Ultrastructural analysis of the intracellular surfactant in lungs of healthy and ovalbumin sensitized and challenged Brown Norway rats.

Introduction: In human and experimentally induced asthma, a dysfunction of the intra-alveolar-surface active agent (surfactant) has been demonstrated. Type II alveolar epithelial cells (AEII) synthesize, secrete and recycle surfactant. Prior to secretion, intracellular surfactant is stored in specific secretory organelles of AEII. The lamellar bodies (Lb) represent its ultrastructural correlate. The aim of this study was to investigate whether disturbances of the intra-alveolar surfactant are accompanied by alterations in the intracellular surfactant.Material and Methods: Brown-Norway rats were sensitized twice with ovalbumin (OVA) and heat killed Bordetella pertussis bacilli. During airway challenge, an aerosol of 5% ovalbumin/saline solution (0.25 l/min) was nebulized. 24 h after airway challenge, lungs were fixed by vascular perfusion. AEII and their Lb were characterized stereologically by light and electron microscopy.Results: In both groups, AEII were structurally intact. The number of AEII per lung and their number-weighted mean volume did not differ (controls: 49 × 106, 393 µm3; asthmatics: 44 × 106, 390 µm3). A mean of 90 Lb in AEII of asthmatics and of 93 Lb in AEII of controls were evaluated. The Lb mean total volume was 59 µm in asthmatics and 68 µm in controls. Values of both parameters did not reach significance. Also, the size distribution and mean volume of Lb was not influenced by asthma induction, because the volume weighted mean volume of Lb (2.18 µm in asthmatics compared to 1.87 µm in controls) and the numerical weighted mean volume (0.96 µm in asthmatics and 0.75 µm in controls) were comparable in both groups.Conclusion: The obtained results suggest that asthma-induced surfactant dysfunction is not related to disturbances in the intracellular surfactant´s ultrastructural correlates.

Bacterial species-specific antimicrobial efficacies of three different body embalming solutions for anatomical studies.

While body decompensation is mainly facilitated by bacteria, investigating the antimicrobial properties of body preservation methods is still a neglected research area. We performed microbiological sampling for potentially pathogenic bacteria species of brain, lung, liver, colon, and subcutis samples obtained from bodies perfused with embalming solutions of variable composition with emphasis on variable formaldehyde concentrations. We, thereby, identified spore-forming aerobic and anaerobic bacteria mainly in the samples obtained from the colon of ethanol- and lower-concentrated formaldehyde formulation embalmed bodies. Moreover, we could identify Enterococcus species in bodies preserved with the latter method. Tissue samples of the subcutis remained sterile. Long-term incubation of special mycobacteria growth indicator tubes revealed no growth of mycobacteria in all 60 samples analyzed. Overall, we show survival of bacterial genera known to be especially environmentally resistant but also include potentially pathogenic members. Knowledge of bactericidal capacities of embalming solutions are therefore critical to assess risk and apply appropriate disinfection routines while working with human bodies. Moreover, new formulations to reduce potentially toxic substances for embalming needs to be evaluated regarding their bactericidal capacities.

The relationship between the subclavian vessels and brachial plexus and the overlying clavicle: Anatomical study with application to plate osteosynthesis.

A subclavian artery aneurysm after clavicle fracture and plate osteosynthesis in a suspected case of a screw that was too long led us to investigate body donor cadavers. The aim was to verify clavicle variability, and the course of the neurovascular bundle in relation to the clavicle and to the osteosynthesis plate, in order to clarify safe zones for plate and screw fixation. We used one fresh frozen and 25 embalmed donors for in situ measurements: (1) length and craniocaudal thickness of the clavicle, (2) distances between the sternal end of the clavicle and the center of parts of the neurovascular bundle. The clavicle was 15.15 cm long. The mean distances from the sternal end of the clavicle were 5.62 cm to the subclavian vein, 6.75 cm to the subclavian artery and 8.42 cm to the cords of the brachial plexus. The subclavius muscle was 1 cm thick. Because of sex differences in length and distances, we recorded the distances between the sternal end and parts of the neurovascular bundle as ratios of clavicle length (at-risk area) to provide sex-independent parameters: 0.379 for the vein, 0.449 for the artery and 0.554 for the nerve. The neurovascular bundle runs below the clavicle between the medial fourth and three fifths of clavicle length. To avoid iatrogenic neurovascular injuries, special caution is necessary during drilling and screwing the osteosynthesis. We also recommend using screws shorter than 1.4 cm.

