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Published as doi: 10.1096/fj.05-4702fje.
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(The FASEB Journal. 2006;20:1245-1247.)
© 2006 FASEB

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{dagger}, François A. Auger* and Lucie Germain*,1

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

{dagger} Institute of Pharmacology, Medical School, Sherbrooke University, Sherbrooke, Canada

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

ABSTRACT

Whether the adventitia component of blood vessels directly participates in the regulation of vascular tone remains to be demonstrated. We have recently developed a human tissue-engineered blood vessel comprising the three tunicae of a native blood vessel using the self-assembly approach. To investigate the role of the adventitia in the modulation of vascular tone, this tissue-engineering method was used to produce three vascular constructs from cells explanted and proliferated from donor vessel tunicae 1) an adventitia + a media, or only 2) an adventitia, or 3) a media. The vasoconstriction responses of these 3 constructs to endothelin, the most potent vasopressor known up-to-date, as well as to nonselective and selective agonists and antagonists, were compared. The adventitia contracted to endothelin-1, -2, whereas the media and the media+adventitia contracted to all three endothelins. Endothelin-induced contraction of the adventitia was dependent on ETA receptors, whereas that of the media and the adventitia+media was ETA and ETB receptor-dependent. RT-PCR studies corroborated these results. SNP induced a dose-dependent relaxation of the three tissue constructs. We also demonstrated that the endothelin-converting enzyme, responsible for the formation of the active endothelin peptides, was present and functional in the adventitia. In conclusion, this is the first direct demonstration that the adventitia has the capacity to contract and relax in response to vasoactive factors. The present study suggests that the adventitia of a blood vessel could play a greater role than expected in the modulation of blood vessel tone.—Laflamme, K., Roberge, C. J., Grenier, G., Rémy-Zolghadri, M., Pouliot, S., Baker, K., Labbé, R., D’Orléans-Juste, P., Auger, F. A., Germain, L. Adventitia contribution in vascular tone: insights from adventitia-derived cells in a tissue-engineered human blood vessel.


Key Words: adventitia • contraction • media • endothelin • blood vessel

THE WALL OF A BLOOD VESSEL is composed of three tunicae: intima, media, and adventitia (1) . The innermost tunica, known as the intima, includes a single layer of endothelial cells lining the vessel lumen and the internal elastic lamina membrane. The middle tunica, termed media, is mainly composed of vascular smooth muscle cells (VSMCs) in an extracellular matrix (ECM) and corresponds to the muscular portion of the blood vessel, whereas the tunica adventitia is mainly composed of vascular fibroblasts (VFs) and ECM.

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, for example, by the endothelial cells of the intima (2) . 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 (1) . Interestingly, recent studies suggest that the adventitia influences vascular function (3 4 5 6 7) . 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) has prevented direct investigations on the possible role of that layer in the regulation of vasomotor tone. For example, the stripping method used in these procedures can result in the injury of the media tunica and does not permit us to obtain functional adventitia isolated from a native blood vessel (6) .

We have recently developed, using the self-assembly technique, a human tissue-engineered blood vessel (TEBV) composed of the layers representing the three tunicae found in a native blood vessel (8) . In the present study, we took advantage of the self-assembly method to produce three independent vascular constructs from amplified VSMCs and VFs isolated from 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 to investigate the role of the adventitia in the modulation of vascular tone by comparing each of these vascular construct responses to endothelin, the most powerful vasopressor agent known to date (9) . Studies in humans have demonstrated the importance of endothelin in the maintenance of vascular tone (10) and blood pressure (11) . Three endogenous isoforms of endothelin have been discovered, endothelin-1 (ET-1), endothelin-2 (ET-2) and endothelin-3 (ET-3) (12) . 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 (13) . The endothelin receptors (ETA and ETB) implicated in the observed responses to the peptide were also investigated in our three different vascular constructs.

In the present study, all of the vascular constructs tested responded to endothelin, although a heterogeneity in the response was observed. Indeed, all three vascular constructs tested contracted to ET-1 and ET-2, but only TEVMA and TEVM responded to ET-3. Furthermore, endothelin-induced contraction of TEVA was found to be dependent on the presence of ETA receptors, while both ETA and ETB receptors were present and functional on TEVMA and TEVM. Finally, the three types of vascular constructs tested also had the capacity of vasodilating in response to a relaxing agent such as sodium nitroprusside (SNP). Our results show that the adventitia may play a greater role than expected in the maintenance of vascular tone and compliance.

