Tissue-Engineered Human Vascular Media With a Functional Endothelin System
http://www.100md.com
循环学杂志 2005年第2期
the Laboratoire d’Organogenèse Experimentale/LOEX, H;pital Saint-Sacrement du CHA
Department of Surgery, Laval University, Quebec, PQ, Canada (K.L., C.J.R., F.A.A., L.G., S.P.)
Institute of Pharmacology, Medical School, Sherbrooke University, Sherbrooke, PQ, Canada (J.L., P.D.J.).
Abstract
Background— Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor’s hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel.
Methods and Results— Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ETA receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ETA but not the ETB receptor was present in these human tissue–engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media.
Conclusions— Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ETA receptors and endothelin-converting enzyme.
Key Words: endothelin ; muscle contraction ; myocytes, smooth muscle
Introduction
Coronary artery disease is the most common cause of morbidity and mortality in Western countries.1 Treatment options include drugs, balloon angioplasty, and CABG surgery. Most artery bypass graft surgery involves harvesting of the long saphenous vein or mammary artery. However, in about a third of patients, this surgery proves inadequate or unsuitable.2 Surgeons have therefore turned to the use of prosthetic materials like polyethylene terephthalate (Dacron), polytetrafluoroethylene, expanded polytetrafluoroethylene, and polyurethane.2–8 However, the results obtained with these prosthetic materials prove to be inferior to those obtained with autologous conduits, especially when the blood vessel diameter is <5 mm. The problems that can arise when these types of materials are used include increased risk of thrombosis and infection, limited durability, lack of compliance both of the graft and around the anastomosis,9 and failure as a result of restenosis. These problems necessitate further interventions.10–12
One approach to the elaboration of a vascular prosthesis would be to construct one that would very closely resemble a natural healthy blood vessel in both composition and characteristics. This prosthesis would be composed of human cells only (devoid of exogenous synthetic material) and would have the capacity to contract and relax in response to vasoactive agents. We have recently developed a tissue-engineering approach to produce a completely biological blood vessel from cultured human cells.13 These human tissue–engineered blood vessels (TEBVs) were designed to be vascular grafts and contained the 3 cell types found in natural vessels: endothelial cells, vascular smooth muscle cells (VSMCs), and fibroblasts.
One of the most important functional characteristics of blood vessels is to help regulate peripheral resistance. This control of peripheral resistance is mediated via the secretion of vasoactive mediators by the endothelium. Some of these vasoactive substances possess their specific receptors on VSMCs where they mediate the contraction or relaxation of the tissue that these cells compose. Among them, 3 endogenous isoforms of endothelin have been discovered: endothelin (ET)-1, ET-2, and ET-3.14 Of these 3 isoforms, ET-1 is ubiquitous in mammal cells and is the most potent endogenous vasopressor agent reported to date.15 Studies in humans have demonstrated the importance of ET-1 in the maintenance of vascular tone16 and blood pressure.17 ET-1 is cleaved from its precursor, big ET-1, by the action of 1 phosphoramidon-sensitive endothelin-converting enzymes (ECEs) to produce a mature biologically active peptide, ET-1.15 Three isoforms of ECE have been identified to date: ECE-1, ECE-2, and ECE-3. ECE-1, however, is the most physiologically implicated because of its wide action and expression.18,19
ET-1 binds 2 different receptor subtypes: endothelin A (ETA) receptors, which have a higher affinity for ET-1 than ET-3, and endothelin B (ETB) receptors, which have equal affinity for ET-1 and ET-3.20,21 The ETA receptors are located on VSMCs and mediate vasoconstriction. The ETB receptors present in the endothelium and the smooth muscle layer mediate different responses, depending on their location. The ETB receptors are clearance receptors and induce endothelium-dependent dilation by release of nitric oxide and prostaglandins.22,23 Contractile ETB receptors localized in smooth muscle of low-resistance vessels have an effect similar to that of their ETA neighbors.24,25
In the present study, we further characterized the suitability of our human TEBV as a vascular prosthesis by investigating the response of its media component to endothelins in vitro. More precisely, a tissue-engineered vascular media (TEVM) was produced through the use of cultured human VSMCs. Our results demonstrate that endothelin receptors are expressed and are functional on the TEVM because a contraction to ET-1 and ET-2 could be observed. Moreover, ECE, the main enzyme responsible for the production of mature ET-1, was also present and functional.