Differential temporal development of alveoli and the alveolar capillary network in the postnatal rat lung.

Quantitative data about the internal lung structure are needed to better understand normal and pathological lung development. Aberrant lung development causes deficits in alveolar and microvascular development; however, the normal temporal relationship between these processes is still not fully understood. We hypothesized that alveolar and capillary development show a differential time pattern. Lungs of rats aged 3, 7, 14, 21 days (d) or 3 mo (n = 8-10 each) were fixed by vascular perfusion and processed for light microscopy. Using design-based stereology number, the surface area and volume of alveoli, septal capillaries, and alveolar septa were quantified. The total number and the total volume of alveoli increased progressively during postnatal development. Interestingly, the numerical density of capillary loops was significantly higher in 14- and 21-d-old rats than before or after this age, causing a duplication of the total number of capillary loops between 1 and 2 wk of age. The mean thickness of alveolar septa started to decline slightly at the age of 14d and more pronounced at later stages. Although the septal epithelial surface area increased in proportion to alveolar number during the first 3 wk, the capillary endothelial surface area grew only slightly compared with the number of capillaries. In conclusion, the number of elements composing the alveolar capillary network expands massively during the first two postnatal weeks and exceeds the formation of alveoli. The thinning of the alveolar septa during further development suggests a reduction of the capillary network during alveolarization.

Lung development and immune status under chronic LPS exposure in rat pups with and without CD26/DPP4 deficiency.

Dipeptidyl-peptidase IV (CD26), a multifactorial integral type II protein, is expressed in the lungs during development and is involved in inflammation processes. We tested whether daily LPS administration influences the CD26-dependent retardation in morphological lung development and induces alterations in the immune status. Newborn Fischer rats with and without CD26 deficiency were nebulized with 1 µg LPS/2 ml NaCl for 10 min from days postpartum (dpp) 3 to 9. We used stereological methods and fluorescence activated cell sorting (FACS) to determine morphological lung maturation and alterations in the pulmonary leukocyte content on dpp 7, 10, and 14. Daily LPS application did not change the lung volume but resulted in a significant retardation of alveolarization in both substrains proved by significantly lower values of septal surface and volume as well as higher mean free distances in airspaces. Looking at the immune status after LPS exposure compared to controls, a significantly higher percentage of B lymphocytes and decrease of CD4+CD25+ T cells were found in both subtypes, on dpp7 a significantly higher percentage of CD4 T+ cells in CD26+ pups, and a significantly higher percentage of monocytes in CD26- pups. The percentage of T cells was significantly higher in the CD26-deficient group on each dpp. Thus, daily postnatal exposition to low doses of LPS for 1 week resulted in a delay in formation of secondary septa, which remained up to dpp 14 in CD26- pups. The retardation was accompanied by moderate parenchymal inflammation and CD26-dependent changes in the pulmonary immune cell composition.

Aging impairs alveolar epithelial type II cell function in acute lung injury.

Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 µg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.

Spermidine supplementation and voluntary activity differentially affect obesity-related structural changes in the mouse lung.

