FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by L’HEUREUX, N.
Right arrow Articles by ANDRIANTSITOHAINA, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by L’HEUREUX, N.
Right arrow Articles by ANDRIANTSITOHAINA, R.
(The FASEB Journal. 2001;15:515-524.)
© 2001 FASEB

A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses

NICOLAS L’HEUREUX*,{dagger},1, JEAN-CLAUDE STOCLET{dagger}, FRANÇOIS A. AUGER*, GUY JEAN-LOUIS LAGAUD{dagger}, LUCIE GERMAIN*2 and RAMAROSON ANDRIANTSITOHAINA{dagger}

* Laboratoire d’Organogénèse Expérimentale, Hôpital du Saint-Sacrement du CHA, 1050, chemin Sainte-Foy, Québec Canada G1S 4L8 and Department of Surgery, Laval University, Québec, Canada; and
{dagger} Laboratoire de Pharmacologie et Physico-Chimie des Intéractions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, UMR CNRS 7034, BP 24, 67401 Illkirch-Cedex, France

2Correspondence: Laboratoire d’Organogenèse Expérimentale, Hôpital du Saint-Sacrement du CHA, 1050, chemin Sainte-Foy, Québec, Canada G1S 4L8. E-mail: Lucie.germain{at}chg.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our method for producing tissue-engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue-engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re-expression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration-response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1, B2, and P2Y1, and pyrimidinoceptors). Sustained agonist-induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine- and caffeine-sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.—L’Heureux, N., Stoclet, J.-C., Auger, F. A., Lagaud, G. J.-L., Germain, L., Andriantsitohaina, R. A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses.


Key Words: tissue engineering • blood vessel • pharmacology • calcium • purinoceptors • cyclic nucleotides • contraction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE VASCULAR MEDIA is the thickest layer of the blood vessel and is comprised of vascular smooth muscle cells (VSMC), elastin, collagen, and other extracellular matrix components. The contraction of VSMC within this tissue allows blood vessels to control blood flow in response to physiological stimuli. VSMC tone is continuously regulated in the body by the opposing influences of contracting and relaxing agents. Some factors are released by perivascular nerve endings (e.g., norepinephrine and ATP) whereas others are generated by the endothelium. Endothelial cells can produce vasoactive molecules either through direct synthesis [e.g., nitric oxide (NO) and prostaglandins] or through controlled diffusion and enzymatic activation of circulating precursors [e.g., angiotensin I and II, endothelins, and bradykinin (BK)] (2) .

VSMC contraction is triggered by activation of receptors on the plasma membrane causing an elevation in cytoplasmic calcium ions concentration ([Ca2+]c). This is achieved by either extracellular Ca2+ entry or Ca2+ release from intracellular stores or both (for a review, see ref 3 ). Increased [Ca2+]c favors the formation of a Ca2+/calmodulin/myosin light chain kinase complex, which phosphorylates the myosin light chain, thus activating the classic actomyosin interaction. In addition, the sensitivity of the contractile machinery to local [Ca2+]c may be enhanced (‘sensitization’) by various phosphorylation/dephosphorylation pathways on activation of some vasoconstrictor receptors (3) . Conversely, VSMC relaxation is the result of two major pathways. The first is the activation of plasma membrane-coupled adenylyl cyclase, which results in cyclic AMP accumulation. The second is the activation of either soluble or plasma membrane-coupled guanylyl cyclases by NO and natriuretic peptides, respectively, causing cyclic GMP accumulation. Both cyclic nucleotides can decrease [Ca2+]c and antagonize the increase in Ca2+ sensitivity caused by some vasoconstrictor agonists. It is noteworthy that many vasoconstrictor agonists possess several receptor subtypes that can contribute to the SMC tone regulation. Receptors for ATP (P2 purinergic receptors) are a classical example of such a family of receptors (for a review, see ref 4 ). Moreover, receptor subtypes can cause either contraction or relaxation and can be differentially expressed depending of the vascular beds or environmental stimuli. For example, bradykinin B2 receptors trigger contraction in veins and relaxation in arteries whereas bradykinin B1 receptors are induced by inflammatory environments (5 , 6) .

In recent years, the new field of tissue engineering has integrated tissue culture principles and cell biology innovations to produce 3-dimensional tissues and assemble these tissues into organs. We have recently developed a new tissue engineering approach for the production of completely biological blood vessels from cultured human cells (1) . These tissue-engineered blood vessels were designed to be vascular grafts and contained the three cell types found in natural vessels: endothelial cells, VSMC, and fibroblasts. Here, our goal was to apply this new technology to create the first human contractile tissue-engineered vascular media (TEVM) specifically designed for pharmacological studies. Classical cardiovascular pharmacology has heavily relied on the study of isolated blood vessel contraction in organ bath. This approach has been instrumental in numerous discoveries and modified to create very elegant experimental designs. However, organ bath studies have been largely restricted to the use of animal tissues because of ethical considerations limiting availability and quantity of human blood vessels and because of variability of responses related to uncontrolled events in patients. Unfortunately, results obtained with animal tissues can sometimes be difficult to extrapolate to humans (7 8 9) .

It is well known that cultured human VSMC undergo a dedifferentiation process and lose their ability to contract on subculturing (10 11 12) . Using our new tissue engineering approach that was shown to promote differentiation of human VSMC, 3-dimensional TEVM were created with the physical shape and mechanical properties of a blood vessel in hopes of producing the first human contractile TEVM. Such vessels would combine the advantages of working with human tissues and the convenience of cell culture. For example, in comparison to the use of dissected tissues, a model based on cultured cells means large and reliable supplies, increased reproducibility, little ethical issues, and offers defined cell populations and culture conditions impossible to establish in vivo. Naturally, the utilization of human cells imparts more clinical relevance to the results. Compared to standard cell cultures, contractile TEVM would also allow actual contraction/relaxation studies in standard organ baths (available in most cardiovascular pharmacology laboratories) as opposed to indirect biochemical measurements. Finally, compared to real-time ‘single cell’ approaches, TEVM would give measurements of ‘tissue contraction’ with no need for extended sampling of individual cell or complex instrumentation.