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 umbilical vein endothelial cells (HUVECs) and VSMCs were obtained with the method of Jaffe et al26 as described previously.13 Briefly, umbilical cords were obtained from healthy newborns and kept in ice-cold culture medium for 12 hours. Veins were cannulated at both ends and washed with calcium-free HEPES buffer (10 mmol/L HEPES, 119 mmol/L NaCl, 6.7 mmol/L KCl, pH 7.35). Warm thermolysin solution (Sigma) (250 μg/mL in HEPES buffer containing 1 mmol/L CaCl2) was injected to rinse and fill the vein. After a 30-minute incubation at 37°C, the veins were perfused with culture medium, and the HUVECs were cultured in M199 medium (Sigma) containing antibiotics (100 U/mL penicillin G and 25 μg/mL gentamicin) (Sigma), ECGS 20 μg/mL (Sigma), and 20% FBS (Hyclone). HUVECs were used at their third to fifth passage for reverse-transcriptase polymerase chain reaction (RT-PCR). To isolate VSMCs, the umbilical cord veins were further injected with warm collagenase H solution (Roche) (0.200 U/mL in HEPES buffer containing 5 mmol/L CaCl2). After 30 minutes of incubation at 37°C, the vein was perfused with medium. The cell solution was centrifuged, and the cell pellet was resuspended in medium for SMC [DMEM:Ham’s F12 modified medium (3:1), 10% FBS, and antibiotics]. SMC identity was confirmed by specific -SM-actin immunostaining. For these experiments, cells were plated at a density of 104 cells/cm2, maintained at 37°C in a humidified atmosphere (92% air/8% CO2), and used between passages 3 and 7.
TEVM Production
TEVM was produced as previously described.27 Briefly, we cultured 104 cells/cm2 human VSMCs in the SMC culture medium supplemented with 50 μg/mL sodium ascorbate to stimulate extracellular matrix synthesis. After 10 to 15 days of culture, cells formed thick sheets comprising cells and the extracellular matrix they secreted that could be peeled off from the culture flask with fine forceps. These sheets were wrapped around a tubular support (inside diameter, 3 mm) to produce a cylinder. After a week of maturation, the tissue was cut into 5-mm-long rings while remaining on the tubular support. These rings were further cultured for 2 weeks.
Contraction Experiments
Umbilical veins were dissected free of surrounding tissues and mechanically denuded of their endothelium. The vein was cut into 5-mm-long rings. Rings of TEVM were also removed from the tubular support used for the culture and rinsed in physiological salt solution (Krebs’ solution): 119 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, and 10 mmol/L glucose. Rings of TEVM and umbilical vein were mounted in isolated organ baths containing Krebs’ solution maintained at 37°C and gassed with a mixture of 95% O2/5% CO2 (pH 7.4). The TEVM and umbilical vein rings were set up between 2 L-shaped wires for isometric force measurements (Radnoti). After being mounted, each tissue was equilibrated for 30 minutes before being passively stretched with a preload of 500 mg for TEVM rings and 2 g for umbilical vein rings. During the next 60 minutes, each tissue was rinsed 3 times, and the tissue tension was readjusted to 500 mg and 2 g for TEVM and umbilical vein rings, respectively, until a stable tension was observed. Before most experiments, the TEVM and umbilical vein rings were challenged with 3 mmol/L ATP and 50 mmol/L KCl, respectively (Sigma) (Fisher) to evaluate the contractile capacity of the vessel. After 3 rinses and the return to a baseline tension, tissues were challenged with increasing concentrations of the indicated vasoconstrictor agonist [ET-1, ET-2, ET-3, big ET-1, big ET-2, big ET-3, IRL-1620 (American Peptides)] added cumulatively in the bath in the absence or presence of selective antagonists BQ-123 (a gift of Witold Neugebauer, Sherbrooke University), BQ-788 (American Peptides), and phosphoramidon (Sigma). Antagonists were added 30 minutes before application of the agonist.
Expression of Results and Statistical Analysis
Contractions were expressed as percentage of the maximal contractile response obtained with 3 mmol/L ATP for TEVM and 50 mmol/L KCl for umbilical vein. Results are expressed as mean±SE of n experiments. Student’s unpaired t test was used for statistical analysis. P0.05 was considered significant.
RT-PCR studies
Tissues were frozen in liquid nitrogen and stored at –70°C until use. Total mRNA was isolated with the RNeasy Mini Kit (Qiagen). Extraction procedures were performed according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed into cDNA with SuperScript One Step RT-PCR with platinum Taq (Invitrogen) according to manufacturer’s recommendations. Sense and antisense for human ETA receptor were 5'-TGGCCTTTTGATCACAATGACTTT-3' and 5'-TTTGATG-TGGCATTGAGCATACAGGTT-3'; for ETB receptor, 5'-ACTGGCCATTTGGAGCTGAGATGT-3' and 5'-CTGCATGC-CACTTTTCTTTCTCAA-3'; and for human cyclophilin A, 5'-GGTCAACCCCACCGTGTTCT-3' and 5'-TTGCCATCCAGC-CACTCAGTC-3'. The conditions for PCR were 95°C for 2 minutes, 65°C for 1 minute, and 72°C for 1 minute. Before the first cycle, a 2-minute denaturation step at 95°C was included. After 30 cycles, specific PCR products were run on ethidium bromide–stained agarose gel (2%) and visualized under UV light.
