Right atrium and superior vena cava pressure measurements in a novel animal model to study one and a half ventricle repair as compared to Fontan type procedure

Background & Objectives: To evaluate the advantages of the one and a half ventricle repair on maintaining a low pressure in the inferior vena cava district. Also evaluate the competition of flows at the superior vena cava – right pulmonary artery anastomosis site, in order to understand the hemodynamic interaction of a pulsatile flow in combination to a laminar one. Materials & Methods: Adult rabbits (n=30) in terminal anaesthesia with a follow up of 8 h were used, randomly distributed in three experimental groups: Group 1: animals with an anastomosis between superior vena cava and right pulmonary artery, as a model of one and one half ventricle repair; Group 2: animals with the cavopulmonary anastomosis followed by clamping of the right pulmonary artery proximal to the anastomosis; and Group 3: sham animals. Pressures of superior vena cava and pulmonary arteries were afterwards measured, in a resting condition as well as after induced pharmacological stress test.Results: In Group 1, superior vena cava pressure was significantly higher, while venous pressure in the inferior vena cava – right atrium district was constant or lower in comparison with the other groups. After stress test, the pressure in the superior vena cava and the heart rate both increased further, but the right ventricular, right atrial and pulmonary artery pressures remained similar to the values in a resting condition. This proved that the inferior vena cava return was well-preserved, and no venous hypertension was present in the inferior vena cava district even after stress test (good exercise tolerance).Conclusion: One and one half ventricle repair can be considered a good surgical strategy for maintaining a low pressure in the inferior vena cava district with potential for right ventricle growth, restoring the more physiological circulation in borderline or failing right ventricle conditions. The experiment presented a positive finding in favour of one and one half ventricle repair, as compared to Fontan type procedure

First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Decellularized diaphragmatic muscle drives a constructive angiogenic response in vivo

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1\u3b1. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31+, \u3b1SMA+, and vWF+ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF+ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration

Right atrium and superior vena cava pressure measurements in a novel animal model to study one and a half ventricle repair as compared to Fontan type procedure

Background &amp; Objectives: To evaluate the advantages of the one and a half ventricle repair on maintaining a low pressure in the inferior vena cava district. Also evaluate the competition of flows at the superior vena cava – right pulmonary artery anastomosis site, in order to understand the hemodynamic interaction of a pulsatile flow in combination to a laminar one. Materials &amp; Methods: Adult rabbits (n=30) in terminal anaesthesia with a follow up of 8 h were used, randomly distributed in three experimental groups: Group 1: animals with an anastomosis between superior vena cava and right pulmonary artery, as a model of one and one half ventricle repair; Group 2: animals with the cavopulmonary anastomosis followed by clamping of the right pulmonary artery proximal to the anastomosis; and Group 3: sham animals. Pressures of superior vena cava and pulmonary arteries were afterwards measured, in a resting condition as well as after induced pharmacological stress test.Results: In Group 1, superior vena cava pressure was significantly higher, while venous pressure in the inferior vena cava – right atrium district was constant or lower in comparison with the other groups. After stress test, the pressure in the superior vena cava and the heart rate both increased further, but the right ventricular, right atrial and pulmonary artery pressures remained similar to the values in a resting condition. This proved that the inferior vena cava return was well-preserved, and no venous hypertension was present in the inferior vena cava district even after stress test (good exercise tolerance).Conclusion: One and one half ventricle repair can be considered a good surgical strategy for maintaining a low pressure in the inferior vena cava district with potential for right ventricle growth, restoring the more physiological circulation in borderline or failing right ventricle conditions. The experiment presented a positive finding in favour of one and one half ventricle repair, as compared to Fontan type procedure.</p

Allogenic tissue-specific decellularized scaffolds promote long-term muscle innervation and functional recovery in a surgical diaphragmatic hernia model

Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFP\u202f+\u202fSchwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

L-citrulline is protective in hyperoxic lung damage and improves matrix remodelling and alveolarization