In this study, the responses of the first contractile human TEVM were assessed on stimulation by various vasoconstrictor and vasodilator agents. Using a standard pharmacological approach, many receptors were identified. Finally, more mechanistic studies were also performed with this TEVM to characterize its calcium handling abilities and cyclic nucleotide sensitivity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue culture
Human VSMC were isolated by the method of Ross as described before(13 ). Briefly, umbilical cords were collected from healthy newborns and kept in ice-cold culture medium for 12 h or less. Veins were slit longitudinally and the endothelium was removed by vigorous scrubbing with gauze, followed by thorough rinsing. Strips of the media were carefully dissected, cut in small biopsies (3 mm3) and kept in culture as explants. Human VSMC migrated from the explant within 2 wk and readily proliferated. Two weeks later, cells were trypsinized (0.05% trypsin and 0.01% EDTA) and plated at a density of 1 x 104 viable cells/cm2 in tissue culture flasks. Cells showed a constant phenotype during subculturing and were used on passage 7. Cells and explants were cultured with Dulbecco-Vogt modification of Eagle’s medium with Ham’s F12 in a ratio 3:1 (Flow Lab., Mississauga, Canada), 10% bovine serum (Fetal Clone II, Hyclone, Logan, Utah), and antibiotics (100 U/ml penicillin and 25 µg/ml gentamicin). Human VSMC identity was confirmed by VSMC specific {alpha}-actin immunostaining (13 , 14) . Cells were tested at different passages for mycoplasmal infection with Hoechst fluorescent staining for cytoplasmic DNA and always found to be negative(15 ).

Production of TEVM
To obtain TEVM, human VSMC were cultured in medium supplemented with 50 µg/ml of sodium ascorbate to stimulate extracellular matrix synthesis (ECM). After 15 to 20 days of culture, cells formed thick sheets comprised of cells and ECM that could be peeled off from the culture flask using fine forceps. These sheets were wrapped around a styrene tubular support (outside diameter=2.5–4.5 mm) to produce a cylinder composed of approximately five concentric sheet layers. After a week of maturation, the layers adhered to one another forming a cohesive tubular tissue. The tissue was cut into {approx} 7 mm long rings while remaining on the tubular support. These rings were further cultured for a period ranging from 3 to 7 wk (average 53 days±16). At the end of the maturation period, tissues were cultured in medium containing 0.5% serum for an average period of 33 ± 13 days before contractile experiments. No correlation was observed between length of maturation and responsiveness in these experiments.

Histological labeling
Immunolabeling was performed as described before (1 , 13) . Briefly, frozen cross sections were fixed in cold acetone and incubated with rabbit polyclonal anti-type I collagen antibody (Chemicon, Temecula, Calif.), mouse anti-desmin, or anti-{alpha}-smooth muscle actin monoclonal antibody (Sigma, St. Louis, Mo.), and Texas red-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, Oreg.). Nuclei were stained blue with Hoechst 33258.

Contraction experiments
Rings of TEVM of 5–7 mm length were removed from the tubular support used for culture and rinsed in physiological salt solution (PSS) with the following composition in M: NaCl 119, KCl 4.7, KH2PO4 0.4, NaHCO3 14.9, MgSO4 1.17, CaCl2 2.5, glucose 5.5. Rings were mounted in a myograph filled with PSS kept at 37°C and continuously gassed with a mixture of 95% O2, 5% CO2 (pH 7.4). Briefly, two tungsten wires were inserted through the lumen. Mechanical activity was recorded isometrically by a force transducer (Kistler-Morse, DSG BE4), connected to one of the two wires.

After being mounted, the vessel was equilibrated for 30 min before being passively stretched with a load of 500 mg. During the next 60 min, the tissue was rinsed three times and the tissue tension readjusted to 500 mg until a stable tension was observed. Before most experiments, the vessel was challenged with 3 mM ATP to evaluate the contractile capacity of the vessel. After three rinses and return to baseline tension (60 min), TEVM rings were challenged with increasing concentrations of the indicated vasoconstrictor agonist added cumulatively in the bath in the absence and the presence of selective antagonists. When the activity of selective antagonists was evaluated, the compounds were added 30 min before the application of the agonist.

Relaxant effect of agents acting on cyclic nucleotides
To study the effect of vasodilator agents acting on cyclic nucleotide contents, TEVM was precontracted with 1 µM bradykinin. When the contraction reached a steady state, increasing concentrations of either the NO donor, sodium nitroprusside (SNP, 10 to 100 µM), or the adenylate cyclase activator forskolin (0.1 to 30 µM) were added cumulatively.

Assay of TEVM cyclic AMP and cyclic GMP content
TEVM were incubated for 5 min in the absence and presence of a single concentration of either forskolin (10 µM) or exogenous NO donors such as 3-morpholinosydnonimine (SIN-1, 100 µM) or SNP (100 µM) in PSS. The incubation buffer also contained isobutyl methylxanthine (IBMX 100 µM), SOD (100 U/ml), and catalase (100 U/ml) bubbled with a 95% O2-5% CO2 mixture and kept at 37°C. IBMX was added to inhibit cyclic AMP or cyclic GMP degradation through nucleotide phosphodiesterases. Superoxide dismutase (SOD) was added to prevent NO degradation by O2-. The reaction was stopped by addition of ice-cold HCl (0.1 N). After homogenization, the cyclic AMP and cyclic GMP contents of the TEVM were determined by radioimmunoassay according to the method of Cailla et al. (16) .

Measurements of [Ca2+]c
Changes in [Ca2+]c were determined by measuring the fluorescence of trapped Fura-2 with a dual excitation wavelength fluorometer (Fluorolog II, SPEX, Edison, N.J.) using the method previously described by Andriantsitohaina et al. (17) . TEVM segments were loaded with Fura-2 by incubation in the dark with PSS containing 5 µM Fura-2 AM (the acetoxymethyl ester of Fura-2) and 2% pluronic acid for 1 h. PSS was kept at 37°C and continuously gassed with a 95% O2-5% CO2 mixture (pH 7.4). At the end of each experiment, the Ca2+ signal was calibrated using ionomycin (20 µM) and CaCl2 (5 mM) for the maximal fluorescence and 20 mM EGTA in Ca2+-free solution for the minimal fluorescence. The ratio of fluorescence (measured at 510 nm) obtained at the two excitation wavelengths (340/380 nm) was calculated after subtraction of the autofluorescence at 340 and 380 nm.

Mechanism of Ca2+ entry and release produced by ATP
The mechanisms of [Ca2+]c increased caused by ATP were further investigated. To study the sources of Ca2+ implicated in the response to ATP, TEVM was challenged with 1 mM ATP in either normal PSS or in Ca2+-free medium. For Ca2+-free PSS, calcium was omitted and 0.5 mM EGTA was added.