MATERIALS AND METHODS

Tissue culture
The study was approved by the CHA institutional review committee for the protection of human subjects. Tissues were obtained after informed consent was given. Human vessel was obtained from adult volunteer during the surgical saphenous vein-stripping procedure. Once collected, vein biopsy was put in a transport medium and processed immediately. Vascular smooth muscle cells (VSMCs) and vascular fibroblasts (VFs) were isolated as described previously (14) . This new isolation technique enables us to isolate from the same small biopsy sample all of the cell types necessary for the reconstruction of our vascular constructs (VSMCs and VFs) (14) .

Veins were kept at room temperature in transport medium: Dulbecco-Vogt modification of Eagle medium with Ham’s F12 (DMEM-Ham; ratio 3:1) (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% fetal calf serum (FCS) (HyClone, Logan, UT) containing antibiotics, penicillin (100 U/ml) (Sigma, Oakville, Ontario, Canada), and gentamicin (25 µg/ml) (Schering, Pointe-Claire, Canada). The veins were washed in PBS, opened longitudinally, and pinned to a wooden dissection board with the luminal surface facing upward. Endothelial cells were gently scraped off the tissue with a #15 scalpel blade (Bard-Parker) taking care not to damage the subendothelial layer. The veins were then cleaned with sterile gauze soaked in PBS to remove any remaining endothelial cells. Fragments of the thin underlying media layer were then collected with sterile tweezers, washed three times with 10 ml of culture media, cut into smaller pieces using a scalpel blade, and carefully placed in a 35-mm gelatin-coated and prewetted petri dish so as to allow their attachment to the plastic. To facilitate the adhesion of explants onto plastic, a small vol of DMEM-Ham (3:1) supplemented with 30% FCS (HyClone), 20 µg/ml endothelial cell growth supplement (ECGS) (Calbiochem, San Diego, CA), and antibiotics were added into the petri dish until cells migrated out of the biopsy samples. The adherent tissue explants were then incubated at 37°C with 95% humidity and 8% CO2 until the VSMCs had migrated from the explants.

Fibroblasts from perivascular connective tissue were obtained using the same procedure as for the media, except that the external component of the veins was used. Explants were cultured in DMEM-Ham (3:1) supplemented with 30% FCS, ECGS, and antibiotics.

Tissue samples from each extraction phase were processed for histology. The obtained results confirmed that the explants were harvested from the adequate layer: media for the VSMCs and adventitia for the VFs (data not shown). Two weeks later, cells were trypsinized (0.05% trypsin (Intergen, Toronto, Ontario, Canada) and 0,01% EDTA (JT. Baker, Phillipsburg, NY)) and plated at a density of 1 x 104 viable cells/cm2 in tissue culture flasks and maintained at 37°C in a humidified atmosphere (92% air and 8% CO2). Cells showed a constant phenotype during subculturing and were used at passage 5. These different cell types (VSMCs and VFs) were well characterized (14) . The time necessary for the migration of VSMC and VFs from human vein explants was 14 and 9 days, respectively. A high percentage of VSMCs expressed {alpha}-SM-actin, whereas only a low number of {alpha}-SM-actin expressing cells was detected in VFs cultured on plastic substrate (14) .

For {alpha}-SM-actin immunofluorescent staining, 5 µm-thick frozen TEVM or TEVA tissue sections were fixed 10 min in methanol at –20°C and processed as described previously (14) . Sections were incubated with a mouse monoclonal antibody directed against {alpha}-SM-actin (Dako, Mississauga, Ontario, Canada), and Alexa Fluor 594-labeled-goat antimouse IgG (Molecular Probes, Eugene, OR). For controls, the primary antibody was omitted. Nuclei were stained blue with Hoechst 33258.