Results
Presence and Functionality of ETA and/or ETB Receptors in TEVM
SMCs isolated from the vein of human umbilical cords were used to reconstruct vascular media by the self-assembly approach of tissue engineering.13,27 Classic vasoconstriction studies were then performed on the reconstructed TEVM to study its response to endothelin. TEVM rings were placed in separate isolated organ baths,27 and increasing concentrations of various agonists were added (Figure 1). ET-1 and ET-2, known to have equal affinity for the ETA receptor, induced similar dose-dependent contractile responses of the TEVM (Figure 1A). The maximal contractile responses were 213% for both agonists. In contrast, the selective ETB receptor agonists ET-3 and IRL-1620 had no effect on the TEVM (Figure 1A).
To evaluate whether the contractile pattern of the reconstructed TEVM was similar to the vein from which the SMCs were isolated or if it resulted from the culture conditions, the endothelin responses of SMCs were compared with that of the umbilical vein from which they were isolated. As demonstrated in Figure 1A and 1B, both the umbilical veins and the reconstructed TEVM contracted to ET-1. However, no contraction was observed in response to the various concentrations of ET-3 tested. These results demonstrate that the umbilical veins and TEVM produced with the SMCs isolated from the same umbilical veins presented similar responses to the endothelins tested.
Pretreatment with the specific ETA receptor antagonist BQ-123 (0.1 μmol/L) inhibited the ET-1–induced contraction of TEVM in a dose-dependent manner. In contrast, the specific ETB receptor antagonist BQ-788 had no effect on the ET-1–induced contraction of TEVM (Figure 2). These results indicate that ET-1 and ET-2 induced contraction via interaction with the ETA receptor.
ETA and ETB mRNA Levels
To determine whether ETB and/or ETA mRNA receptors were expressed by the SMCs comprising the TEVM, our constructs were analyzed by RT-PCR with specific mRNA probes. mRNA was extracted from TEVM with the same maturation time (3 weeks) used for the vasocontractile experiments. As expected, ETB receptor mRNA but not ETA receptor mRNA was detected in HUVECs cultured on plastic used as control (Figure 1C). The TEVM expressed mRNA for the ETA receptor but failed to express detectable levels of ETB receptor mRNA. These results indicated that TEVM expressed mRNA for the ETA receptor but not for the ETB receptor.
Characterization of the Functionality of ECE in TEVM
To further characterize the TEVM, we sought to determine whether the ECE, responsible for the cleavage of big ET-1 to ET-1 in vivo, was present and functional in these in vitro reconstructed tissues. The contraction of blood vessels in response to big ET-1 requires its cleavage and conversion to ET-1 by a functional ECE. TEVM was thus incubated with big ET-1 or big ET-2, and contraction was monitored in an isolated organ bath. A contraction was observed when big ET-1 was added, indicating that an ECE was present, had been activated, and was functional in the TEVM (Figure 3). The contractions observed were 5 to 10 times less potent in response to big ET-1 and big ET-2 compared with that observed for the respective related mature peptide. Furthermore, the contraction detected in response to big ET-1 was at least twice as strong as that of big ET-2 (15.2 to 30 nmol/L, respectively). As expected from the results obtained with ET-3, big ET-3 did not induce contraction of TEVM. As demonstrated in Figure 4, the concentration-response curve of big ET-1 was shifted in the presence of the ECE inhibitor phosphoramidon (10–5 mol/L). This effect of phosphoramidon was concentration dependent and completely blocked the big ET-1 effect at a high concentration (10–4 mol/L). Therefore, these results demonstrate that a phosphoramidon-sensitive ECE is present and functional in the SMCs of the TEVM.
Discussion
A 3D small-diameter blood vessel has recently been developed in vitro through the use of the self-assembly approach of tissue engineering. This TEBV offers the advantage of containing no exogenous collagen or synthetic material because it is composed exclusively of human cells and the extracellular matrix that the cells have synthesized. Furthermore, studies with this TEBV revealed that it has several properties of a native small-diameter human blood vessel.13 Indeed, the burst strength of this TEBV13 was beyond the known burst level of a human saphenous vein; the media component of this TEBV contracts and relaxes in response to vasoactive mediators such as histamine and sodium nitroprusside, respectively.27 In this report, the vasoactive response to endothelin of the media component of this TEBV was investigated. Our results suggest that the media component of our TEBV has the potential to control vascular resistance via the presence of functional endothelin ETA receptors and ECE.