Ca2+ entry blockers were used to study the Ca2+ entry component of the response to ATP. They were applied at maximally active concentrations, being 10 µM for the voltage-operated Ca2+ channel blocker nitrendipine and 0.2 mM for the nonselective Ca2+ channel blocker La3+. Cationic permeant pathway was also tested using Mn2+ as a rapid diminishment (or quench) of Fura-2 fluorescence method in the absence and presence of either 1 mM ATP or the dihydropyridine the voltage-operated Ca2+ channel agonist BAY K 8644 (1 µM).

To study the component of ATP-induced increase in [Ca2+]c due to internal Ca2+ release, caffeine (10 mM) or ryanodine (10 µM), an activator and inhibitor of the Ca2+-induced Ca2+ release channels, was used. The experiments were performed in Ca2+-free medium; caffeine and ryanodine for 10 min before addition of ATP (1 mM).

Expression of results and statistical analysis
Contractions were expressed either in mg or as percentage of the maximal contractile response obtained with 3 mM ATP. The change in [Ca2+]c was calculated using the equation described by Grynkiewicz et al. and expressed in nM (18) . Cyclic AMP and cyclic GMP contents were expressed as fentomoles per micrograms of DNA.

Results are expressed as means ± SE of n experiments. Student’s unpaired t test was used for statistical analysis. Analysis of variance was used to compare the concentration-response curves to vasoconstrictors in the absence and presence of the antagonists. P<= 0.05 was considered significant.

Drugs
Angiotensin II, ATP disodium salt, ADP, bradykinin, caffeine, mepyramin, norepinephrine bitartrate, PGF2{alpha}, ranitidine, reactive blue 2, SNP, and UTP sodium salt were purchased from Sigma (Grenoble, France). 2-MeSATP and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic sodium salt (PPADS) were purchased from Research Biochemicals International (Natick, Mass.). Nitrendipine and suramin were a generous gift from Bayer AG (Wuppertal, Germany; Paris, France). HOE 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) was from Hoechst (Paris, France). Stock solutions were prepared in distilled water (Q10, Millipore) except for norepinephrine (dissolved in 5% NaHCO3). Nitrendipine was dissolved in absolute ethanol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Structure of the TEVM
Human smooth muscle was cultured with ascorbic acid to produce sheets, which were wrapped around a cylinder to produce a cohesive tubular tissue after maturation (TEVM). Figure 1A shows a series of six rings of TEVM in their culture container. These rings have an internal diameter of 4.5 mm and a width of 5 to 7 mm. The diameter can be modified by using a different tubular support. Since rings are produced by cutting a contiguous segment of TEVM during maturation, their width can be adapted to the experimental needs. At the end of the maturation period, these rings are stable for more than a month. Assuming sterile techniques are used, rings can be independently slid off the tubular support and mounted on a standard organ bath/force transducer apparatus as would a natural blood vessel (Fig. 1B ). As with a normal blood vessel, TEVM rings can repeatedly be stretched to a fixed pre-load (0.5 to 1 g) and be submitted to multiple stimulations and rinses for a period of up to 8 h.



View larger version (105K):
[in this window]
[in a new window]
  		Figure 1. Macroscopic views of the human TEVM. A) Rings of TEVM on a 4.5 mm tubular support during maturation. B) A TEVM ring mounted on a force transducer in an organ bath.

Histological analysis of TEVM revealed VSMC as elongated cells in an orientation resembling human media (Fig. 2A ). Positive immunostainings for muscular contractile markers {alpha}-smooth muscle actin and desmin were obtained in TEVM (Fig. 2B , Fig. 2C ). Vascular VSMC density, although high for an in vitro model, was still lower than in a normal vascular media.



View larger version (74K):
[in this window]
[in a new window]
  		Figure 2. Histology of the human TEVM. Top: Cross section of the TEVM stained with Masson’s trichrome shows collagen in blue and cells in purple. Middle: Cross section of the TEVM immunolabeled for smooth muscle specific {alpha}-actin (red). Nucleus are stained blue. Bottom: Cross section of the TEVM immunolabeled for desmin (red) and type I collagen (green).

Contraction and relaxation studies
As shown in Fig. 3 , classic vein constrictor agonists such as histamine, bradykinin, and ATP induced contractile responses in a concentration-dependent manner. The maximal contractile responses observed were 182 ± 19.4, 104 ± 6.1 and 80 ± 3.7 mg, respectively (n=47). To demonstrate that TEVM can be used for pharmacological studies, a classic pharmacological methodology was use to identify the various receptors present on the VSMC. In the presence of mepyramine, an H1 receptor antagonist, the concentration-response curve to histamine was shifted to the right in a concentration-dependent manner without a change in the maximal response. However, the contractile response was not affected by ranitidine, an H2 receptor antagonist. The bradykinin B2 receptor antagonist, HOE 140, produced a rightward shift of the concentration response curve to bradykinin without a change in the maximal response. Together, these results suggest that TEVM possess functional H1 and B2 receptor subtypes. It should be noted that a small but significant contraction was observed with PGF2{alpha} (result not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 3. Concentration-effect curves of agonists producing contraction (% maximal response, Emax obtained with 3 mM ATP) of human TEVM in the absence and presence of selective antagonists. Mean of 4 to 6 experiments. Vertical bars stand for SE. **P < 0.01, ***P < 0.001 significantly different from control using analysis of variance.

In the case of purinergic receptors, the use of different purinoceptor agonists showed that ATP, 2-MeSATP, and UTP produced concentration-dependent contraction with the following order of potency: 2-MeSATP>ATP=UTP (Fig. 4A ). The strong contraction induced by UTP suggests the presence of either P2Y2 or P2Y4 pyrimidinoceptors. This is supported by the absence of cross desensitization observed between ATP and UTP in mediating contraction (Fig. 4B ). Indeed, ATP (1 mM) was able to increase TEVM contraction even after stimulation with a saturating dose of UTP (3 mM). This is consistent with the fact that P2Y1 receptors are insensitive to UTP. Conversely, UTP could still produce an increase in contraction after stimulation of the vessels with ATP. The specific P2X purinoceptor agonist {alpha},ß-MeATP was not able to induce contraction of the TEVM at concentrations of up to 10 µM and did not modify the contractile response to ATP (Fig. 5A ). This is consistent with the inability of the relative P2X antagonists suramin and PPADS to significantly modify the ATP dose-response curve (Fig. 5B C ). However, the ATP induced contractions were greatly reduced by the P2Y1 antagonist reactive blue 2. These data suggest that ATP induces contraction of the VSMC through its interaction with the P2Y1, but not the P2X, purinoceptor subtype. Thus, pharmacological characterization of purinoceptors shows the existence of at least two receptor subtypes activated by ATP or UTP, P2Y1, and probably P2Y2 or P2Y4.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 4. Effects of ATP analogs (ATP (open circles), 2-MeSATP (filled circles), and UTP (filled triangle) in human TEVM. A)mean contractions (% maximal response, obtained with 3 mM ATP) ± SE; 4 to 6 experiments. B) representative traces of contractile tension (in mg) of 4 experiments.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Effects of selective antagonists on contraction elicited by ATP in human TEVM media layer. Mean of 4–6 experiments ± SE. **P < 0.01 significantly different from control using analysis of variance.