Construct production
Tissue-engineered vascular constructs composed of a both media and adventitia (TEVMA), only a media (TEVM) or solely adventitia (TEVA) were produced using the tissue-engineering method previously described (8) . Briefly, 104 cells/cm2 human VSMCs or VFs were cultured in DMEM-Ham (3:1) (30% FCS, ECGS, and antibiotics) supplemented with 50 µg/ml of sodium ascorbate (Sigma) to stimulate ECM synthesis. After 10 to 15 days of culture, cells formed thick living tissue sheets, comprising cells embedded in the ECM they secreted, which 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) until a four-layer thick construct of VSMCs or VFs were respectively obtained. For the TEVMA constructs, two layers of VSMCs sheet and two layers of VFs sheet were wrapped over the tubular support to obtain a construct, which had the same number of layers (four) as the final TEVM and the TEVA constructs. These different constructs were cultivated in DMEM-Ham (3:1) supplemented with 10% bovine FetalClone II serum (HyClone), antibiotics and 50 µg/ml of sodium ascorbate. After a week of maturation, each construct was cut into {approx}5-mm-long rings, while remaining on the tubular support. These rings were further cultured for 2 wk, and the medium was changed 3 times a week.

Contraction experiments
Rings of TEVM, TEVA, and TEVMA were removed from their respective tubular support used for the culture and rinsed in physiological salt solution (Kreb’s solution): 119 mM NaCl (Fisher, Nepean, Canada), 4.7 mM KCl (Fisher), 1.2 mM KH2PO4 (Fisher), 25 mM NaHCO3 (Fisher), 1.2 mM MgSO4 (Sigma), 2.5 mM CaCl2 (Fisher), 10 mM glucose (VWR, Mississauga, Ontario). Rings of TEVM, TEVA, and TEVMA were separately 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). The TEVM, TEVA, and TEVMA rings were set up between 2 L-shaped wires for isometric force measurements (Radnoti, Harvard Apparatus, Ville St-Laurent, Quebec, Canada). After being mounted, each tissue was equilibrated for 30 min before being passively stretched with a preload of 500 mg. During the next 60 min, each tissue was rinsed 3 times and the tissue tension was readjusted to 500 mg until a stable tension was observed. Before most experiments, the TEVM, TEVA, and TEVMA rings were challenged with 3 mM ATP (Sigma) to evaluate the contractile capacity of each construct. After three rinses and the return to a baseline tension, tissues were challenged with increasing concentrations of the indicated vasoconstrictor agent (ET-1, ET-2, ET-3, big ET-1, big ET-2, big ET-3) (American Peptides, Sunnyvale, CA) or SNP (Sigma) added cumulatively in the bath in the absence or presence of selective antagonists BQ-123 and BQ-788 (American Peptides), and phosphoramidon (Sigma). Antagonists were added 30 min before application of the various agonists. For relaxation studies, U46619 (Sigma) was added to precontract the different constructs. Each condition tested was done in triplicate. Contractions were expressed as percentage of the maximal contractile response obtained with 3 mM ATP for contraction studies and 1 µM U46619 for relaxation studies performed with SNP.

RT-PCR studies
Tissues were frozen in liquid nitrogen and stored at –70°C until used. Total mRNA was isolated with RNeasy Mini Kit (Qiagen). Extraction procedures were performed according to the manufacturer’s instructions. Total RNA (1 µg) was reverse transcribed into cDNA using SuperScript One Step RT-PCR with platinum Taq (Invitrogen), according to the manufacturer’s recommendations. Sense and antisense for human ETA receptor were 5'-TGGCCTTTTGATCACAATGACTTT-3' and 5'-TTTGATGTGGCATTGAGCATACAGGTT-3', for ETB receptor, 5'-ACTGGCCATTTGGAGCTGAGATGT-3' and 5'-CTGCATGCCACTTTTCTTTCTCAA-3' and for human cyclophilin A, 5'-GGTCAACCCCACCGTGTTCT-3' and 5'-TTGCCATCCAGCCACTCAGTC-3'. The conditions for PCR were 95°C for 2 min, 65°C for 1 min, and 72°C for 1 min. Before the first cycle, a 2-min denaturation step 95°C was included. After 30 cycles, specific PCR products were run on ethidium bromide-stained agarose gel (2%) and visualized under UV light.

Statistical analysis
Results are expressed as means ± SEM of n experiments. Student’s unpaired t-test was used for statistical analysis. P ≤ 0.05 was considered significant.

