Puncturing of lyophilized tissue engineered vascular matrices enhances the efficiency of their recellularization

Data on in vitro engineered "off the shelf" matrices support the concept of endogenous cellular repopulation driving the graft's remodeling via immune-mediated response. This seems important to further accelerate the cell reconstitution and may play a crucial role when mononuclear cells are used. Nevertheless, studies on decellularized xenogeneic grafts showed only limited host cell repopulation post-implantation. This study aims at a systematic comparison of reseeding methods (dripping, injection, bathing in a cell suspension and combined puncturing-dripping method) to define the most efficient technique enhancing recellularization of tissue engineered vascular matrices (patches, vessels, small diameter and standard size valves) prior implantation. The constructs were analyzed histologically, biochemically and biomechanically. Various preconditioning treatments (wet, lyophilized and air-dried) combined with reseeding methods demonstrated the highest cell loading efficiency, despite applied crimping and flow stress, of lyophilization followed by puncturing-dripping technique. This novel seeding method allows for an efficient, time saving graft reseeding that can be used within a one-step cardiovascular clinical intervention. STATEMENT OF SIGNIFICANCE The concept of living tissue engineered, self-repairing, autologous cardiovascular replacements, was proposed alternatively to existing synthetic/xenogeneic prostheses. Recent studies in animal models demonstrate faster in vivo recellularization after grafts pre-seeding with cells prior implantation. Pre-seeded cells hold either, the ability to differentiate directionally or attract host cells, crucial for graft integration and remodeling. It is unclear, however, how efficient the pre-loading is and how well cells withstand the flow. The study presents a systematic overview on cell loading techniques of different cardiovascular constructs, tested under static and dynamic conditions. Comparison illustrates a significantly higher efficiency of cells loading in lyophilized tissues punctured before their standard seeding. This technique may beneficially accelerate remodeling of cardiovascular grafts in further in vivo studies

A genetic analysis of nitric oxide-mediated signaling during chronological aging in the yeast

In mammals, NO•, a signaling molecule is implicated in the regulation of vasodilation, neurotransmission and immune response. It is believed that NO• is a signaling molecule also in unicellular organism like yeast and may be involved in the regulation of apoptosis and sporulation. It has been reported that NO• is produced during chronological aging (CA) leading to an increase of the superoxide level, which in turn mediates apoptosis. Since this conclusion was based on indirect measurements of NO• by the Griess reaction, the role of NO• signaling during CA in the yeast remains uncertain. We investigated this issue more precisely using different genetic and biochemical methodologies. We used cells lacking the factors influencing nitrosative stress response like flavohemoglobin metabolizing NO•, S-nitrosoglutathione reductase metabolizing S-nitrosoglutathione and the transcription factor Fzf1p mediating NO• response. We measured the standard parameters describing CA and found an elevation in the superoxide level, percentage of death cells, the level of TUNEL positive cells and a decrease in proliferating potential. These observations showed no significant differences between wild type cells and the disruptants except for a small elevation of the superoxide level in the Δsfa1 mutant. The intracellular NO• level and flavohemoglobin expression decreased rather than increased during CA. Products of general nitrogen metabolism and protein tyrosine nitration were slightly decreased during CA, the magnitude of changes showing no differences between the wild type and the mutant yeast. Altogether, our data indicate that apoptosis during yeast CA is mediated by superoxide signaling rather than NO• signaling

Celiprolol but not losartan improves the biomechanical integrity of the aorta in a mouse model of vascular Ehlers–Danlos syndrome

AIMS: Antihypertensive drugs are included in the medical therapy of vascular Ehlers-Danlos syndrome (vEDS). The β-blocker celiprolol has been suggested to prevent arterial damage in vEDS, but the underlying mechanism remains unclear. It is also unknown whether the widely used angiotensin II receptor type 1 antagonist losartan has a therapeutic effect in vEDS. Here, we evaluated the impact of celiprolol and losartan on the biomechanical integrity of the vEDS thoracic aorta. METHODS AND RESULTS: We established a new approach to measure the maximum tensile force at rupture of uniaxially stretched murine thoracic aortic rings. In a vEDS model, which we (re-)characterized here at molecular level, heterozygous mice showed a significant reduction in the rupture force compared to wild-type mice, reflecting the increased mortality due to aortic rupture. For the assessment of treatment effects, heterozygous mice at 4 weeks of age underwent a 4-week treatment with celiprolol, losartan, and, as a proof-of-concept drug, the matrix metalloproteinase inhibitor doxycycline. Compared to age- and sex-matched untreated heterozygous mice, treatment with doxycycline or celiprolol resulted in a significant increase of rupture force, whereas no significant change was detected upon losartan treatment. CONCLUSIONS: In a vEDS model, celiprolol or doxycycline, but not losartan, can improve the biomechanical integrity of the aortic wall, thereby potentially reducing the risk of dissection and rupture. As doxycycline is a broad-spectrum antibiotic with considerable side effects, celiprolol may be more suitable for a long-term therapy and thus rather indicated for the medication of patients with vEDS