The ability of TEVM to relax in response to vasodilating agents was investigated in rings precontracted with 1 µM bradykinin. Sodium nitroprusside, a NO donor, produced concentration-dependent relaxation of TEVM (Fig. 6 ). The observed vasorelaxation was associated with an increase in cyclic GMP but not cyclic AMP. Another NO donor, SIN-1 like sodium niroprusside, produced an increase in the cyclic GMP but not cyclic AMP content of TEVM (Fig. 6) . Forskolin, a vasorelaxant agent acting through the activation of adenylyl cyclase, produced relaxation in a concentration-dependent manner (Fig. 6) . This relaxation was associated with an increase in the cyclic AMP but not cyclic GMP content of TEVM.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. Relaxation of human TEVM elicited by forskolin (A) and by sodium nitroprusside (SNP) in the absence and presence of methylene blue (MB) (C), and elevation in cyclic AMP (B) or cyclic GMP (D) produced by either forskolin (10 µM, black columns), the two NO donors SIN-1 (10 µM, hatched columns), and SNP (10 µM, gray columns). Results are means of 6–8 experiments. **P < 0.01, ***P < 0.001 significantly different from control using Student’s unpaired t test.

Mechanisms of calcium handling in TEVM
To demonstrate that the TEVM is a versatile pharmacological model, mechanisms of calcium handling in response to vasoconstrictors agonists were also investigated. Figure 7 shows typical traces for the change in [Ca2+]c and contraction produced by histamine (10 µM), bradykinin (1 µM) and ATP (3 mM) in normal PSS. Responses to the vasoconstrictor agonists comprised an initial phase, during which the increase in tension was associated with an increase in [Ca2+]c, and a sustained phase during which increased tension persisted although [Ca2+]c returned to baseline. The sustained phase remained stable for the three agonists tested for the 30 min period of measurement. Together, these results suggest that sustained contraction involved agonist-induced increased sensitivity of myofilaments to Ca2+, and no differences in the long-term contractile response to the agonists can be demonstrated under the experimental conditions used. A difference between vascular responses to the agonists cannot be excluded for a longer period of measurement (more than 30 min). Furthermore, the responses to these agonists were not significantly different within 30 days after the maturation period of TEVM. The physiologically important agonist angiotensin II (up to 100 nM) also produced an increase in [Ca2+]c, with a lower magnitude and slower time course than the other agonists tested, but it failed to produce contraction. Thus, TEVM possess functional angiotensin II receptors whose activation produced an increase in [Ca2+]c.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 7. Representative traces (of 3 to 5 experiments) of the increase in tension (upper panel) and [Ca2+]c (lower panel) produced by vasoconstrictor agonists in human TEVM.

The mechanisms of [Ca2+]c increase caused by ATP were further investigated. ATP (3 mM) produced an increase in [Ca2+]c in both normal PSS and Ca2+-free medium. However, the ATP response was significantly lower in Ca2+-free medium, suggesting that ATP induced both extracellular Ca2+ entry and Ca2+ release from internal sources (Fig. 8 ).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 8. Effects of ATP on [Ca2+]c and Ca2+ influx in human TEVM media layer. Top: representative traces (of 4 experiments) showing [Ca2+]c in the presence (normal physiological salt solution, PSS) and absence (Ca2+-free PSS) of extracellular Ca2+. Bottom: manganese quench (at 350 nm) produced by ATP in a Fura-2 loaded preparation (representative trace of 3 experiments).

The increase in [Ca2+]c produced by ATP was reduced by the nonselective calcium channel blocker La3+ (1 mM), but not the L-type calcium channel blocker nitrendipine (1 µM) (not shown). KCl (100 mM) depolarization failed to produce an increase in [Ca2+]c of TEVB. ATP, but not the dihydropyridine agonist BAY K 8644 (1 µM), caused Mn2+ influx as detected by rapid quench of Fura-2 fluorescence at 350 nm (Fig. 8) . Altogether, these results suggest that ATP causes Ca2+ influx primarily through a nonselective divalent cation-permeant pathway blocked by La3+.

In Ca2+-free medium, caffeine (10 mM) produced a fast increase in [Ca2+]c and almost completely prevented a subsequent response to ATP. Ryanodine (10 µM) produced a slow and weak increase in [Ca2+]c and reduced partially the response to subsequent addition of ATP (Fig. 9 ). These results suggest that ATP triggers the release of Ca2+ mainly from caffeine- and ryanodine-sensitive intracellular stores.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 9. Representative traces (of 3 experiments) showing the effects of ATP (3 mM) in the absence and presence of caffeine (10 mM) or ryanodine (10 µM) of human TEVM in Ca2+-free physiological salt solution (PSS).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human umbilical vein VSMC contained in these TEVM displayed histological organization and differentiation markers ({alpha}-smooth muscle actin, desmin) typical of differentiated VSMC found in normal blood vessels. Moreover, the above findings show that these VSMC were able to perform such functions as contraction and relaxation in response to vasoactive drugs, which are associated with a high level of differentiation. The amplitude of the contractile responses was less in TEVM than in umbilical vein rings, which can be explained by a lower density of VSMC in the engineered tissue. However, the profile of these contraction/relaxation responses and the data on intracellular calcium handling suggest that VSMC in the TEVM displayed many signaling cascades observed in fully differentiated VSMC. In fact, the TEVM proved to be a good pharmacological model as it was compatible with standard pharmacological methodology. A first pharmacological characterization of receptors to vasocontractile agonists revealed that VSMC of the TEVM express bradykinin B2 and histamine H1 receptors as well as P2 purinoceptors, all of which are present on VSMC of intact human umbilical veins (19 20 21 22) .