RESULTS

Implication of the adventitia in endothelin-induced contraction.
Tissue constructs were prepared by the self-assembly approach previously described (8 , 15) . Tissue-engineered vascular media (TEVM) and tissue-engineered vascular adventitia (TEVA) constructs were composed of four layers of VSMC sheets or VF sheets, respectively. TEVMA construct contained two layers of VSMC sheets and two layers of VF sheets to obtain the same total number, four layers, as TEVM and TEVA constructs. These sheets were produced with VSMCs and VFs isolated from native media and adventitia, respectively, and then amplified in vitro. TEVM contained a high percentage of cells expressing {alpha}-SM-actin (Figure 1 A). In contrast, only a low number of {alpha}-SM-actin expressing cells was detected in TEVA (Figure 1B ), reflecting that the differences between VFs and VSMCs were preserved in the reconstructed vascular tissues.


Figure 1
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  		Figure 1. Immunofluorescence staining of {alpha}-SM-actin on tissue-engineered vascular media (TEVM) (A) composed of a four-layer-rolled sheet of vascular smooth muscle cells (VSMCs) and tissue-engineered vascular adventitia (TEVA) (C) composed of a four-layer-rolled sheet of fibroblast cells. B and D) nuclei staining with Hoechst 33258 of (A and C), respectively. Scale bars: 100 µm.

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. 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 after precontracting the tissue rings with 3 mM ATP. The ATP and endothelin induced contractile responses of TEVA, as well as TEVM and TEVMA (Fig. 2 ). The maximal contractions induced by ATP were the same for the different vascular constructs (Figure 2A ). In contrast, a heterogeneity was noted in the maximal contractile response to ET-1 (10 nM), TEVA contracting less than TEVMA and TEVM (Figure 2B ). TEVMAs dose-dependently contracted to ET-1, ET-2, or ET-3 (Fig. 3 ). The maximal contractile responses being 192%, 190%, and 193%, respectively. When taken separately both the media (TEVM) and the adventitia (TEVA) component of the TEVMA also contracted to endothelin.


Figure 2
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  		Figure 2. Effects of ATP 3 mM (A) and ET-1 10 nM (B) on the different vascular constructs. Typical traces illustrating the contractile response of rings from TEVM composed of a four-layer-rolled sheet of VSMCs, TEVA composed of a four-layer-rolled sheet of fibroblast cells, and tissue-engineered vascular media + adventitia (TEVMA) constructs.


Figure 3
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  		Figure 3. Vasocontractile responses to endothelin showing the presence of different receptors on the various vascular constructs. TEVM composed of a four-layer-rolled sheet of VSMCs, TEVA composed of a four-layer-rolled sheet of fibroblast cells, and 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.

A heterogeneity in the response was, however, observed. ET-1 and ET-2 were found to induce a similar dose-dependent contractile response in these two constructs (Figure 3A, 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. ET-3 induced a contraction of the TEVMs, but failed to affect the tone of the TEVAs (Figure 3C ). Taken together, these results show that the reconstructed adventitia display a vasoconstrictor response to endothelin ET-1 and ET-2, but not to ET-3.

Implication of ETA and/or ETB receptor in the endothelin-induced contraction of our TEVA.
Selective ETA (BQ-123) and ETB (BQ-788) receptor antagonists were used to pharmacologically characterize the response to the endothelins. As seen in Fig. 4 , the ET-1-induced contraction of TEVMs and TEVAs was not affected by pretreatment with the ETA receptor antagonist BQ-123 or the ETB receptor antagonist BQ-788. Combination of the two receptor antagonists however shifted to the right the ET-1-induced-dose-response curve. In contrast, BQ-123 but not BQ-788 shifted the response to ET-1 in TEVAs. In addition, pretreatment of the TEVA with a mixture of the two receptor antagonists also displaced the dose-response curve to the right. Overall, these results indicate that ET-1-induced contraction occurs via the ETA receptor in the TEVA, while it is via ETA and ETB receptors in TEVM and TEVMA.