Moderate hyperoxia alters alveolar and vascular lung morphogenesis. Nitric oxide (NO) and matrix metalloproteinases (MMP) have a crucial role in the homeostasis of the matrix and bronchoalveolar structure and may be regulated abnormally by exposure to hyperoxia. Disruption of vascular endothelial growth factor (VEGF)-NO signaling impairs vascular growth and contributes to hyperoxia-induced vascular disease in bronchopulmonary dysplasia (BPD). We hypothesize that L-citrulline, by raising the serum levels of L-arginine and enhancing endogenous NO synthesis, might attenuate hyperoxia-induced lung injury in an experimental model of BPD. Neonatal rats (1 day old) were exposed to 60% oxygen or room air for 14 days and administered L-citrulline or a vehicle (sham). Lung morphometry were performed; Serum was tested for arginine level; Matrix metalloproteinases2 (MMP2) gene expression, VEGF gene and protein expression and endothelial NO synthase (eNOS) protein expression were compared. Mean linear intercept was higher in the hyperoxia and sham groups when compared with the room air (RA) and L-citr+hyperoxia treated group (p&lt;0.02). Secondary crests number was higher in L-citrulline treated and RA when compared to hyperoxia and sham group (p&lt;0.02). L-Arginine level rose in the L-citrulline-treated group (p&lt;0.05). L-citrulline did not affect MMP2 gene expression, but it regulated the MMP2 active protein, which rose in bronchoalveolar lavage fluid (p&lt;0.05), presumably due to a post-transductional effect. Compared with RA controls, hyperoxia significantly decreased VEGF and eNOS protein expression. At the same time, an increased lung VEGF gene and protein expression (p&lt;0.05) were also seen in the rats treated with L-citrulline. We conclude that: (i) hyperoxia decreases growth and disrupts VEGF-NO signaling of lung; (ii) the main effects of L-citrulline are an increased serum level of arginine, as a promoter and a substrate of the nitric oxide synthase; and (ii) a better alveolar growth and matrix control than in hyperoxia-induced lung damage seems promising

Administration of L-citrulline in an animal model of perinatal lung damage

Moderate hyperoxia and induced chorioamnionitis by intrauterine administration of endotoxin LPS into near-term pregnant rats cause alveolar and vascular lung derangement in the newborns. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of L-arginine to L-citrulline in endothelial cells. We investigated whether administering L-citrulline by raising the serum levels of L-arginine and enhancing NO endogenous synthesis, attenuates lung injury in a chorioamnionitis and/or moderate hyperoxia-induced model. Material and Methods. Newborn rats (receiving or not intrauterine LPS) were exposed to FiO2=0.6 or room air till 14 days after birth and were administered L-citrulline. Serum and lung tissues were collected for further analysis. The lung sections were subsequently stained with H&E and photomicrographs were obtained at 10X magnification. For vessel density assessment, sections were stained to reveal the presence of von Willebrand Factor Antigen. VEGF and eNOS protein expression was examined by Western blot. High performance liquid chromatography-mass spectrometry was used for simultaneous determination and quantification of ADMA, SDMA, L-arginine, L-citrulline, NMMA and homo-arginine in the serum. Results. The lung histopathology analysis of the Hyperoxia group showed a pattern typically emphysematous, similar to the LPS exposure group, when compared to controls. Exposure to hyperoxia was associated with an arrested alveolarization, inducing a change in lung morphology with patchy areas of parenchymal thickening interspersed with areas of enlarged air spaces. The lung sections of the CITR+hyperoxia and LPS+CITR rats contained smaller and more numerous air spaces, and were more similar to the control lungs. The mean alveolar size was higher in Hyperoxia group vs. controls, or LPS+CITR, in a post hoc comparison unchanged with respect to CITR+hyperoxia, or LPS, or CITR. The secondary crests were higher in the Control and CITR+hyperoxia and LPS+CITR groups than in the Hyperoxia only, or LPS only groups. VEGF gene expression evaluated by real-time quantitative PCR was lower in the Hyperoxia group, than in the CITR+hyperoxia or Control groups. Also, lung sections from Control and CITR+hyperoxia animals showed a similar vWF expression, whereas staining was weaker in the Hyperoxia group. In the CITR+hyperoxia sections there was also evidence of a better organization of the vessel network than in animals exposed to hyperoxia. The amount of eNOS protein normalized in the lung tissue from the L-citrulline treated animals was higher than in the tissues from the Hyperoxia group. Serum assessment with mass spectrometry did not show major differences in the time course and treatment groups. Conclusions. Our main findings were that: (i) administering L-citrulline proved effective in improving alveolar growth after oxygen-induced and antenatal endotoxin exposure lung damage; (ii) VEGF gene and protein were over-expressed in the group treated with L-citrulline. There may have been further protective effects on the alveolar vascular network and, consequently, on matrix maturation in our model and this may be promising with a view to BPD prevention strategies.La corioamnionite indotta dalla somministrazione intrauterina dell’endotossina LPS e da una moderata iperossia nei primi giorni di vita causano uno squilibrio alveolare e vascolare del polmone nel ratto neonato. L’ossido nitrico (NO) endogeno, che promuove la crescita polmonare, viene prodotto nelle cellule endoteliali dal metabolismo del L-arginina verso il suo prodotto, la L-citrullina. Abbiamo studiato l’efficacia della somministrazione di L-citrullina in un modello di danno indotto da corioamnionite e/o da iperossia nei ratti neonati nell’attenuare il danno polmonare intervenendo sulla sintesi del NO endogeno aumentando i livelli di L-arginina. Materiali e Metodi. I ratti neonati (che ricevono o no LPS nella loro fase intrauterina) vengono esposti a un FiO2=0.6, o ad aria ambiente, per 14 giorni dopo la nascita con la somministrazione, per alcuni di loro, della L-citrullina. A vari time-points sperimentali siero e tessuto polmonare vengono raccolti per ulteriori analisi. Le sezioni polmonari vengono colorate con ematossilina & eosina e fotografate a 10X. Per una valutazione della densità vascolare le sezioni sono colorate per la presenza dell’antigene del Fattore di von Willebrand. La VEGF e l’espressione proteica eNOS vengono esaminati con il Western blot. La HPLC Spettrometria di Massa viene usata per determinare e quantificare nel siero ADMA, SDMA, L-arginina, L-citrullina, NMMA e omo-arginina. Risultati. L’esposizione a moderati regimi di iperossia era associata istologicamente con aree estese di tipo enfisematoso, simile al quadro del gruppo esposto al LPS e, inoltre, con un arresto dell’alveolarizzazione e contestuale variazione eterogenea della morfologia polmonare, e ha indotto un cambiamento nella morfometria polmonare con aree irregolari di inspessimento parenchimatoso intervallate da aree con spazi aumentati. Il gruppo ricevente il farmaco presentava un grado di alveolarizzazione più sviluppata con un incremento del numero degli alveoli per mm2, statisticamente significativo rispetto al gruppo con iperossia. Le sezioni polmonari dei gruppi CITR+iperossia e LPS+CITR contenevano spazi più piccoli e più numerosi, simili ai controlli. Il numero delle creste secondarie era più alto nei controlli e nei gruppi CITR+iperossia e LPS+CITR, che nei gruppi con iperossia solo, o LPS sola. L’espressione genica del VEGF era più bassa nel gruppo dell’iperossia, rispetto al gruppo CITR+iperossia, o ai controlli. Inoltre, le sezioni polmonari da animali di controllo o da trattati con CITR+iperossia presentavano un’espressione vWF simile, mentre la colorazione era più bassa nel gruppo con iperossia. Nei campioni da animali trattati con CITR+iperossia era evidente anche un organizzazione migliore della rete vascolare rispetto agli animali esposti solo all’iperossia. La quantità delle proteine eNOS normalizzate nei tessuti polmonari da animali trattati con L-citrullina era più alta che nei tessuti del gruppo con sola iperossia. La valutazione con spettrometria di massa dei campioni di siero non ha mostrato grandi differenze tra i gruppi trattati. Conclusioni. In conclusione abbiamo provato che: (i) la somministrazione della L-citrullina aiuta la crescita alveolare nel danno polmonare da ossigeno, o da esposizione antenatale a endotossina; (ii) il gene e la proteina VEGF sono over-espressi nel gruppo trattato con L-citrullina. Ulteriori effetti protettivi potranno essere manifesti sul network alveolare e vascolare del polmone e, di conseguenza, sulla maturazione della matrice nel nostro modello di danno polmonare; tutto questo potrà essere promettente in vista di una strategia della prevenzione della broncodisplasia polmonare