To further demonstrate the potential of the TEVM as a pharmacological model, a study to discriminate between the different subtypes of purinoceptors was performed. Results showed the presence of purinoceptors of the P2Y1 subtype and of pyrimidinoceptors, as well as the absence of the P2X subtype. The absence of cross desensitization between ATP and UTP is consistent with the presence of P2Y1 purinoceptors since the latter is known to be insensitive to UTP (4) . These results are consistent with the literature, since the P2X subtype has been shown to be rapidly lost in culture (23) although it is expressed in umbilical veins (22) . The presence of P2Y1 purinoceptors has also been reported in human blood vessels (24) and even up-regulated in culture (25) . The absence of cross desensitization between ATP and UTP, along with the fact that ATP and UTP displayed similar potency, implies the presence of pyrimidinoceptors in the TEVM. Pyrimidinoceptors have been shown in human veins and arteries (23 , 24) . Among the various pyrimidinoceptors identified, only subtype P2Y6 has been shown to be insensitive to ATP. However, it has also been shown to be slow-acting and only weakly activated by UTP (26 27 28) . Pyrimidinoceptor subtypes P2Y2 and P2Y4 have also been identified in VSMC (29) ; however, the P2Y2 subtype was shown to be equally sensitive to ATP and UTP (30) . On the other hand, the cloned human pyrimidinoceptor P2Y4 subtype has been shown to have little or no affinity for ATP (31 , 32) . Thus, it is most likely that the receptors activated by UTP involved P2Y4 receptor subtype, although P2Y2 receptors may also participate in the response. The P2Y4 subtype has been reported to play a role in VSMC contractility (33) and is present in cultured VSMC (29 , 34) . Although pharmacological results were not conclusive, this model should be an interesting tool when specific agonists for P2Y2 and/or P2Y4 receptor subtypes become available.

Cyclic AMP and cyclic GMP are both intracellular mediators of vasodilatation induced by endogenous compounds, like catecholamines (via ß-adrenergic receptors) and NO, respectively. They have also been shown to inhibit VSMC migration and proliferation in vitro (35 , 36) . Activation of cyclic AMP-dependent protein kinase or cyclic GMP-dependent protein kinase (PKG) accounts for the relaxing effect of cyclic nucleotides (37) and cross activation of PKG by cyclic AMP has also been described (38) . Dedifferentiation of VSMC in culture is associated with impaired expression of PKG (39) , together with down-regulation of contractile proteins. Moreover, transfection of PKG-deficient VSMC lines with a PKG vector results in increased expression of contractile phenotype marker proteins and restores the capacity of cyclic AMP and cyclic GMP analogs to inhibit cell migration. This suggests that a functional cyclic GMP pathway is critical for the modulation of VSMC phenotype. In our study, drugs able to increase either cyclic GMP (NO donors) or cyclic AMP (forskolin) caused relaxation of TEVM. These results not only show that important regulatory mechanisms of vascular tone are functional in the VSMC, including adenylyl and guanylyl cyclase, but also are indicative of highly differentiated VSMC expressing a contractile phenotype. They also have implications for the future use of a recently described human tissue-engineered blood vessel, which includes a similar multilayer of VSMC (1 , 40) . Since hyperproliferation of dedifferentiated VSMC is the major cause of mid- and long-term graft failure, it is reassuring to know that the VSMC present in our tissue-engineered vascular graft are highly differentiated and sensitive to the effects of NO. Indeed, it has been suggested that the vasoprotective effects of NO is linked to its anti-proliferative and anti-dedifferentiation activities on VSMC (41) .

An increase in the intracellular concentration of Ca2+ ions is determinant for contraction of vascular smooth muscle in response to vasoconstrictor agonists. Although intracellular mobilization of Ca2+ from different stores is often a major pathway, the contribution of Ca2+ entry is also clearly important (for a review, see refs 42 , 43 ). In the present study, it is shown that Ca2+ entry and Ca2+ release from intracellular stores were both involved in agonist-induced contraction of TEVM. In addition to the increase of [Ca2+]c, the data suggest that Ca2+ sensitization of contractile machinery also accounted for agonist-induced sustained contraction. These results suggest that TEVM VSMC possess many of the intracellular signaling pathways observed during contraction of native human umbilical vein.

In VSMC, Ca2+ entry may occur either through cationic nonselective channels or voltage-dependent Ca2+ channels, or both. In the present study, the increase in [Ca2+]c produced by ATP was blocked by the nonselective calcium channel blocker La3+, but not by the L-type calcium channel blocker nitrendipine. Furthermore, ATP, but neither KCl depolarization nor the dihydropyridine agonist BAY K 8644, caused Mn2+ influx as detected by rapid quench of Fura-2 fluorescence. Altogether, these results suggest that TEVB do not possess functional voltage-dependent Ca2+ channels and that ATP causes Ca2+ influx through a nonselective divalent cation-permeant pathway. Further studies including electrophysiology experiments are required to identify the involved ionic channels. An ATP-induced rise in [Ca2+]c in freshly isolated VSMC from human saphenous vein involved the activation of nonselective cation conductance (44) . However, the observed response was mediated by the activation of P2X receptors whereas P2Y1 receptors were implicated in ATP-induced Ca2+ entry in TEVM. The fact that TEVM apparently do not possess functional voltage-dependent Ca2+ entry pathway was intriguing. The present results contrasts with those in cultured aortic VSMCs showing that loss of dihydropyridine-sensitive high voltage-activated Ca2+ channels is associated with proliferation and lack of contractility (45) . It might be possible that TEVM VSMCs possess voltage-dependent Ca2+ channels whose functionality is down regulated by regulatory proteins such as phosphoprotein phosphatases type 2. Such a mechanism has been reported in VSMCs isolated from human umbilical vein (46) . Finally, TEVM VSMCs might possess voltage-dependent Ca2+ channels that were not expressed in a sufficient amount to allow the detection of their functionality with the experimental procedures performed in the present study. Nevertheless, results clearly show that TEVM possess nonselective cationic channels that allow Ca2+ to enter to promote both an increase in [Ca2+]c and contraction in response to vasoconstrictor agonists.

The present report also shows that in addition to inducing Ca2+ entry, vasoconstrictor agonist such as ATP produced an increase in [Ca2+]c in Ca2+-free medium suggesting that the agonist can promote the release of Ca2+ from intracellular stores, presumably in the sarcoplasmic reticulum. The experiments with caffeine and ryanodine show that VSMC possessed calcium stores releasable by these two compounds. Such stores are generally related to the Ca2+-induced Ca2+-release (CICR) mechanism via the opening of the ryanodine receptor. However, in some vascular smooth muscle cells, they may also contribute to the inositol 3',4,5-trisphosphate (IP3) induced Ca2+-release (47) . Since both caffeine and ryanodine inhibited ATP responses in Ca2+-free medium, it is unlikely that this response involved CICR activated by Ca2+ entry through the nonselective channels (44) because the inhibition was observed in the absence of extracellular Ca2+. Hence, Ca2+ release from intracellular stores was more probably elicited by IP3 formed upon activation of phospholipase Cß by Gq/11-coupled P2Y receptors (48) .