Figure 4
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  		Figure 4. Effects of selective endothelin receptor antagonists on contraction elicited by ET-1 on the different vascular constructs (A–C) and RT-PCR studies showing that ETA and ETB receptor mRNA are expressed in TEVM and TEVMA, whereas only ETA receptor mRNA is expressed in TEVA (D). BQ-123 and BQ-788 were used to selectively block ETA and ETB receptor respectively on TEVM (A), TEVA (B), and TEVMA (C). (% maximal response, Emax obtained with 3 mM ATP). Data points are 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. D) RT-PCR studies showing that ETA and ETB receptor mRNA are expressed in TEVM and TEVMA, whereas only ETA receptor mRNA is expressed in TEVA. mRNAs were also extracted from human umbilical vein endothelial cells (HUVECs) as a known positive ETB receptor expression control. RT-PCR products were then fractionated by denaturing agarose gel electrophoresis and visualized under UV lamp. Lanes 1 to 4 are the mRNA for ETA receptor in TEVM, TEVA, TEVMA, and HUVEC, respectively. Lanes 5 to 8 are the mRNA for ETB receptor in TEVM, TEVA, TEVMA, and HUVEC, respectively. Cyclophilin A is the internal control. One representative experiment of five experiments is presented.

The selective ETA and ETB receptor antagonists were also tested in the ET-3-induced contraction response observed in TEVM and TEVMA. TEVA was not used since it was not affected by ET-3 (Fig. 3) . The dose-response curve to ET-3 was displaced to the right after pretreatment with BQ-788, whereas BQ-123 had no effect on these constructs (Fig. 5 ). These results indicate that ET-3 induces contraction in the TEVM and TEVMA via interaction with the ETB receptor.


Figure 5
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  		Figure 5. Effects of selective endothelin receptors antagonist on contraction elicited by ET-3 on the different vascular constructs. BQ-123 and BQ-788 were used to selectively block ETA and ETB receptors, respectively, on TEVM (A) and TEVMA (B). (% maximal response, Emax obtained with 3 mM ATP). Data points are mean results of five separate rings from different TEVM or TEVMA constructs. Vertical bars represent SEM. *P < 0.01, significantly different from control using Student’s unpaired t test.

Heterogeneity of endothelin receptor mRNA expression.
The various constructs were analyzed by RT-PCR with specific mRNA probes for ETA and ETB receptors to determine the type of endothelin receptors (ETA and/or ETB) expressed by the cells composing our various constructs. mRNA was extracted from TEVM, TEVA, and TEVMA constructs at the same maturation period (3 wk) as that used for the vasoconstriction experiments. Human umbilical vein endothelial cells (HUVECs) cultured on plastic were used as a positive control for the presence of ETB receptor. As expected, only the mRNA for the ETB receptor was detected in HUVEC (Figure 4D ). Both mRNA for ETA and ETB receptors were expressed by the TEVM as well as the TEVMA constructs. In contrast, the TEVA expressed mRNA for the ETA, but failed to express detectable levels of mRNA for the ETB receptor. These results indicated that TEVM and TEVMA expressed mRNA for the ETA and ETB receptors, whereas only the ETA receptor mRNA was expressed in TEVA.

Characterization of the functionality of ECE in TEVM, TEVA, and TEVMA.
To further characterize the contribution of the adventitia in the contractile response of blood vessels, we assessed whether the enzyme responsible for the cleavage of big ET to ET in vivo, the endothelin converting enzyme (ECE), was present and functional in these in vitro reconstructed tissues. The TEVM, TEVA, and TEVMA constructs were thus incubated in the presence of big ET-1, big ET-2, or big ET-3, and their contraction monitored in isolated organ baths. Big ET-1 or big ET-2 triggered vasoconstriction in TEVMA, TEVM, and TEVA, indicating the presence of an ECE (Fig. 6 ). In contrast, no contractile response was observed following addition of big ET-3 on the TEVA consistent with the absence of ETB receptors (Figure 6C ). Furthermore, TEVM and TEVMA also did not respond to big ET-3 probably because the specificity of ECE-1 for big ET-3 was too low (big ET-1>big ET-2>big ET-3) (16) . As demonstrated in Figure 6A and B , the concentration-response curves of big ET-1 and big ET-2 were abolished by the ECE inhibitor, phosphoramidon (0.1 mM). These results demonstrate that a phosphoramidon-sensitive ECE is present and is functional in the various tissue-constructs tested.


