Tissue-engineered Vascular Media Research Articles

Interleukin-10 controls the protective effects of circulating microparticles from septic shock patients on tissue-engineered vascular media

Interleukin-10 controls the protective effects of circulating microparticles from septic shock patients on tissue-engineered vascular media

Interleukin-10 controls the protective effects of circulating microparticles from patients with septic shock on tissue-engineered vascular media

During sepsis, inflammation can be orchestrated by the interaction between circulating and vascular cells that, under activation, release MPs (microparticles). Previously, we reported that increased circulating MPs in patients with sepsis play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity. The present study was designed to investigate the effects of MPs from patients with sepsis on the contractile response of TEVM (tissue-engineered vascular media). TEVM that were composed only of a media layer were produced by tissue engineering from human arterial SMCs (smooth muscle cells) isolated from umbilical cords. TEVM was incubated with MPs isolated from whole blood of 16 patients with sepsis. TEVM were incubated for 24 h with MPs and used for the study of vascular contraction, direct measurements of NO and O2- (superoxide anion) production by EPR and quantification of mRNA cytokine expression. MPs from patients with sepsis increased contraction induced by histamine in TEVM. This effect was not associated with inflammation, neither linked to the activation of NF-κB (nuclear factor κB) pathway nor to the increase in iNOS (inducible NO synthase) and COX (cyclo-oxygenase)-2 expression. In contrast, mRNA expression of IL (interleukin)-10 was enhanced. Then, we investigated the effect of IL-10 on vascular hyporeactivity induced by LPS (lipopolysaccharide). Although IL-10 treatment did not modify the contractile response in TEVM by itself, this interleukin restored contraction in LPS-treated TEVM. In addition, IL-10 treatment both prevented vascular hyporeactivity induced by LPS injection in mice and improved survival of LPS-injected mice. These findings show an association between the capacity of MPs from patients with sepsis to restore vascular hyporeactivity induced by LPS and their ability to increase IL-10 in the tissue-engineered blood vessel model.

Human fibroblast-derived ECM as a scaffold for vascular tissue engineering

The self-assembly approach is based on the capability of mesenchymal cells to secrete and organize their own extracellular matrix (ECM). This tissue engineering method allows for the fabrication of autologous living tissues, such as tissue-engineered blood vessels (TEBV) and skin. However, the secretion of ECM by smooth muscle cells (SMCs), required to produce the vascular media, may represent a long process in vitro. The aim of this work was to reduce the time required to produce a tissue-engineered vascular media (TEVM) and extend the production of TEVM with SMCs from all patients without compromising its mechanical and functional properties. Therefore, we developed a decellularized matrix scaffold (dMS) produced from dermal fibroblasts (DF) or saphenous vein fibroblasts (SVF), in which SMCs were seeded to produce a TEVM. Mechanical and contractile properties of these TEVM (referred to as nTEVM) were compared to standard self-assembled TEVM (sTEVM). This approach reduced the production time from 6 to 4 weeks. Moreover, nTEVM were more resistant to tensile load than sTEVM and their vascular reactivity was also improved. This new fabrication technique allows for the production of a vascular media using SMCs isolated from any patient, regardless of their capacity to synthesize ECM. Moreover, these scaffolds can be stored to be available when needed, in order to accelerate the production of the vascular substitute using autologous vascular cells.

A Computer-Controlled Apparatus for the Characterization of Mechanical and Viscoelastic Properties of Tissue-Engineered Vascular Constructs

Tissue-engineered blood vessels can be partly characterized by analyzing their mechanical properties using burst pressure testing, compliance measurement, creep and cyclic testing. Studying these parameters provides information on the capability of a fabrication method to produce tissue-engineered blood vessels (TEBV) and allow for the optimization of their resistance and viscoelastic properties. This study presents the design and fabrication of an apparatus allowing accurate and reliable measurements of the mechanical properties of tissue-engineered vascular constructs. A computer-controlled system was designed to monitor pressure and diameter variations of vascular constructs submitted to hydrostatic loading. The system was programmed to control the motorized portion of the setup and allow simultaneous data acquisition, analysis and real-time display. Data acquisition cards allow for synchronous monitoring of pressure and diameter of the constructs through a pressure transducer and a CCD camera. Image analysis and pressure data computation resulted in compliance, creep and dynamic characterization of the tested tissues. This experimental setup succeeded in measuring the burst pressure, compliance, creep and cyclic behavior of tissue-engineered vascular media (TEVM), adventitia (TEVA) and a combination of a media and an adventitia (TEVMA) reconstructed by the self-assembly method. Our apparatus has proven to be a precise and reliable tool for the characterization of the mechanical properties of vascular constructs.