Finally, Ca2+ sensitization of contractile proteins appears to be involved in the sustained contractions of TEVM in response to agonists. The latter mechanism plays a crucial role in contractile response of human blood vessels to vasoconstrictor agonists in addition to the direct effects of increase [Ca2+]c. Ca2+ sensitization can be controlled by inhibition of myosin light chain phosphatase through the small GTP binding protein RhoA or PKC (49) . The mechanism involved in Ca2+ sensitization has not been investigated here, but the results suggest that the VSMC present in the TEVM possess at least one of the transduction pathways cited above.

In the last 20 years, progress in cell culture has made available large supplies of cultured human VSMC, while progress in cell biology has given rise to a wide array of methodologies to quantify cytosolic concentrations of various second messengers involved in VSMC contraction. Although cultured human VSMC do not contract in vitro under standard culture conditions, changes in second messenger concentrations are widely used as indirect measurements of human VSMC ‘contraction’. However, these methods have some serious limitations such as the need for expensive and delicate instruments. Furthermore, most of these methods are limited to the study of either short-term temporal variations in isolated cells or end point measurements of large cell populations. Although these methods provide precise second messenger measurements, these are not necessarily a good indication of the VSMC contraction or relaxation response since they only provide information on one of many regulatory mechanisms of muscle contraction. Finally, these methods cannot predict the level of contractile forces generated by VSMC.

Various groups have proposed methods to measure actual contraction of animal VSMC in culture. Methods based on image analysis and changes of length or area during contraction of isolated single VSMC have been developed (50 51 52) . Although the actual contraction can be observed in isolated single VSMC, changes in cell dimensions are not directly indicative of force generation since the cells are not exposed to a controlled mechanical load. Indeed, when adhered cells contract, they progressively lose their anchorage points as they round up. Alternatively, if a drug causes disassembly of the adhesion complexes, cells would lose their anchorage points, which would give the appearance of cell contraction. Another method is based on the ability of contracting cells cultured on a thin layer of silicone to create visible ruffles in the silicone (53 54 55 56) . However, ruffle formation is difficult to quantify, which explains why this method is usually used qualitatively. Though these single cell systems can give information, they are not optimal for contraction studies.

Using a classic tissue engineering approach, several groups have embedded various cell types in biochemically purified animal collagen gels and measured contractile forces with specially designed force transducers (57 58 59 60) . These tissues were bulky and lacked the physical shape and mechanical strength necessary to be used in a standard organ bath/force transducer apparatus found in cardiovascular pharmacology laboratories. Recently, a tubular construct showing measurable contractile responses to pharmacological agents was reported, but it was obtained by seeding animal VSMC in a biomaterial scaffolding (40) . Using a radically different approach (1) , we have produced the first human contractile TEVM. Moreover, this TEVM is completely biological, which avoids possible mechanical or chemical artifacts due to exogenous materials.

In this report, we provide evidence that TEVM display fundamental histological, functional, and many pharmacological characteristics of human vessels from which the cells were originally isolated. Moreover, this new in vitro model can be a substitute to animal tissues in contraction/relaxation studies performed in classical organ bath. Results showed that our TEVM possesses many receptor-mediated responses and second messengers involved in calcium handling. Although TEVM do not have all the characteristics of human vessels, the fact that they are tissue engineered from human cells allows for some experimental designs that otherwise could not be achieved. For instance, we recently reported that adventitial cells have a crucial role in the impairment of smooth muscle contractility in the rat aorta exposed to endotoxin (61) . In the future it will be possible to include other cell types in the preparation of TEVM in order to investigate their cross talks with VSMC. These cells will include not only endothelial cells or fibroblasts, as we recently reported (1) , but also other cells, like macrophages, which play a major physiopathological role in blood vessels (62) . In addition, VSMC from other vascular beds could be used to produce TEVM to study differential pharmacological responses. In conclusion, this study clearly demonstrates the potential of this new model as a tool for better understanding human vascular biology.


   ACKNOWLEDGMENTS
 
We thank Dr. Eric Petitclerc for invaluable discussions and suggestions. We thank Christa Schott for technical assistance with contraction/relaxation experiments and Dr. Bernard Muller for helpful suggestions for the nucleotide assays. This study was supported by the Medical Research Council (MRC) of Canada (to F.A. and L.G.), ‘Canadian Heart and Stroke Foundation’ (to F.A. and L.G.), Action concerté Sciences du vivant no 9 du Ministère de la Recherche et Technologie de la France, and a ‘France-Québec’ exchange program. L.G. was a recipient of Scholarships from ‘Fonds de la Recherche en Santé du Québec’ (FRSQ) and MRC, F.A.A. was the recipient of a Scholarship from FRSQ, and N.L’H. was the recipient of a Studentship from the ‘Fonds FCAR du Québec’.