Figure 6
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  		Figure 6. Effects of different big endothelins and inhibitor of ECE on the three vascular constructs. Big ET-1 (A), big ET-2 (B) and big ET-3 (C) were incubated in presence or in absence of phosphoramidon with the different vascular constructs. Data points are mean results of five separate rings from different TEVM, TEVA or TEVMA constructs. (% maximal response, Emax obtained with 3 mM ATP). Vertical bars represent SEM. *P < 0.01, significantly different from control using Student’s unpaired t test.

Relaxation of the TEVM, TEVA, and TEVMA.
To evaluate the relaxing potential of the TEVA, the effect of SNP, a pharmacological agent widely used in relaxation studies of blood vessels since it forms NO in solution, was tested (17) . A dose-dependent relaxation was observed in TEVMA, TEVM, and TEVA in response to SNP (Fig. 7 ) which reduced vascular tone by close to 80% in the three reconstituted vessels.


Figure 7
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  		Figure 7. 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.

DISCUSSION

The role of the media in the control of peripheral blood resistance is well known. However, a long-standing question is whether the adventitia can directly participate in the regulation of blood vessel tone. In the present study, we combined the use of tissue-engineered blood vessel model with the most powerful pressor agent known to date, endothelin, to demonstrate that the adventitia can directly respond to vasocontractile agents.

Endothelin plays a major role in the control of peripheral blood resistance in human (9 10 11) . The vast majority of blood vessels have been demonstrated to respond to endothelin. For example, vasoconstriction studies performed on human native saphenous veins demonstrated that the presence of specific endothelin receptors are responsible for the observed contraction (18 19 20) . 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 vessel (3 4 5 6 7) . 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 (6) . Moreover, all the 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.

The human reconstructed tissue-engineered vascular media composed of VSMCs isolated from human umbilical cord veins is a well-characterized pharmacological model to study the vasoactive response. They contract in response to vasoactive mediators such as histamine (14 , 15) and endothelin (21) . We took advantage of this tissue engineering method to produce human reconstructed adventitia to overcome the difficulties in obtaining functional adventitia from native blood vessels for vasocontractile studies.

TEVMA constructs, made of VSMCs and VFs derived from human saphenous veins and then amplified in vitro, contracted in response to endothelin. The use of separate TEVM and TEVA constructs permitted us to demonstrate that the adventitia of the TEVMA, in addition to media component, has the capacity to contract in response to ET-1 or ET-2. Calculated from the observed dose–response contraction curve of the three vascular constructs, the relative affinity of ET-1, was found to be similar to the EC50 reported in the literature for intact human blood vessels (18) . ET-2 also contracted the TEVA, as well as the TEVM and the TEVMA tested with the same affinity as that of ET-1 (Table 1 ). As reported in the literature, ET-1 and ET-2 have the same affinity for the ETA receptor (13) suggesting the presence of this receptor on the various vascular constructs. ET-3 had no contracting effect on TEVA, whereas TEVM and TEVMA had the capacity to contract in response to that particular peptide. Because it is known that ET-3 has a greater affinity for ETB receptor, (13) it suggested that this receptor was absent on the TEVA, a result corroborated by the RT-PCR studies.


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  		Table 1. EC50 and Emax of different endothelin and big endothelin on the TEVA, TEVM, and TEVMA

The maximal contractions obtained in response to ET-1 and ET-2 were greater for TEVM than for TEVA (Table 1) . However, the increased maximal contractile response observed for TEVM compared to the one obtained with TEVMA is likely due to the number of VSMCs layers present in each construct. The TEVMA was composed of two layers of VSMCs and two layers of VFs (four layers total), whereas the TEVM contained four layers of VSMCs.

The presence of endothelin receptors on each of the vascular constructs was confirmed by the use of selective endothelin receptor antagonists. The contractile response of the TEVM and TEVMA to ET-1 was found to take place via ETA and ETB receptors since the contraction observed in response to ET-1 was shifted to the right following pretreatment with the selective ETA antagonist BQ-123 and with the ETB receptor antagonist, BQ-788. Furthermore, the contractile response of the TEVM and TEVMA to ET-3 was almost completely blocked after pretreatment with BQ-788 at 1 µM. The contractile response of the TEVA to ET-1 was found to take place only via ETA receptor since the contraction observed in response to ET-1 was shifted to the right in the same way after pretreatment with BQ-123 and the mixture of BQ-123 and BQ-788.

Furthermore, mRNAs for the ETA and ETB receptors were detected in TEVM and TEVMA, whereas only the mRNA for the ETA receptor was detected in these TEVA. Taken together, these results indicated that ETA and ETB receptors were present in TEVM and TEVMA and ligation of these receptors by ET-1 and ET-3 led to the contraction of these vascular constructs. In contrast, only ETA receptors were present in TEVA, indicating that ligation of these receptors by ET-1 led to the contraction of the adventitia construct.

The relaxation response observed after administration of SNP in both TEVA and TEVMA indicated that the adventitia also responded to vasorelaxing agents. These results demonstrate the presence of a cGMP-dependant functional mechanism in the adventitia. The adventitia layer of a blood vessel is surrounded and penetrated by a network of nutritional vessels, anatomically identified as the vasa vasorum, which normally provides supply to the outer media and adventitial layers of conductance as well as of large resistance vessels, and known to respond to vasomotor stimuli (adenosine, ET-1, bradykinin, and substance P) and endothelium-dependent and neuron-derived agents (22 23 24) . NO secreted by endothelial cells of the vasa vasorum network could eventually diffuse toward the adventitia and participate in its relaxation because the diffusion radius of NO is 150–300 µm (25) . It is noteworthy that our reconstructed vessel does not possess a vasa vasorum. Nonetheless, our results show that the adventitia possesses a functional guanylate cyclase that is involved in vasorelaxation (26) .

Our results have shown that several differences clearly exist between the TEVM and TEVA constructs tested in terms of contraction and endothelin receptor expression. These variations are representative of the cells derived from the explants of the vascular media and adventitia. Although there may have been a selection during the derivation procedure in the cells that migrated and proliferated in comparison to the total native cells present in the tissue of origin, clear differences were observed between the isolated VSMCs and VFs in terms of the time necessary for the migration out of the explants, doubling time, {alpha}-SM-actin expression, and the thickness of the TEVM constructs in comparison to its TEVA counterpart (14) .

High levels of circulating ET-1 have been demonstrated in some pathologies (27) . Furthermore, beneficial effects have been demonstrated when ECE inhibitors treatment is given (27 28 29 30) . Because the existence of ECE had previously been demonstrated in smooth muscle cells and fibroblasts, (31 32 33 34 35) it was thus of interest to determine whether a biologically active ECE was present in our vascular constructs. The results presented here clearly demonstrate the contraction of the TEVM, TEVA, and TEVMA constructs tested when big ET-1 or big ET-2 was added, and these observed contractions were inhibited in the presence of the ECE inhibitor phosphoramidon. These results suggested that one or more phosphoramidon-sensitive ECE was present and functional in our adventitia constructs as shown for the media of TEVM (21) . Furthermore, ECE-1 was found not to convert big ET-2 as efficiently as big ET-1 (16) , as found for these precursor molecules in the present study. Furthermore, ECE-1 was demonstrated to have a very low affinity for the conversion of big ET-3 to ET-3, (16) , and no contraction was observed following incubation of our TEVM, TEVA, and TEVMA with ET-3.

A variety of studies have recently suggested that the adventitia is activated during the development of atherosclerosis and hypertension (36 37 38 39) . The adventitia is also increasingly being considered a highly active segment of vascular tissue that contributes to a variety of disease pathologies. The present study demonstrates that a tissue-engineered vascular adventitia reconstructed with vascular fibroblasts has the capacity of contracting and relaxing. 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. 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.

Finally, the selective characterization of the adventitia with regards to its responsiveness to humoral or biomechanical stimuli may contribute to the development of more efficient extravascular stents used as aorto-coronary bypass grafts (40 41) .

ACKNOWLEDGMENTS

The authors are thankful to Danielle Larouche, LOEX, for her assistance with photography and pictures. This work was supported by the Canadian Institutes of Health Research (CIHR). L. Germain is recipient of the Canadian Research Chair on Stem Cells and Tissue Engineering from CIHR. P. D’Orleans-Juste is recipient of a National Scholarship from the Fonds de la Recherche en Santé du Québec (FRSQ), K. Laflamme was recipient of studentships from FRSQ and CIHR and G. Grenier was a recipient of a studentship from FRSQ.

Received for publication October 12, 2005. Accepted for publication January 17, 2006.

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