Obesity is associated with lung function impairment and respiratory diseases; however, the underlying pathophysiological mechanisms are still elusive, and therapeutic options are limited. This study examined the effects of prolonged excess fat intake on lung mechanics and microstructure and tested spermidine supplementation and physical activity as intervention strategies. C57BL/6N mice fed control diet (10% fat) or high-fat diet (HFD; 60% fat) were left untreated or were supplemented with 3 mM spermidine, had access to running wheels for voluntary activity, or a combination of both. After 30 wk, lung mechanics was assessed, and left lungs were analyzed by design-based stereology. HFD exerted minor effects on lung mechanics and resulted in higher body weight and elevated lung, air, and septal volumes. The number of alveoli was higher in HFD-fed animals. This was accompanied by an increase in epithelial, but not endothelial, surface area. Moreover, air-blood barrier and endothelium were significantly thicker. Neither treatment affected HFD-related body weights. Spermidine lowered lung volumes as well as endothelial and air-blood barrier thicknesses toward control levels and substantially increased the endothelial surface area under HFD. Activity resulted in decreased volumes of lung, septa, and septal compartments but did not affect vascular changes in HFD-fed mice. The combination treatment showed no additive effect. In conclusion, excess fat consumption induced alveolar capillary remodeling indicative of impaired perfusion and gas diffusion. Spermidine alleviated obesity-related endothelial alterations, indicating a beneficial effect, whereas physical activity reduced lung volumes apparently by other, possibly systemic effects.

Bone marrow stem cells accelerate lung maturation and prevent the LPS-induced delay of morphological and functional fetal lung development in the presence of ErbB4.

ErbB4 is a regulator in lung development and disease. Prenatal infection is an important risk factor for the delay of morphologic lung development, while promoting the maturation of the surfactant system. Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to prevent lung injury. We hypothesized that BMSCs in comparison with hematopoietic control stem cells (HPSCs) minimize the lipopolysaccharide (LPS)-induced lung injury only when functional ErbB4 receptor is present. We injected LPS and/or murine green fluorescent protein-labeled BMSCs or HPSCs into the amniotic cavity of transgenic ErbB4heart mothers at gestational day 17. Fetal lungs were analyzed 24 h later. BMSCs minimized significantly LPS-induced delay in morphological lung maturation consisting of a stereologically measured increase in mesenchyme and septal thickness and a decrease of future airspace and septal surface. This effect was more prominent and significant in the ErbB4heart+/- lungs, suggesting that the presence of functioning ErbB4 signaling is required. BMSC also diminished the LPS induced increase in surfactant protein (Sftp)a mRNA and decrease in Sftpc mRNA is only seen if ErbB4 is present. The reduction of morphological delay of lung development and of levels of immune-modulating Sftp was more pronounced in the presence of the ErbB4 receptor. Thus, ErbB4 may be required for the protective signaling of BMSCs.

Characterization of surfactant alterations in pigs infected with Actinobacillus pleuropneumoniae.

PURPOSE/AIM OF THE STUDY: Surfactant, a surface active complex of phospholipids and proteins located at the inner surface of alveoli and small conducting airways is necessary for breathing. Bacterial respiratory tract infections frequently lead to surfactant alterations and to an increase in surface tension. Pigs, often used in experimental lung research, could suffer from severe pleuropneumonia, a highly contagious disease often characterized by sudden onset, short clinical course, high morbidity, and high mortality. It is caused by the gram-negative bacterium Actinobacillus pleuropneumoniae (A.pp.). This study tests the hypothesis that also in the subacute stage pathomorphological lung alterations are accompanied with increased inactive surfactant components. Clinical lung scores, functional and ultrastructural analysis of porcine surfactant were performed in pigs before infection and in the subacute state of infection. Clinical signs were determined using inter alia different subscores. Surfactant was isolated from the BALF for functional and quantitative ultrastructural studies. RESULTS: In the subacute stage clinical, ultrosonographic and radiographic scores as well as the overall Respiratory Health Score showed significantly higher values than before infection. However, surfactant surprisingly contained more active surfactant subtypes and significantly less inactive subtypes such as unilamellar vesicles. The quantity of multilamellar vesicles with unclear function did not differ. The minimal surface tension of surfactant before and after infection was comparable. CONCLUSIONS: Thus, in spite of continued severe lung tissue alterations the surfactant system show signs of recovery. This may be the result of an effective adaption to inflammatory lung disorders caused by swine-specific pathogens.

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice.

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Morphological and molecular aspects of lung development.

Healthy breathing relies on normal morphological and functional development of the lung. This includes different prenatal and postnatal developmental stages. Depending on species and postnatal behavior as nest escapers or nest squatters, the duration of individual developmental phases and the state of differentiation of the lungs at birth differ. However, the sequence and morphology of the lung developmental stages are similar in all mammals, so knowledge gained from animal models about development-specific genetic control and regulatory mechanisms can be translated in principle to the human lung. Functional lung development comprises the maturation of the surfactant system, which is closely linked to the morphological development of the pulmonary acini. Although a number of reviews are found in the literature, a presentation that integrates the morphological and molecular regulatory mechanisms is missing. Therefore, the aim of this article was to provide an up-to-date comprehensive review of the main morphological steps and regulatory mechanisms of lung development, including clinical aspects related to developmental disorders.

The expression of the surfactant proteins SP-A and SP-B during postnatal alveolarization of the rat lung.

OBJECTIVE: Surfactant-specific proteins (SP) are responsible for the functional and structural integrity as well as for the stabilization of the intra-alveolar surfactant. Morphological lung maturation starts in rat lungs after birth. The aim of this study was to investigate whether the expression of the hydrophilic SP-A and the hydrophobic SP-B is associated with characteristic postnatal changes characterizing morphological lung maturation. METHODS: Stereological methods were performed on the light microscope. Using immunohistochemical and molecular biological methods (Western Blot, RT-qPCR), the SP-A and SP-B of adult rat lungs and of those with different postnatal developmental stages (3, 7, 14 and 21 days after birth) were characterized. RESULTS: As signs of alveolarization the total septal surface and volume increased and the septal thickness decreased. The significantly highest relative surface fraction of SP-A labeled alveolar epithelial cells type II (AEII) was found together with the highest relative SP-A gene expression before the alveolarization (3th postnatal day). With the downregulation of SP-A gene expression during and after alveolarization (between postnatal days 7 and 14), the surface fraction of the SP-A labeled AEII also decreased, so they are lowest in adult animals. The surface fraction of SP-B labeled AEII and the SP-B gene expression showed the significantly highest levels in adults, the protein expression increased also significantly at the end of morphological lung maturation. There were no alterations in the SP-B expression before and during alveolarization until postnatal day 14. The protein expression as well as the gene expression of SP-A and SP-B correlated very well with the total surface of alveolar septa independent of the postnatal age. CONCLUSION: The expression of SP-A and SP-B is differentially associated with morphological lung maturation and correlates with increased septation of alveoli as indirect clue for alveolarization.

The Use of Plantaris Muscle Flap for Facial Reanimation: A Comprehensive Anatomical Study.

Background: Functional facial reanimation remains challenging and the quest for optimization continues. Objective: To characterize the anatomical conditions of the plantaris muscle for facial reanimation. Study Design and Methods: Forty-two plantaris muscle specimens were obtained from 23 post-mortem chemically fixed cadavers. The muscles were dissected, evaluated, and measured. Mock facial reanimation was performed on three cadaver heads. Results: The plantaris muscle was a consistently available muscle. Mean muscle belly length was 10.1 cm (standard deviation [SD] 1.4), and mean width was 1.7 cm (SD 0.4). The mean tendon length of 30.1 cm (SD 2.8) is unique in the human body. The main artery supplying the muscle had a mean length of 1.4 cm (SD 0.4). The mean nerve length was 2.2 cm (SD 0.7). Sixteen variations of vascular supply were identified. Mock facial reanimations demonstrated a good size match, and great versatility of the long tendon for oral fixation. Conclusions: The plantaris muscle as a free flap for facial reanimation could offer new possibilities in terms of oral fixation and volumetric aesthetic conditions.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Teaching gross anatomy during the Covid-19 pandemic: Effects on medical students' gain of knowledge, confidence levels and pandemic-related concerns.