   FOOTNOTES
 
1 Present address: Department of Bioengineering, University of California, San Diego, La Jolla, USA.

Received for publication April 24, 2000. Revision received August 2, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. L’Heureux, N., Paquet, S., Labbe, R., Germain, L., Auger, F. A. (1998) A completely biological tissue-engineered human blood vessel. FASEB J 12,47-56[Abstract/Free Full Text]
  2. Ross, R. (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (London) 362,801-809[Medline]
  3. Somlyo, A. P., Somlyo, A. V. (1994) Signal transduction and regulation in smooth muscle. Nature (London) 372,231-236[Medline]
  4. Kunapuli, S. P., Daniel, J. L. (1998) P2 receptor subtypes in the cardiovascular system. Biochem. J. 336,513-523
  5. Drummond, G. R., Cocks, T. M. (1995) Endothelium-dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery. Br. J. Pharmacol. 116,2473-2481[Medline]
  6. Marceau, F. (1995) Kinin B1 receptors: a review. Immunopharmacology 30,1-26[Medline]
  7. Zhao, M., Bo, X., Burnstock, G. (1996) Distribution of [3H] alpha, beta-methylene ATP binding sites in pulmonary blood vessels of different species. Pulm. Pharmacol. 9,167-174[Medline]
  8. Delaey, C., Van de Voorde, J. (1997) Heterogeneity of the inhibitory influence of sulfonylureas on prostanoid-induced smooth muscle contraction. Eur. J. Pharmacol. 325,41-46[Medline]
  9. Fukuroda, T., Kobayashi, M., Ozaki, S., Yano, M., Miyauchi, T., Onizuka, M., Sugishita, Y., Goto, K., Nishikibe, M. (1994) Endothelin receptor subtypes in human versus rabbit pulmonary arteries. J. Cell. Physiol. 76,1976-1982
  10. Chamley-Campbell, J., Campbell, G. R., Ross, R. (1979) The smooth muscle cell in culture. Physiol. Rev. 59,1-61[Free Full Text]
  11. Thyberg, J., Hedin, U., Sjolund, M., Palmberg, L., Bottger, B. A. (1990) Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 10,966-990[Free Full Text]
  12. Owens, G. K. (1995) Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev. 75,487-517[Abstract/Free Full Text]
  13. L’Heureux, N., Germain, L., Labbe, R., Auger, F. A. (1993) In vitro construction of a human blood vessel from cultured vascular cells: a morphologic study. J. Vasc. Surg. 17,499-509[Medline]
  14. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., Gabbiani, G. (1986) A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103,2787-2796[Abstract/Free Full Text]
  15. Chen, T. R. (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104,255-262[Medline]
  16. Cailla, H. L., Vannier, C. J., Delaage, M. A. (1976) Guanosine 3', 5'-cyclic monophosphate assay at 10(-15)-mole level. Anal. Biochem. 70,195-202[Medline]
  17. Andriantsitohaina, R., Lagaud, G. J., Andre, A., Muller, B., Stoclet, J. C. (1995) Effects of cGMP on calcium handling in ATP-stimulated rat resistance arteries. Am. J. Physiol. 268,H1223-H1231[Abstract/Free Full Text]
  18. Grynkiewicz, G., Poenie, M., Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260,3440-3450[Abstract/Free Full Text]
  19. Gessi, S., Rizzi, A., Calo, G., Agnello, G., Jorizzo, G., Mollica, G., Borea, P. A., Regoli, D. (1997) Human vascular kinin receptors of the B2 type characterized by radioligand binding. Br. J. Pharmacol. 122,1450-1454[Medline]
  20. Feletou, M., Martin, C. A., Molimard, M., Naline, E., Germain, M., Thurieau, C., Fauchere, J. L., Canet, E., Advenier, C. (1995) In vitro effects of HOE 140 in human bronchial and vascular tissue. Eur. J. Pharmacol. 274,57-64[Medline]
  21. Altura, B. M., Malaviya, D., Reich, C. F., Orkin, L. R. (1972) Effects of vasoactive agents on isolated human umbilical arteries and veins. Am. J. Physiol. 222,345-355
  22. Bo, X., Sexton, A., Xiang, Z., Nori, S. L., Burnstock, G. (1998) Pharmacological and histochemical evidence for P2X receptors in human umbilical vessels. Eur. J. Pharmacol. 353,59-65[Medline]
  23. Erlinge, D. (1998) Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen. Pharmacol. 31,1-8[Medline]
  24. Strobaek, D., Olesen, S. P., Christophersen, P., Dissing, S. (1996) P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells. Br. J. Pharmacol. 118,1645-1652[Medline]
  25. Pacaud, P., Malam-Souley, R., Loirand, G., Desgranges, C. (1995) ATP raises [Ca2+]i via different P2-receptor subtypes in freshly isolated and cultured aortic myocytes. Am. J. Physiol. 269,H30-H36[Abstract/Free Full Text]
  26. Robaye, B., Boeynaems, J. M., Communi, D. (1997) Slow desensitization of the human P2Y6 receptor. Eur. J. Pharmacol. 329,231-236[Medline]
  27. Nicholas, R. A., Lazarowski, E. R., Watt, W. C., Li, Q., Boyer, J., Harden, T. K. (1996) Pharmacological and second messenger signalling selectivities of cloned P2Y receptors. J. Auton. Pharmacol. 16,319-323[Medline]
  28. Nicholas, R. A., Watt, W. C., Lazarowski, E. R., Li, Q., Harden, K. (1996) Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol. Pharmacol. 50,224-229[Abstract]
  29. Harper, S., Webb, T. E., Charlton, S. J., Ng, L. L., Boarder, M. R. (1998) Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. Br. J. Pharmacol. 124,703-710[Medline]
  30. Parr, C. E., Sullivan, D. M., Paradiso, A. M., Lazarowski, E. R., Burch, L. H., Olsen, J. C., Erb, L., Weisman, G. A., Boucher, R. C., Turner, J. T. (1994) Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy [published erratum appears in Proc. Natl. Acad. Sci. USA, 1994, Vol. 91, p. 13067]. Proc. Natl. Acad. Sci. USA 91,3275-3279[Abstract/Free Full Text]
  31. Communi, D., Pirotton, S., Parmentier, M., Boeynaems, J. M. (1995) Cloning and functional expression of a human uridine nucleotide receptor. J. Biol. Chem. 270,30849-30852[Abstract/Free Full Text]
  32. Nguyen, T., Erb, L., Weisman, G. A., Marchese, A., Heng, H. H., Garrad, R. C., George, S. R., Turner, J. T., O’Dowd, B. F. (1995) Cloning, expression, and chromosomal localization of the human uridine nucleotide receptor gene. J. Biol. Chem. 270,30845-30848[Abstract/Free Full Text]
  33. Charlton, S. J., Brown, C. A., Weisman, G. A., Turner, J. T., Erb, L., Boarder, M. R. (1996) Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP. Br. J. Pharmacol. 119,1301-1303[Medline]
  34. Erlinge, D., Hou, M., Webb, T. E., Barnard, E. A., Moller, S. (1998) Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR. Biochem. Biophys. Res. Commun. 248,864-870[Medline]
