Environmental Influences on Endothelial to Mesenchymal Transition in Developing Implanted Cardiovascular Tissue-Engineered Grafts

Tissue-engineered grafts for cardiovascular structures experience biochemical stimuli and mechanical forces that influence tissue development after implantation such as the immunological response, oxidative stress, hemodynamic shear stress, and mechanical strain. Endothelial cells are a cell source of major interest in vascular tissue engineering because of their ability to form a luminal antithrombotic monolayer. In addition, through their ability to undergo endothelial to mesenchymal transition (EndMT), endothelial cells may yield a cell type capable of increased production and remodeling of the extracellular matrix (ECM). ECM is of major importance to the mechanical function of all cardiovascular structures. Tissue engineering approaches may employ EndMT to recapitulate, in part, the embryonic development of cardiovascular structures. Improved understanding of how the environment of an implanted graft could influence EndMT in endothelial cells may lead to novel tissue engineering strategies. This review presents an overview of biochemical and mechanical stimuli capable of influencing EndMT, discusses the influence of these stimuli as found in the direct environment of cardiovascular grafts, and discusses approaches to employ EndMT in tissue-engineered constructs

Matrix Production and Organization by Endothelial Colony Forming Cells in Mechanically Strained Engineered Tissue Constructs

<div><p>Aims</p><p>Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an <i>in vitro</i> cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs) are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT). We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor β1 (TGFβ1).</p><p>Methods and Results</p><p>A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFβ1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFβ1 before exposing them to strain led to more homogenous matrix production.</p><p>Conclusions</p><p>Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both <i>in vitro</i> and <i>in situ</i> cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.</p></div

Antibodies and corresponding secondary antibodies used for immunofluorescence.

<p>Antibodies and corresponding secondary antibodies used for immunofluorescence.</p

Elasin and collagen staining on slices of 3D constructs (A) and quantification of elastin (B).

<p>Representative images are shown for the Verhoeff van Gieson staining (A), with elastin in blue-black and collagen in red. On this global scale results for all ECFC groups (all medium groups, static or cyclic strain) were similar. (i) ECFCs showed both collagen and elastin, (ii) HVSCs showed only collagen. (iii) Native human tissue was used as a positive control for collagen and elastin. Scale bar 200 µm. Elastin quantification by Fastin Elastin assay (B) showed significant differences, where cyclic full medium+TGFβ1 is significantly higher than primed static and static bare medium+TGFβ1, and HVSC static is significantly higher than primed static, static bare medium +TGFβ1 and static full medium +TGFβ1; * indicates p<0.05, N = 3.</p

EndoMT markers and ECM genes and corresponding primer sequences used for qPCR.

<p>EndoMT markers and ECM genes and corresponding primer sequences used for qPCR.</p

Schematic overview of the experimental set up.

<p>Top view shows 2D cell layer and 3D constructs on Bioflex plates with 2 (static strain protocol) or 4 (cyclic strain protocol) rectangular Velcro strips glued to the flexible membrane. Scale bar indicates 3 mm. Velcro strips leave space for cell-populated fibrin gels. Side views show schematic cross-section of the Flexcell setup used for cyclic strain application. When vacuum is applied, the flexible membrane is deformed over a rectangular loading post resulting in a uniaxial strain of the membrane and, thus, of the tissue. ECFCs were cultured in full medium, full medium +TGFβ1, bare medium and bare medium +TGFβ1. The bottom row shows analyses performed on the samples.</p

Phase contrast and immunofluorescent images of ECFCs on coverglasses in different media.

<p>Phase contrast pictures, after 14 days of culture, at 10×magnification show the complete change in ECFC morphology in bare medium +TGFβ1, where cells are elongated and they have lost the cobblestone morphology, still retained by ECFCs cultured in the other media. Immunostaining for CD31 (red), αSMA (green) and DAPI (blue), confirmed the differences observed by optical imaging, with loss of CD31 in part of the cells cultured in full medium +TGFβ1 and bare medium +TGFβ1, and αSMA stress fibers only detected in bare medium +TGFβ1. Immunostaining for collagen type III (green), IV (red) and DAPI (blue) showed a decrease in matrix production in bare medium with or without TGFβ1. This result was also confirmed for collagen type I (red). The scalebar represents 100 µm.</p

In situ tissue engineering of functional small-diameter blood vessels by host circulating cells only

Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent matrix formation. Typically, vascular regeneration in small animals is governed by transanastomotic cell ingrowth. However, this process is very rare in humans and hence less relevant for clinical translation. Therefore, a novel rat model was developed, in which cell ingrowth from the adjacent tissue is inhibited using Gore-Tex sheathing. Using this model, our aim here was to prove that functional blood vessels can be formed in situ through the host inflammatory response, specifically by blood-borne cells. The model was validated by implanting sex-mismatched aortic segments on either anastomoses of an electrospun poly(ε-caprolactone) (PCL) graft, filled with fibrin gel, into the rat abdominal aorta. Fluorescent in situ hybridization analysis revealed that after 1 and 3 months in vivo, over 90% of infiltrating cells originated from the bloodstream, confirming the effective shielding of transanastomotic cell ingrowth. Using the validated model, PCL/fibrin grafts were implanted, either or not loaded with monocyte chemotactic protein-1 (MCP-1), and cell infiltration and tissue development were investigated at various key time points in the healing cascade. A phased healing response was observed, initiated by a rapid influx of inflammatory cells, mediated by the local release of MCP-1. After 3 months in vivo, the grafts consisted of a medial layer with smooth muscle cells in an oriented collagen matrix, an intimal layer with elastin fibers, and confluent endothelium. This study proves the regenerative potential of cells in the circulatory system in the setting of in situ vascular tissue engineering

qPCR of ECFCs and primed ECFCs under static and cyclic strain in 3D.

<p>Expression of ECM genes is shown relative to primed ECFCs before seeding into the 3D gels. (A) Cyclic strain significantly decreased expression of collagen type I and III in ECFCs for all medium groups. The expression of collagen IV was not detected. (B) Cyclic strain significantly upregulated expression of collagen type I and III in ECFCs primed with TGFβ1. Elastin expression was reduced in these cells in response to cyclic strain. * = p<0.05, ** = p<0.01, N = 3.</p