For medical students the dissection course is the preferred method to learn gross anatomy. However, the added value of active cadaver dissection on knowledge gain in multimodal curricula offering a diversity of e-learning resources is unknown. The Covid-19-related lockdown forced educators to replace the dissection course by e-learning resources. At the end of the summer term 2020 loosening of pandemic-related regulations allowed offering a compact, voluntary active dissection course of the head-neck region to first-year medical students at Hannover Medical School. A study was conducted comparing a dissection group (G1, n = 115) and a non-dissection group (G2, n = 23). Knowledge gain and confidence level were measured with a multiple-choice (MC-)test. The use of e-learning resources was recorded. A questionnaire measured motivation, interest and level of concern regarding Covid-19 and anatomy teaching. No differences between groups were found regarding motivation and interest in anatomy of the head-neck region. G2, however, had significantly higher concerns regarding the Covid-19 pandemic than G1. Neither before nor after the educational intervention, differences in the scores of the MC-test were found. However, after the course G1 answered more MC-questions with highest confidence level than G2 (6.7 ± 6.0 vs. 3.6 ± 4.6, p < 0.05) and demonstrated by trend an increased improvement in the scores of image-based questions (30.8 ± 18.2 % vs. 17.1 ± 14.8 %, p = 0.06). In general, frequent users of online quizzes, a part of the e-learning resources, scored significantly better in the knowledge test. Active dissection improves self-assurance to identify anatomical structures and should be re-implemented in multimodal, blended-learning-based anatomical curricula in the post-pandemic era.

Neuregulin receptor ErbB4 functions as a transcriptional cofactor for the expression of surfactant protein B in the fetal lung.

Sufficient pulmonary surfactant production is required for the fetal-neonatal transition, especially in preterm infants. Neuregulin (NRG) and its transmembrane receptor ErbB4 positively regulate the onset of fetal surfactant synthesis. Details of this signaling process remain to be elucidated. ErbB4 is known to regulate gene expression in the mammary gland, where the receptor associates with the signal transducer and activator of transcription Stat5a to transactivate the ß-casein gene promoter. We hypothesized that in the fetal lung, ErbB4 functions as a transcriptional regulator for surfactant protein B (Sftpb), the most critical surfactant protein gene. Re-expressing full-length ErbB4 in primary fetal ErbB4-depleted Type II epithelial cells led to an increased expression of Sftpb mRNA. This stimulatory effect required the nuclear translocation of ErbB4 and association with Stat5a, with the resultant binding to and activation of the Sftpb promoter. We conclude that ErbB4 directly regulates important aspects of fetal lung maturation that help prepare for the fetal-neonatal transition.

ErbB4 regulates the timely progression of late fetal lung development.

The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.

Lipopolysaccharide-induced injury is more pronounced in fetal transgenic ErbB4-deleted lungs.

Pulmonary ErbB4 deletion leads to a delay in fetal lung development, alveolar simplification, and lung function disturbances in adult mice. We generated a model of intrauterine infection in ErbB4 transgenic mice to study the additive effects of antenatal LPS administration and ErbB4 deletion during fetal lung development. Pregnant mice were treated intra-amniotically with an LPS dose of 4 µg at E17 of gestation. Lungs were analyzed 24 h later. A significant influx of inflammatory cells was seen in all LPS-treated lungs. In heterozygote control lungs, LPS treatment resulted in a delay of lung morphogenesis characterized by a significant increase in the fraction of mesenchyme, a decrease in gas exchange area, and disorganization of elastic fibers. Surfactant protein (Sftp)b and Sftpc were upregulated, but mRNA of Sftpb and Sftpc was downregulated compared with non-LPS-treated controls. The mRNA of Sftpa1 and Sftpd was upregulated. In ErbB4-deleted lungs, the LPS effects were more pronounced, resulting in a further delay in morphological development, a more pronounced inflammation in the parenchyma, and a significant higher increase in all Sftp. The effect on Sftpb and Sftpc mRNA was somewhat different, resulting in a significant increase. These results imply a major role of ErbB4 in LPS-induced signaling in structural and functional lung development.

The Sphingosine-1 Phosphate receptor agonist FTY720 dose dependently affected endothelial integrity in vitro and aggravated ventilator-induced lung injury in mice.

