in response to SNP (Fig. 2 ), which reduced vascular tone by close to 80% in the three reconstructed vascular models.

View larger version (10K): [in this window] [in a new window]   Figure 2. Effect of sodium nitroprusside (SNP) on the different vascular constructs. Relaxation is expressed as the % of a previous contraction to 1 µM U46619. Vertical bars represent SEM. *P < 0.01, significantly different from control using Student’s unpaired t test.

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrated for the first time that a tissue-engineered vascular adventitia reconstructed with vascular fibroblasts derived from human adventitia has the capacity of contracting and relaxing. Endothelin plays a major role in the control of peripheral blood resistance in humans. The vast majority of blood vessels have been demonstrated to respond to endothelin. These types of studies, however, did not pinpoint whether the adventitia, in addition to the media, could also be involved in the endothelin-induced response. The indirect evidence on a potential role of the adventitia in vasoconstriction was based on the decreased contraction obtained after the removal of the adventitia from native blood vessels. However, the stripping method used in most of these studies to physically remove the adventitia may have induced tissue damage to the media resulting in a decrease of its response. These separation methods did not allow the preservation of an isolated adventitia for vasocontractile experiments to study directly whether the adventitia component of the blood vessel can participate in the regulation of blood vessel tone.

To our knowledge, this is the first direct demonstration that the adventitia has the capacity of contracting and relaxing in response to known vasoactive and vasodilating agents. The results of the present study suggest that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone. The involvement of the adventitia in regulation of vasomotor tone process may require the development of strategies allowing for the administration of potentially active compounds not only targeting the media but also the outer layer of the vessel wall. Moreover, our tissue-engineered vascular adventitia could also serve as a new model to study the vascular function of the adventitia in various pathologies.

View larger version (33K): [in this window] [in a new window]   Figure 3. Schematic representation of the experimental strategy we used to show that in addition to the media, the adventitia of the blood vessel also presents a vasoactive response. VSMCs and vascular fibroblasts (VFs) were isolated and amplified from human saphenous vein. After 10 to 15 days of culture in appropriate medium supplemented with sodium ascorbate to stimulate extracellular matrix (ECM) synthesis, cells formed thick living tissue sheets, comprising cells embedded in the ECM they secreted, that could be peeled off from the culture flask using fine forceps. TEVM and TEVA constructs were obtained by wrapping a tissue sheet of VSMCs or VFs around a tubular support (inside diameter = three mm) in order to obtain a four-layer thick construct of VSMCs or VFs, respectively. For the TEVMA constructs, a sheet was wrapped over the tubular support with two layers of VSMCs and two layers of VFs to obtain a construct that had the same number of layers (four) as the TEVM and the TEVA constructs. After a week of maturation, each construct was cut into 5-mm-long rings, while remaining on the tubular support. These rings were further cultured for two weeks. For vasoconstriction studies, rings of TEVM, TEVA, and TEVMA were removed from their support, rinsed in physiological salt solution (Kreb’s solution) and mounted in isolated organ baths containing Kreb’s solution maintained at 37°C and gassed with a mixture of 95% O2, 5% CO2 (pH 7.4). After three rinses and the return to a baseline tension, tissues were challenged with different concentrations of endothelins added in the bath. The response of the constructs is indicated. (The authors are thankful to Danielle Larouche, LOEX, for this picture.)

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4702fje

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