To the best of our knowledge, the present report is the first to characterize the response to endothelin of the media component of a human TEBV prosthesis. Although Niklason et al31 have previously demonstrated that their TEBV composed of bovine vascular cells seeded into a synthetic scaffolding material (polyglycolic acid) contracted to ET-1, no characterization of the response was performed.
The results shown here demonstrated that the TEVM made from cultured human vascular smooth muscle cells isolated from several human umbilical cord veins contract after exposure to endothelin. These results are important because the media component of a blood vessel and the vasoactive agent endothelin are known to play a major role in the control of peripheral resistance in humans.
Of the 3 isoforms of endothelin, only ET-1 and ET-2 induced a contractile response of the different TEVM tested. The dose-response contraction curve obtained in response to ET-1 demonstrated that the relative affinity of ET-1 was 3.1 nmol/L, which is similar to the EC50 reported in the literature for intact blood vessels.32 As expected, ET-2 contracted the TEVM with the same affinity obtained with ET-1.
The endothelin receptors ETA and/or ETB implicated in the contraction of the TEVM in response to ET-1 and ET-2 were investigated next. The contractile response of the TEVM to ET-1 and ET-2 was found to take place via ETA receptors because the contraction observed in response to both of these agonists was inhibited after pretreatment with the selective ETA receptor antagonist BQ-123 but not with the ETB receptor antagonist BQ-788. Furthermore, only the mRNA for the ETA receptor was detected in these TEVM. Taken together, these results indicate that only ETA receptors are present in the TEVM and that ligation of this receptor by ET-1 and ET-2 leads to the contraction of the TEVM tested.
Before mature ET-1 can carry out its biological activity, it must be cleaved from a precursor molecule, big ET-1, by the action of 1 ECE. The physiological importance of the conversion of big ET-1 to the mature ET-1 peptide by ECE resides in the fact that this mature peptide has a much higher affinity (140-fold more) than its precursor molecule (big ET-1).33 Because the existence of ECE had previously been demonstrated in SMCs,31 it was important to determine whether a biologically active ECE was present in our TEVM. Knowing that contraction of blood vessels in response to big ET-1 requires its cleavage and conversion to ET-1 by a functional ECE, our TEVM was thus incubated with big ET-1 or big ET-2, and the resulting effects on contraction were monitored. The results presented here clearly demonstrated the contraction of the TEVM tested when big ET-1 or big ET-2 was added and that these observed contractions were inhibited in the presence of the ECE inhibitor phosphoramidon. These results suggest that 1 phosphoramidon-sensitive ECE is present and functional in our TEVM. Although the ECE responsible for the conversion of big ET-1 to ET-1 was not determined in this study, the ECE isoform ECE-1 could be a candidate. This isoform has been localized at the surface of cultured SMCs and demonstrated to convert exogenously added big ET-1 to mature ET-1.34–37 Furthermore, ECE-1 was found not to convert big ET-2 as efficiently as big ET-1. Similar results were obtained when our TEVM was incubated in the presence of these 2 precursor molecules.
The pharmacology of endothelin has been studied mostly with blood vessels obtained from various animal species and animal cells isolated from blood vessels. Few studies have been performed on human tissues because of the difficulty in obtaining healthy human blood vessels and tissue specimens and because of the intrinsic variability that exists between tissues from different individuals. Our reconstructed media could provide a new pharmacological human vascular model to study the endothelin system and its modulation by pharmacological agents because it responds to ET-1 and ET-2 via ETA receptors and has functional ECE activity. Furthermore, our model is made from SMCs isolated from human veins and has the advantage of presenting an endothelin response profile similar to the blood vessel from which the cells were derived.
Acknowledgments
This work was supported by the Canadian Institutes of Health Research. L. Germain was a recipient of the Canadian Research Chair on Stem Cells and Tissue Engineering and Scholarships from Canadian Institutes of Health Research. K. Laflamme was a recipient of studentships from the Fonds de la Recherche en Sante du Quebec and Canadian Institutes of Health Research. J. Labonte is a recipient of studentships of the Heart Stroke Foundation of Canada and of the Fonds de la Recherche en Sante du Quebec. We gratefully thank Dr Witold Neugebauer, Sherbrooke University, for BQ-123.
References
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Department of Surgery, Laval University, Quebec, PQ, Canada (K.L., C.J.R., F.A.A., L.G., S.P.)
Institute of Pharmacology, Medical School, Sherbrooke University, Sherbrooke, PQ, Canada (J.L., P.D.J.).
Abstract
Background— Cardiovascular diseases remain a major cause of death and disability in the Western world. Among the various approaches adopted to counteract the morbidity associated with these diseases, surgical procedures and cardiac and vascular xenotransplantations or allotransplantations are routinely performed. The suitable vascular graft would be as close as possible to the native and healthy vessel composed exclusively of human components provided by the patient and would adapt to the donor’s hemodynamics. We have developed such a tissue-engineered human blood vessel reconstructed with human cells. Because endothelin is the most potent vasopressor known to date, we were interested in investigating the functionality of the endothelinergic system in our reconstructed human blood vessel.
Methods and Results— Vasoconstriction studies were performed with nonselective and selective agonists and antagonists to demonstrate that ETA receptors were present and functional in tissue-engineered human vascular media constructed with the self-assembly method. Reverse-transcriptase polymerase chain reaction studies demonstrated that mRNA of the ETA but not the ETB receptor was present in these human tissue–engineered blood vessels. Furthermore, we demonstrated that the endothelin-converting enzyme, the main enzyme responsible for the formation of the biologically active endothelin peptides, was present and functional in these same bioengineered vascular media.
Conclusions— Our results suggest that the media component of our tissue-engineered blood vessel has the potential of controlling vascular resistance via the presence of functional endothelin ETA receptors and endothelin-converting enzyme.
Key Words: endothelin ; muscle contraction ; myocytes, smooth muscle
Introduction
Coronary artery disease is the most common cause of morbidity and mortality in Western countries.1 Treatment options include drugs, balloon angioplasty, and CABG surgery. Most artery bypass graft surgery involves harvesting of the long saphenous vein or mammary artery. However, in about a third of patients, this surgery proves inadequate or unsuitable.2 Surgeons have therefore turned to the use of prosthetic materials like polyethylene terephthalate (Dacron), polytetrafluoroethylene, expanded polytetrafluoroethylene, and polyurethane.2–8 However, the results obtained with these prosthetic materials prove to be inferior to those obtained with autologous conduits, especially when the blood vessel diameter is <5 mm. The problems that can arise when these types of materials are used include increased risk of thrombosis and infection, limited durability, lack of compliance both of the graft and around the anastomosis,9 and failure as a result of restenosis. These problems necessitate further interventions.10–12
One approach to the elaboration of a vascular prosthesis would be to construct one that would very closely resemble a natural healthy blood vessel in both composition and characteristics. This prosthesis would be composed of human cells only (devoid of exogenous synthetic material) and would have the capacity to contract and relax in response to vasoactive agents. We have recently developed a tissue-engineering approach to produce a completely biological blood vessel from cultured human cells.13 These human tissue–engineered blood vessels (TEBVs) were designed to be vascular grafts and contained the 3 cell types found in natural vessels: endothelial cells, vascular smooth muscle cells (VSMCs), and fibroblasts.
One of the most important functional characteristics of blood vessels is to help regulate peripheral resistance. This control of peripheral resistance is mediated via the secretion of vasoactive mediators by the endothelium. Some of these vasoactive substances possess their specific receptors on VSMCs where they mediate the contraction or relaxation of the tissue that these cells compose. Among them, 3 endogenous isoforms of endothelin have been discovered: endothelin (ET)-1, ET-2, and ET-3.14 Of these 3 isoforms, ET-1 is ubiquitous in mammal cells and is the most potent endogenous vasopressor agent reported to date.15 Studies in humans have demonstrated the importance of ET-1 in the maintenance of vascular tone16 and blood pressure.17 ET-1 is cleaved from its precursor, big ET-1, by the action of 1 phosphoramidon-sensitive endothelin-converting enzymes (ECEs) to produce a mature biologically active peptide, ET-1.15 Three isoforms of ECE have been identified to date: ECE-1, ECE-2, and ECE-3. ECE-1, however, is the most physiologically implicated because of its wide action and expression.18,19
ET-1 binds 2 different receptor subtypes: endothelin A (ETA) receptors, which have a higher affinity for ET-1 than ET-3, and endothelin B (ETB) receptors, which have equal affinity for ET-1 and ET-3.20,21 The ETA receptors are located on VSMCs and mediate vasoconstriction. The ETB receptors present in the endothelium and the smooth muscle layer mediate different responses, depending on their location. The ETB receptors are clearance receptors and induce endothelium-dependent dilation by release of nitric oxide and prostaglandins.22,23 Contractile ETB receptors localized in smooth muscle of low-resistance vessels have an effect similar to that of their ETA neighbors.24,25
In the present study, we further characterized the suitability of our human TEBV as a vascular prosthesis by investigating the response of its media component to endothelins in vitro. More precisely, a tissue-engineered vascular media (TEVM) was produced through the use of cultured human VSMCs. Our results demonstrate that endothelin receptors are expressed and are functional on the TEVM because a contraction to ET-1 and ET-2 could be observed. Moreover, ECE, the main enzyme responsible for the production of mature ET-1, was also present and functional.