Lung barrier protection by Sphingosine-1 Phosphate (S1P) has been demonstrated experimentally, but recent evidence suggests barrier disruptive properties of high systemic S1P concentrations. The S1P analog FTY720 recently gained an FDA approval for treatment of multiple sclerosis. In case of FTY720 treated patients experiencing multiple organ dysfunction syndrome the drug may accumulate due to liver failure, and the patients may receive ventilator therapy. Whereas low doses of FTY720 enhanced endothelial barrier function, data on effects of increased FTY720 concentrations are lacking. We measured transcellular electrical resistance (TER) of human umbilical vein endothelial cell (HUVEC) monolayers, performed morphologic analysis and measured apoptosis by TUNEL staining and procaspase-3 degradation in HUVECs stimulated with FTY720 (0.01-100 µM). Healthy C57BL/6 mice and mice ventilated with 17 ml/kg tidal volume and 100% oxygen for 2 h were treated with 0.1 or 2 mg/kg FTY720 or solvent, and lung permeability, oxygenation and leukocyte counts in BAL and blood were quantified. Further, electron microscopic analysis of lung tissue was performed. We observed barrier protective effects of FTY720 on HUVEC cell layers at concentrations up to 1 µM while higher concentrations induced irreversible barrier breakdown accompanied by induction of apoptosis. Low FTY720 concentrations (0.1 mg/kg) reduced lung permeability in mechanically ventilated mice, but 2 mg/kg FTY720 increased pulmonary vascular permeability in ventilated mice accompanied by endothelial apoptosis, while not affecting permeability in non-ventilated mice. Moreover, hyperoxic mechanical ventilation sensitized the pulmonary vasculature to a barrier disrupting effect of FTY720, resulting in worsening of ventilator induced lung injury. In conclusion, the current data suggest FTY720 induced endothelial barrier dysfunction, which was probably caused by proapoptotic effects and enhanced by mechanical ventilation.

Tissue engineering human small-caliber autologous vessels using a xenogenous decellularized connective tissue matrix approach: preclinical comparative biomechanical studies.

Suggesting that bioartificial vascular scaffolds cannot but tissue-engineered vessels can withstand biomechanical stress, we developed in vitro methods for preclinical biological material testings. The aim of the study was to evaluate the influence of revitalization of xenogenous scaffolds on biomechanical stability of tissue-engineered vessels. For measurement of radial distensibility, a salt-solution inflation method was used. The longitudinal tensile strength test (DIN 50145) was applied on bone-shaped specimen: tensile/tear strength (SigmaB/R), elongation at maximum yield stress/rupture (DeltaB/R), and modulus of elasticity were determined of native (NAs; n = 6), decellularized (DAs; n = 6), and decellularized carotid arteries reseeded with human vascular smooth muscle cells and human vascular endothelial cells (RAs; n = 7). Radial distensibility of DAs was significantly lower (113%) than for NAs (135%) (P < 0.001) or RAs (127%) (P = 0.018). At levels of 120 mm Hg and more, decellularized matrices burst (120, 160 [n = 2] and 200 mm Hg). Although RAs withstood levels up to 300 mm Hg, ANOVA revealed a significant difference from NA (P = 0.018). Compared with native vessels (NAs), SigmaB/R values were lower in DAs (44%; 57%) (P = 0.014 and P = 0.002, respectively) and were significantly higher in RAs (71%; 83%) (both P < 0.001). Similarly, DeltaB/R values were much higher in DAs compared with NAs (94%; 88%) (P < 0.001) and RAs (87%; 103%) (P < 0.001), but equivalent in NAs and RAs. Modulus of elasticity (2.6/1.1/3.7 to 16.6 N/mm(2)) of NAs, DAs, RAs was comparable (P = 0.088). Using newly developed in vitro methods for small-caliber vascular graft testing, this study proved that revitalization of decellularized connective tissue scaffolds led to vascular graft stability able to withstand biomechanical stress mimicking the human circulation. This tissue engineering approach provides a sufficiently stable autologized graft.