Christine F. Maurus

Living, autologous pulmonary artery conduits tissue engineered from human umbilical cord cells

BACKGROUND Tissue engineering represents a promising approach to in vitro creation of living, autologous replacements with the potential to grow, repair, and remodel. Particularly in a congenital operation, there is a substantial need for such implantation materials. We previously demonstrated fabrication of completely autologous, functional heart valves on the basis of peripheral vascular cells. Presently the feasibility of creating pulmonary artery conduits from human umbilical cord cells was investigated. METHODS Human umbilical cord cells were harvested and expanded in culture. Pulmonary conduits fabricated from rapidly bioabsorbable polymers were seeded with human umbilical cord cells and grown in vitro in a pulse duplicator bioreactor. Morphologic characterization of the generated neo-tissues included histology, transmission, and scanning electron microscopy. Characterization of extracellular matrix was comprised of immunohistochemistry. Extracellular matrix protein content and cell proliferation were quantified by biochemical assays. Biomechanical testing was performed using stress-strain and burst-stress tests. RESULTS Histology of the conduits revealed viable, layered tissue and extracellular matrix formation with glycosaminoglycans and collagens I and III. Cells stained positive for vimentin and alpha-smooth muscle actin. Scanning electron microscopy showed confluent, homogenous tissue surfaces. Transmission electron microscopy demonstrated elements typical of viable myofibroblasts, such as collagen, fibrils, and elastin. Extracellular matrix proteins were significantly lower compared with native tissue; the cell content was increased. The mechanical strength of the pulsed constructs was comparable with native tissue; the static controls were significantly weaker. CONCLUSIONS In vitro fabrication of tissue-engineered human pulmonary conduits was feasible utilizing human umbilical cord cells and a biomimetic culture environment. Morphologic and mechanical features approximated human pulmonary artery. Human umbilical cord cells demonstrated excellent growth properties representing a new, readily available cell source for tissue engineering without necessitating the sacrifice of intact vascular donor structures.

A new source for cardiovascular tissue engineering: human bone marrow stromal cells

OBJECTIVE Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering. METHODS Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays. RESULTS Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs. CONCLUSIONS Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.

Dextran sulfate acts as an endothelial cell protectant and inhibits human complement and natural killer cell-mediated cytotoxicity against porcine cells.

Background. The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an “EC-protectant” preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium. Methods. Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays. Results. DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-&agr; stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively. Conclusions. DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an “EC protectant.”

Chlamydia pneumoniae induces aponecrosis in human aortic smooth muscle cells

BackgroundThe intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.ResultsChlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria.ConclusionThis study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity.

Background. Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-&agr; and hypoxia/reoxygenation in an in vitro model of I/R. Methods. MVEC were stimulated in vitro for 8 h with TNF-&agr;, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using 51Cr-release assays. Results. Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-&agr; but unaffected by hypoxia/reoxygenation in the absence of TNF-&agr;. ICAM-1 expression was further increased by a combination of TNF-&agr; and hypoxia/reoxygenation, whereas TNF-&agr;-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-&agr; and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-&agr; and slighty reduced by hypoxia/reoxygenation. Conclusions. Endothelial activation with TNF-&agr;, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro

OBJECTIVES Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha, TNF-alpha), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. METHODS Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-alpha (1000 U/ml) and exposed to hypoxia (1% O(2)), reoxygenation (21% O(2)) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. RESULTS Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-alpha significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-alpha, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-alpha stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. CONCLUSIONS Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-alpha but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.