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 umbilical vein endothelial cells (HUVECs) and VSMCs were obtained with the method of Jaffe et al26 as described previously.13 Briefly, umbilical cords were obtained from healthy newborns and kept in ice-cold culture medium for 12 hours. Veins were cannulated at both ends and washed with calcium-free HEPES buffer (10 mmol/L HEPES, 119 mmol/L NaCl, 6.7 mmol/L KCl, pH 7.35). Warm thermolysin solution (Sigma) (250 μg/mL in HEPES buffer containing 1 mmol/L CaCl2) was injected to rinse and fill the vein. After a 30-minute incubation at 37°C, the veins were perfused with culture medium, and the HUVECs were cultured in M199 medium (Sigma) containing antibiotics (100 U/mL penicillin G and 25 μg/mL gentamicin) (Sigma), ECGS 20 μg/mL (Sigma), and 20% FBS (Hyclone). HUVECs were used at their third to fifth passage for reverse-transcriptase polymerase chain reaction (RT-PCR). To isolate VSMCs, the umbilical cord veins were further injected with warm collagenase H solution (Roche) (0.200 U/mL in HEPES buffer containing 5 mmol/L CaCl2). After 30 minutes of incubation at 37°C, the vein was perfused with medium. The cell solution was centrifuged, and the cell pellet was resuspended in medium for SMC [DMEM:Ham’s F12 modified medium (3:1), 10% FBS, and antibiotics]. SMC identity was confirmed by specific -SM-actin immunostaining. For these experiments, cells were plated at a density of 104 cells/cm2, maintained at 37°C in a humidified atmosphere (92% air/8% CO2), and used between passages 3 and 7.
TEVM Production
TEVM was produced as previously described.27 Briefly, we cultured 104 cells/cm2 human VSMCs in the SMC culture medium supplemented with 50 μg/mL sodium ascorbate to stimulate extracellular matrix synthesis. After 10 to 15 days of culture, cells formed thick sheets comprising cells and the extracellular matrix they secreted that could be peeled off from the culture flask with fine forceps. These sheets were wrapped around a tubular support (inside diameter, 3 mm) to produce a cylinder. After a week of maturation, the tissue was cut into 5-mm-long rings while remaining on the tubular support. These rings were further cultured for 2 weeks.
Contraction Experiments
Umbilical veins were dissected free of surrounding tissues and mechanically denuded of their endothelium. The vein was cut into 5-mm-long rings. Rings of TEVM were also removed from the tubular support used for the culture and rinsed in physiological salt solution (Krebs’ solution): 119 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, and 10 mmol/L glucose. Rings of TEVM and umbilical vein were mounted in isolated organ baths containing Krebs’ solution maintained at 37°C and gassed with a mixture of 95% O2/5% CO2 (pH 7.4). The TEVM and umbilical vein rings were set up between 2 L-shaped wires for isometric force measurements (Radnoti). After being mounted, each tissue was equilibrated for 30 minutes before being passively stretched with a preload of 500 mg for TEVM rings and 2 g for umbilical vein rings. During the next 60 minutes, each tissue was rinsed 3 times, and the tissue tension was readjusted to 500 mg and 2 g for TEVM and umbilical vein rings, respectively, until a stable tension was observed. Before most experiments, the TEVM and umbilical vein rings were challenged with 3 mmol/L ATP and 50 mmol/L KCl, respectively (Sigma) (Fisher) to evaluate the contractile capacity of the vessel. After 3 rinses and the return to a baseline tension, tissues were challenged with increasing concentrations of the indicated vasoconstrictor agonist [ET-1, ET-2, ET-3, big ET-1, big ET-2, big ET-3, IRL-1620 (American Peptides)] added cumulatively in the bath in the absence or presence of selective antagonists BQ-123 (a gift of Witold Neugebauer, Sherbrooke University), BQ-788 (American Peptides), and phosphoramidon (Sigma). Antagonists were added 30 minutes before application of the agonist.
Expression of Results and Statistical Analysis
Contractions were expressed as percentage of the maximal contractile response obtained with 3 mmol/L ATP for TEVM and 50 mmol/L KCl for umbilical vein. Results are expressed as mean±SE of n experiments. Student’s unpaired t test was used for statistical analysis. P0.05 was considered significant.