Biaxial biomechanical properties of self-assembly tissue-engineered blood vessels

Along with insights into the potential for graft success, knowledge of biomechanical properties of small diameter tissue-engineered blood vessel (TEBV) will enable designers to tailor the vessels' mechanical response to closer resemble that of native tissue. Composed of two layers that closely mimic the native media and adventitia, a tissue-engineered vascular adventitia (TEVA) is wrapped around a tissue-engineered vascular media (TEVM) to produce a self-assembled tissue-engineered media/adventia (TEVMA). The current study was undertaken to characterize the biaxial biomechanical properties of TEVM, TEVA and TEVMA under physiological pressures as well as characterize the stress-free reference configuration. It was shown that the TEVA had the greatest compliance over the physiological loading range while the TEVM had the lowest compliance. As expected, compliance of the SA-TEBV fell in between with an average compliance of 2.73 MPa(-1). Data were used to identify material parameters for a microstructurally motivated constitutive model. Identified material parameters for the TEVA and TEVM provided a good fit to experimental data with an average coefficient of determination of 0.918 and 0.868, respectively. These material parameters were used to develop a two-layer predictive model for the response of a TEVMA which fit well with experimental data.

A Novel Single-Step Self-Assembly Approach for the Fabrication of Tissue-Engineered Vascular Constructs

There is a clinical need for a functional tissue-engineered blood vessel because small-caliber arterial graft (<5 mm) applications are limited by the availability of suitable autologous vessels and suboptimal performances of synthetic grafts. This study presents an analysis of the mechanical properties of tissue-engineered vascular constructs produced using a novel single-step self-assembly approach. Briefly, the tissue-engineered vascular media were produced by culturing smooth muscle cell in the presence of sodium l-ascorbate until the formation of a cohesive tissue sheet. This sheet was then rolled around a tubular support to create a media construct. Alternatively, the tissue-engineered vascular adventitia was produced by rolling a tissue sheet obtained from dermal fibroblasts or saphenous vein fibroblasts. The standard self-assembly approach to obtain the two-layer tissue-engineered vascular constructs comprising both media and adventitia constructs consists of two steps in which tissue-engineered vascular media were first rolled on a tubular support and a tissue-engineered vascular adventitia was then rolled on top of the first layer. This study reports an original alternative method for assembling tissue-engineered vascular constructs comprising both media and an adventitia in a single step by rolling a continuous tissue sheet containing both cell types contiguously. This tissue sheet was produced by growing smooth muscle cells alongside fibroblasts (saphenous vein fibroblasts or dermal fibroblasts) in the same culture dish separated by a spacer, which is removed later in the culture period. The mechanical strength assessed by uniaxial tensile testing, burst pressure measurements, and viscoelastic behavior evaluated by stepwise stress relaxation tests reveals that the new single-step fabrication method significantly improves the mechanical properties of tissue-engineered vascular construct for both ultimate tensile strength and all the viscoelastic moduli.

Applications of Human Tissue-Engineered Blood Vessel Models to Study the Effects of Shed Membrane Microparticles from T-Lymphocytes on Vascular Function

Microparticles (MPs) are membrane vesicles harboring cell surface proteins and containing cytoplasmic components of the original cell. High levels of circulating MPs have been detected in pathological states associated with vascular dysfunction. We took advantage of the self-assembly method of tissue engineering to produce in vitro three vascular constructs from human vascular smooth muscle cells and fibroblasts to investigate the role of the adventitia in the modulation of vascular tone by MPs, comparing the contractile response of each of these constructs to histamine. The first two were composed of an adventitia (tissue-engineered vascular adventitia (TEVA)) or a media (tissue-engineered vascular media (TEVM)) solely, and the third one contained a media and an adventitia (tissue-engineered vascular media and adventitia (TEVMA)). In the three constructs, the results show that histamine induces contraction insensitive to blockade of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) and not affected by MP treatment. MPs decreased NO production and nuclear factor (NF)-kappaB expression but did not affect superoxide anion (O(2)(-)) release in TEVA. MPs enhanced NF-kappaB expression but did not affect iNOS and COX-2 expression or NO or O(2)(-) release in TEVM. In TEVMA, MPs did not enhance NF-kappaB expression, but COX-2 expression was higher, and O(2)(-) release was lower. Thus, MPs affected NO, O(2)(-), NF-kappaB, and COX-2 in a subtle fashion to maintain the contractile response to histamine. The use of tissue-engineered vascular constructs results in a better understanding of the effect of MPs on human adventitia and media.

Polyphenols modulate calcium-independent mechanisms in human arterial tissue-engineered vascular media

In the present study, an arterial tissue-engineered vascular media (TEVM) was produced from cultured human smooth muscle cells of the umbilical artery and we took advantage of this model to evaluate the regulation of contraction and the signalling pathways of polyphenols in arteries. Cultured human smooth muscle cells of the umbilical artery were used to produce arterial TEVMs. Contraction experiments were performed to determine intracellular targets involved in the modulation of contraction by polyphenols extract from red wine, Provinols (SEPPIC Groupe Air Liquide, Paris, France). Smooth muscle cells in arterial TEVM displayed a differentiated phenotype as demonstrated by the expression of alpha-smooth muscle actin, a vascular smooth muscle-specific marker, and tissue contraction in response to vasoconstrictor and vasodilator agents. Contractions caused by histamine were associated with an increase in [Ca(2+)](i) and a Ca(2+)-independent signalling pathway. The latter pathway involved mechanisms sensitive to protein kinase C, myosin light chain kinase, and Rho-associated protein kinase inhibitors. The regulation of contraction induced by Provinols shows that treatment of arterial TEVM with this compound significantly decreased histamine-induced contraction. This effect was associated with the inhibition of the Rho-associated protein kinase pathway and the decrease in alpha-smooth muscle actin expression. The use of arterial TEVM, brings new insights into the mechanisms by which polyphenols regulate vascular contraction in the human artery.