  35. Brown, C., Pan, X., Hassid, A. (1999) Nitric oxide and C-type atrial natriuretic peptide stimulate primary aortic smooth muscle cell migration via a cGMP-dependent mechanism: relationship to microfilament dissociation and altered cell morphology. Circ. Res. 84,655-667[Abstract/Free Full Text]
  36. Dubey, R. K., Jackson, E. K., Luscher, T. F. (1995) Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell. Role of cyclic-nucleotides and angiotensin1 receptors. J. Clin. Invest. 96,141-149
  37. Lincoln, T. M., Cornwell, T. L. (1991) Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. Blood Vessels 28,129-137[Medline]
  38. Jiang, H., Colbran, J. L., Francis, S. H., Corbin, J. D. (1992) Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. J. Biol. Chem. 267,1015-1019[Abstract/Free Full Text]
  39. Cornwell, T. L., Soff, G. A., Traynor, A. E., Lincoln, T. M. (1994) Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells. J. Vasc. Res. 31,330-337[Medline]
  40. Niklason, L. E., Gao, J., Abbott, W. M., Hirschi, K. K., Houser, S., Marini, R., Langer, R. (1999) Functional arteries grown in vitro. Science 284,489-493[Abstract/Free Full Text]
  41. Boerth, N. J., Dey, N. B., Cornwell, T. L., Lincoln, T. M. (1997) Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. J. Vasc. Res. 34,245-259[Medline]
  42. Allen, B. G., Walsh, M. P. (1994) The biochemical basis of the regulation of smooth-muscle contraction. Trends Biochem. Sci. 19,362-368[Medline]
  43. Osol, G. (1995) Mechanotransduction by vascular smooth-muscle. J. Vasc. Res. 32,275-292[Medline]
  44. Loirand, G., Pacaud, P. (1995) Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein. Pfluegers Arch 430,429-436[Medline]
  45. Richard, S., Neveu, D., Carnac, G., Bodin, P., Travo, P., Nargeot, J. (1992) Differential expression of voltage-gated Ca2+-currents in cultivated aortic myocytes. Biochim. Biophys. Acta 1160,95-104[Medline]
  46. Groschner, K., Schumann, K., Mieskes, G., Baumgartner, W., Romanin, C. (1996) A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells. Biochem. J. 318,513-517
  47. Lagaud, G. J., Stoclet, J. C., Andriantsitohaina, R. (1996) Calcium handling and purinoceptor subtypes involved in ATP-induced contraction in rat small mesenteric arteries. J. Physiol. (London) 492,689-703[Medline]
  48. Murthy, K. S., Makhlouf, G. M. (1998) Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-beta1 via Galphaq/11 and to PLC-beta3 via Gbetagammai3. J. Biol. Chem. 273,4695-4704[Abstract/Free Full Text]
  49. Pfitzer, G., Arner, A. (1998) Involvement of small GTPases in the regulation of smooth muscle contraction. Acta Physiol. Scand. 164,449-456[Medline]
  50. Okada, K., Tsai, P., Briner, V. A., Caramelo, C., Schrier, R. W. (1991) Effects of extra- and intracellular pH on vascular action of arginine vasopressin. Am. J. Physiol. 260,F39-F45[Abstract/Free Full Text]
  51. Dubey, R. K., Roy, A., Overbeck, H. W. (1992) Culture of renal arteriolar smooth muscle cells. Mitogenic responses to angiotensin II. Circ. Res. 71,1143-1152[Abstract/Free Full Text]
  52. Briner, V. A., Tsai, P., Wang, X., Schrier, R. W. (1993) Divergent effects of acute and chronic ethanol exposure on contraction and Ca2+ mobilization in cultured vascular smooth muscle cells. Am. J. Hypertens. 6,268-275[Medline]
  53. Murray, T. R., Marshall, B. E., Macarak, E. J. (1990) Contraction of vascular smooth muscle in cell culture. J. Cell. Physiol. 143,26-38[Medline]
  54. Shirinsky, V. P., Birukov, K. G., Sobolevsky, A. V., Vedernikov, Y. P., Posin, E., Popov, E. G. (1992) Contractile rabbit aortic smooth muscle cells in culture. Preparation and characterization. Am J Hypertens. 5,124S-130S[Medline]
  55. Birukov, K. G., Frid, M. G., Rogers, J. D., Shirinsky, V. P., Koteliansky, V. E., Campbell, J. H., Campbell, G. R. (1993) Synthesis and expression of smooth muscle phenotype markers in primary culture of rabbit aortic smooth muscle cells: influence of seeding density and media and relation to cell contractility. Exp. Cell Res. 204,46-53[Medline]
  56. Boswell, C. A., Majno, G., Joris, I., Ostrom, K. A. (1992) Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts. Microvasc. Res. 43,178-191[Medline]
  57. Eastwood, M., McGrouther, D. A., Brown, R. A. (1994) A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling. Biochim. Biophys. Acta 1201,186-192[Medline]
  58. Kolodney, M. S., Wysolmerski, R. B. (1992) Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study. J. Cell Biol. 117,73-82[Abstract/Free Full Text]
  59. Pilcher, B. K., Levine, N. S., Tomasek, J. J. (1995) Thrombin promotion of isometric contraction in fibroblasts: its extracellular mechanism of action. Plast. Reconstr. Surg. 96,1188-1195[Medline]
  60. Moy, A. B., Van Engelenhoven, J., Bodmer, J., Kamath, J., Keese, C., Giaever, I., Shasby, S., Shasby, D. M. (1996) Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. J. Clin. Invest. 97,1020-1027[Medline]
  61. Kleschyov, A. L., Muller, B., Schott, C., Stoclet, J. C. (1998) Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta. Br. J. Pharmacol. 124,623-626[Medline]
  62. Petitclerc, E., Levesque, L., Grose, J. H., Poubelle, P. E., Marceau, F. (1996) Pathologic leukocyte infiltration of the rabbit aorta confers a vasomotor effect to chemotactic peptides through cyclooxygenase-derived metabolites. J. Immunol. 156,3426-3434[Abstract]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
P. J. Pagano and D. D. Gutterman
The adventitia: The outs and ins of vascular disease
Cardiovasc Res, September 1, 2007; 75(4): 636 - 639.
[Full Text] [PDF]

Home page
 Cardiovasc ResHome page
F. A. Auger, P. D'Orleans-Juste, and L. Germain
Adventitia contribution to vascular contraction: Hints provided by tissue-engineered substitutes
Cardiovasc Res, September 1, 2007; 75(4): 669 - 678.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. Y. Liu, D. D. Swartz, H. F. Peng, S. F. Gugino, J. A. Russell, and S. T. Andreadis
Functional tissue-engineered blood vessels from bone marrow progenitor cells
Cardiovasc Res, August 1, 2007; 75(3): 618 - 628.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
K.-E. Andersson and G. J. Christ
REGENERATIVE PHARMACOLOGY: THE FUTURE IS NOW
Mol. Interv., April 1, 2007; 7(2): 79 - 86.
[Abstract] [Full Text] [PDF]