RT-PCR studies
Tissues were frozen in liquid nitrogen and stored at –70°C until use. Total mRNA was isolated with the RNeasy Mini Kit (Qiagen). Extraction procedures were performed according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed into cDNA with SuperScript One Step RT-PCR with platinum Taq (Invitrogen) according to manufacturer’s recommendations. Sense and antisense for human ETA receptor were 5'-TGGCCTTTTGATCACAATGACTTT-3' and 5'-TTTGATG-TGGCATTGAGCATACAGGTT-3'; for ETB receptor, 5'-ACTGGCCATTTGGAGCTGAGATGT-3' and 5'-CTGCATGC-CACTTTTCTTTCTCAA-3'; and for human cyclophilin A, 5'-GGTCAACCCCACCGTGTTCT-3' and 5'-TTGCCATCCAGC-CACTCAGTC-3'. The conditions for PCR were 95°C for 2 minutes, 65°C for 1 minute, and 72°C for 1 minute. Before the first cycle, a 2-minute denaturation step at 95°C was included. After 30 cycles, specific PCR products were run on ethidium bromide–stained agarose gel (2%) and visualized under UV light.
Results
Presence and Functionality of ETA and/or ETB Receptors in TEVM
SMCs isolated from the vein of human umbilical cords were used to reconstruct vascular media by the self-assembly approach of tissue engineering.13,27 Classic vasoconstriction studies were then performed on the reconstructed TEVM to study its response to endothelin. TEVM rings were placed in separate isolated organ baths,27 and increasing concentrations of various agonists were added (Figure 1). ET-1 and ET-2, known to have equal affinity for the ETA receptor, induced similar dose-dependent contractile responses of the TEVM (Figure 1A). The maximal contractile responses were 213% for both agonists. In contrast, the selective ETB receptor agonists ET-3 and IRL-1620 had no effect on the TEVM (Figure 1A).
To evaluate whether the contractile pattern of the reconstructed TEVM was similar to the vein from which the SMCs were isolated or if it resulted from the culture conditions, the endothelin responses of SMCs were compared with that of the umbilical vein from which they were isolated. As demonstrated in Figure 1A and 1B, both the umbilical veins and the reconstructed TEVM contracted to ET-1. However, no contraction was observed in response to the various concentrations of ET-3 tested. These results demonstrate that the umbilical veins and TEVM produced with the SMCs isolated from the same umbilical veins presented similar responses to the endothelins tested.
Pretreatment with the specific ETA receptor antagonist BQ-123 (0.1 μmol/L) inhibited the ET-1–induced contraction of TEVM in a dose-dependent manner. In contrast, the specific ETB receptor antagonist BQ-788 had no effect on the ET-1–induced contraction of TEVM (Figure 2). These results indicate that ET-1 and ET-2 induced contraction via interaction with the ETA receptor.
ETA and ETB mRNA Levels
To determine whether ETB and/or ETA mRNA receptors were expressed by the SMCs comprising the TEVM, our constructs were analyzed by RT-PCR with specific mRNA probes. mRNA was extracted from TEVM with the same maturation time (3 weeks) used for the vasocontractile experiments. As expected, ETB receptor mRNA but not ETA receptor mRNA was detected in HUVECs cultured on plastic used as control (Figure 1C). The TEVM expressed mRNA for the ETA receptor but failed to express detectable levels of ETB receptor mRNA. These results indicated that TEVM expressed mRNA for the ETA receptor but not for the ETB receptor.
Characterization of the Functionality of ECE in TEVM
To further characterize the TEVM, we sought to determine whether the ECE, responsible for the cleavage of big ET-1 to ET-1 in vivo, was present and functional in these in vitro reconstructed tissues. The contraction of blood vessels in response to big ET-1 requires its cleavage and conversion to ET-1 by a functional ECE. TEVM was thus incubated with big ET-1 or big ET-2, and contraction was monitored in an isolated organ bath. A contraction was observed when big ET-1 was added, indicating that an ECE was present, had been activated, and was functional in the TEVM (Figure 3). The contractions observed were 5 to 10 times less potent in response to big ET-1 and big ET-2 compared with that observed for the respective related mature peptide. Furthermore, the contraction detected in response to big ET-1 was at least twice as strong as that of big ET-2 (15.2 to 30 nmol/L, respectively). As expected from the results obtained with ET-3, big ET-3 did not induce contraction of TEVM. As demonstrated in Figure 4, the concentration-response curve of big ET-1 was shifted in the presence of the ECE inhibitor phosphoramidon (10–5 mol/L). This effect of phosphoramidon was concentration dependent and completely blocked the big ET-1 effect at a high concentration (10–4 mol/L). Therefore, these results demonstrate that a phosphoramidon-sensitive ECE is present and functional in the SMCs of the TEVM.
Discussion
A 3D small-diameter blood vessel has recently been developed in vitro through the use of the self-assembly approach of tissue engineering. This TEBV offers the advantage of containing no exogenous collagen or synthetic material because it is composed exclusively of human cells and the extracellular matrix that the cells have synthesized. Furthermore, studies with this TEBV revealed that it has several properties of a native small-diameter human blood vessel.13 Indeed, the burst strength of this TEBV13 was beyond the known burst level of a human saphenous vein; the media component of this TEBV contracts and relaxes in response to vasoactive mediators such as histamine and sodium nitroprusside, respectively.27 In this report, the vasoactive response to endothelin of the media component of this TEBV was investigated. Our results suggest that the media component of our TEBV has the potential to control vascular resistance via the presence of functional endothelin ETA receptors and ECE.
To the best of our knowledge, the present report is the first to characterize the response to endothelin of the media component of a human TEBV prosthesis. Although Niklason et al31 have previously demonstrated that their TEBV composed of bovine vascular cells seeded into a synthetic scaffolding material (polyglycolic acid) contracted to ET-1, no characterization of the response was performed.
The results shown here demonstrated that the TEVM made from cultured human vascular smooth muscle cells isolated from several human umbilical cord veins contract after exposure to endothelin. These results are important because the media component of a blood vessel and the vasoactive agent endothelin are known to play a major role in the control of peripheral resistance in humans.
Of the 3 isoforms of endothelin, only ET-1 and ET-2 induced a contractile response of the different TEVM tested. The dose-response contraction curve obtained in response to ET-1 demonstrated that the relative affinity of ET-1 was 3.1 nmol/L, which is similar to the EC50 reported in the literature for intact blood vessels.32 As expected, ET-2 contracted the TEVM with the same affinity obtained with ET-1.
The endothelin receptors ETA and/or ETB implicated in the contraction of the TEVM in response to ET-1 and ET-2 were investigated next. The contractile response of the TEVM to ET-1 and ET-2 was found to take place via ETA receptors because the contraction observed in response to both of these agonists was inhibited after pretreatment with the selective ETA receptor antagonist BQ-123 but not with the ETB receptor antagonist BQ-788. Furthermore, only the mRNA for the ETA receptor was detected in these TEVM. Taken together, these results indicate that only ETA receptors are present in the TEVM and that ligation of this receptor by ET-1 and ET-2 leads to the contraction of the TEVM tested.
Before mature ET-1 can carry out its biological activity, it must be cleaved from a precursor molecule, big ET-1, by the action of 1 ECE. The physiological importance of the conversion of big ET-1 to the mature ET-1 peptide by ECE resides in the fact that this mature peptide has a much higher affinity (140-fold more) than its precursor molecule (big ET-1).33 Because the existence of ECE had previously been demonstrated in SMCs,31 it was important to determine whether a biologically active ECE was present in our TEVM. Knowing that contraction of blood vessels in response to big ET-1 requires its cleavage and conversion to ET-1 by a functional ECE, our TEVM was thus incubated with big ET-1 or big ET-2, and the resulting effects on contraction were monitored. The results presented here clearly demonstrated the contraction of the TEVM tested when big ET-1 or big ET-2 was added and that these observed contractions were inhibited in the presence of the ECE inhibitor phosphoramidon. These results suggest that 1 phosphoramidon-sensitive ECE is present and functional in our TEVM. Although the ECE responsible for the conversion of big ET-1 to ET-1 was not determined in this study, the ECE isoform ECE-1 could be a candidate. This isoform has been localized at the surface of cultured SMCs and demonstrated to convert exogenously added big ET-1 to mature ET-1.34–37 Furthermore, ECE-1 was found not to convert big ET-2 as efficiently as big ET-1. Similar results were obtained when our TEVM was incubated in the presence of these 2 precursor molecules.
The pharmacology of endothelin has been studied mostly with blood vessels obtained from various animal species and animal cells isolated from blood vessels. Few studies have been performed on human tissues because of the difficulty in obtaining healthy human blood vessels and tissue specimens and because of the intrinsic variability that exists between tissues from different individuals. Our reconstructed media could provide a new pharmacological human vascular model to study the endothelin system and its modulation by pharmacological agents because it responds to ET-1 and ET-2 via ETA receptors and has functional ECE activity. Furthermore, our model is made from SMCs isolated from human veins and has the advantage of presenting an endothelin response profile similar to the blood vessel from which the cells were derived.
Acknowledgments
This work was supported by the Canadian Institutes of Health Research. L. Germain was a recipient of the Canadian Research Chair on Stem Cells and Tissue Engineering and Scholarships from Canadian Institutes of Health Research. K. Laflamme was a recipient of studentships from the Fonds de la Recherche en Sante du Quebec and Canadian Institutes of Health Research. J. Labonte is a recipient of studentships of the Heart Stroke Foundation of Canada and of the Fonds de la Recherche en Sante du Quebec. We gratefully thank Dr Witold Neugebauer, Sherbrooke University, for BQ-123.
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