Mechanical Loading Modulates the Differentiation State of Vascular Smooth Muscle Cells

The cause underlying the onset of stenosis after vascular reconstruction is not well understood. In the present study, we evaluated the effect of mechanical unloading on the differentiation state of human vascular smooth muscle cells (hVSMCs) using a tissue-engineered vascular media (TEVM). hVSMCs cultured in a mechanically loaded three-dimensional environment, known as a living tissue sheet, had a higher differentiated state than cells grown on plastic. When the living tissue sheet was detached from its support, the release of the residual stress resulted in a mechanical unloading and cells within the extracellular matrix (ECM) dedifferentiated as shown by downregulation of differentiation markers. The relaxed living tissue sheet can be rolled onto a tubular mandrel to form a TEVM. The rolling procedure resulted in the reintroduction of a mechanical load leading to a cohesive compacted tissue. During this period, cells gradually redifferentiated and aligned circumferentially to the tubular support. Our results suggest that differentiation of hVSMCs can be driven by mechanical loading and may occur simultaneously in the absence of other cell types. The extrapolation of our results to the clinical context suggests the hypothesis that hVSMCs may adopt a proliferative phenotype resulting from the mechanical unloading of explanted blood vessels during vascular reconstruction. Therefore, we propose that this mechanical unloading may play an important role in the onset of vascular graft stenosis.

Tissue-Engineered Human Vascular Media Produced in Vitro by the Self-Assembly Approach Present Functional Properties Similar to Those of Their Native Blood Vessels

We have developed a tissue-engineering approach for the production of a completely biological blood vessel from cultured human cells. In the present study, we took advantage of this tissue-engineering method to demonstrate that it can be used to reproduce the subtle differences in the expression of receptors present on the media of native human blood vessels. Indeed, a small percentage (3 of 18) of native human umbilical cord veins (HUCVs) responded to endothelin, the most powerful vasopressor agent known to date, via both endothelin A (ET(A)) and endothelin B (ET(B)) receptor activation. In contrast, most HUCVs tested responded to ET via ET(A) receptor activation only. Tissue-engineered vascular media (TEVM) were next reconstructed by using vascular smooth muscle cells (VSMCs) isolated and cultured from HUCVs expressing both ET(A) and ET(B) receptors to determine the functional integrity of our TEVM model. The reconstructed TEVM presents an endothelin response similar to that of respective HUCVs from which VSMCs were isolated. Reverse transcriptase polymerase chain reaction on TEVM reconstructed in vitro correlated these vasocontractile profiles by showing the presence of messenger RNA for both ET(A) and ET(B) receptors. Taken together with recently published results on TEVM expressing only ET(A) receptor, these results show that our reconstructed TEVM present a similar ET response profile as the blood vessel from which the VSMCs were isolated and cultured. These findings indicate that subtle differences, such as receptor expression, are preserved in the reconstructed tissue. Therefore, our TEVM offers a valuable human in vitro model with which to study the functionality of human blood vessels, such as their vasoactive response, or to perform pharmacologic studies.

Mechanism of potentiation by polyphenols of contraction in human vein-engineered media

The potential of natural dietary polyphenols in the treatment of vascular diseases originating from veins has been suggested in the literature. However, the mechanisms involved to explain the effects of polyphenols are not yet elucidated. Therefore, the aim of this study was to investigate the mechanisms by which polyphenols from red wine (Provinols) modulated contraction in human veins. We took advantage of a human model previously reported as a new tool for pharmacological research, using tissue-engineered techniques allowing the production of vascular media based exclusively on human smooth muscle cells. Thus human tissue-engineered vascular media (TEVM) were produced with cells originating from umbilical cord vein. TEVM were treated with either vehicle or Provinols. Results showed that treatment of TEVM with Provinols significantly potentiated the contractile responses induced by histamine and bradykinin. The potentiating effect of Provinols was not associated with an enhancement of histamine-induced increase in cytosolic calcium; rather, it implied the presence of a Ca(2+)-independent signaling pathway. Pharmacological studies indicated that action of Provinols took place at the level of phospholipase A(2)-Rho-kinase pathway and was associated with an enhancement of myosin light chain kinase activity. These results, obtained using the human TEVM, bring new insights to explain the regulation of venous contraction by polyphenols.

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

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.