Differentiation potential of cardiac stem and progenitor cells in a model of heterotopic heart transplantation

Cardiac stem cells were identified in mammalian hearts and they were found to regenerate cardiomyocytes and vessels when injected into an infarcted heart. We used the model of heterotopic heart transplantation to investigate spontaneous potential of cardiac stem and progenitor cells to differentiate properly after tissue damage and remodeling. In a previous study, in which hearts from normal rat donors were heterotopically transplanted into GFP+ transgenic rat hosts, we found abundant extracardiac GFP+ cells that did not contribute to de novo cardiogenesis, but generated rare hybrid cardiomyocytes by cell fusion with resident cardiac cells. In the same transplants we recently identified a robust population of GFP 12 cells expressing markers of stem and mesenchymal cells. Markers of cardiac commitment and neural stem cells identified different populations of cell progenitors. The overall number of these cells increased 5-fold in heart transplants as compared to normal hearts. The majority of these cells did not progress towards maturation, however cardiomyocytes expressing phosphohistone- H3 were occasionally found. A major issue is whether cardiac stem cells can detach from a damaged heart, circulate and from the bloodstream home again into the heart. To address this question the heart of GFP+ transgenic rats was transplanted into a normal host, which had been previously treated with isoproterenol, to induce damage in the native heart. Native hearts, retrieved 15 days after surgery, did not show any significant engraftment by cardiac stem cells mobilized from the heart transplant

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes.

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium