Mitotically inactivated embryonic stem cells can be used as an in vivo feeder layer to nurse damaged myocardium after acute myocardial infarction: a preclinical study.

RATIONALE: Various types of viable stem cells have been reported to result in modest improvement in cardiac function after acute myocardial infarction. The mechanisms for improvement from different stem cell populations remain unknown. OBJECTIVE: To determine whether irradiated (nonviable) embryonic stem cells (iESCs) improve postischemic cardiac function without adverse consequences. METHODS AND RESULTS: After coronary artery ligation-induced cardiac infarction, either conditioned media or male murine or male human iESCs were injected into the penumbra of ischemic myocardial tissue of female mice or female rhesus macaque monkeys, respectively. Murine and human iESCs, despite irradiation doses that prevented proliferation and induced cell death, significantly improved cardiac function and decreased infarct size compared with untreated or media-treated controls. Fluorescent in situ hybridization of the Y chromosome revealed disappearance of iESCs within the myocardium, whereas 5-bromo-2'-deoxyuridine assays revealed de novo in vivo cardiomyocyte DNA synthesis. Microarray gene expression profiling demonstrated an early increase in metabolism, DNA proliferation, and chromatin remodeling pathways, and a decrease in fibrosis and inflammatory gene expression compared with media-treated controls. CONCLUSIONS: As a result of irradiation before injection, ex vivo and in vivo iESC existence is transient, yet iESCs provide a significant improvement in cardiac function after acute myocardial infarction. The mechanism(s) of action of iESCs seems to be related to cell-cell exchange, paracrine factors, and a scaffolding effect between iESCs and neighboring host cardiomyocytes.

Apoptosis of airway epithelial cells in response to meconium.

Meconium aspiration syndrome (MAS) is common among newborn children but its mechanism is unclear. The syndrome is known to produce a strong inflammatory reaction in the lungs resulting in massive cell death. In this work we studied lung cell death by apoptosis after meconium aspiration in forty two-week-old rabbit pups. Analyzing lung samples by ISEL-DNA end labeling demonstrated the specific spread of apoptotic bodies throughout the lungs. These bodies were shrunken and smaller in size compared to normal cells and many of them were lacking cell membranes. About 70% of all apoptotic bodies were found among the airway epithelium cell eight hours after meconium instillation. In comparison, among lung alveolar cells, only about 20% cells were apoptotic in the same animals. In meconium-treated lungs and A549 cells, a significant increase of angiotensinogen mRNA and Caspase-3 expression were observed. The pretreatment of cells with Caspase-3 inhibitor ZVAD-fmk significantly inhibited meconium-induced lung cell death by apoptosis. These findings demonstrate the apoptotic process in meconium-instilled lungs or A549 cells in culture. Our results show lung airway epithelial and A549 cell apoptosis after meconium instillation. We suggest that studies of lung airway epithelial cell death are essential to understanding the pathophysiology of MAS and may present a key point in future therapeutic applications.

Developing a long-term surviving piglet model of neonatal hypoxic-ischemic encephalopathy.

OBJECTIVE: This study was designed to develop a piglet model of neonatal hypoxic-ischemic encephalopathy, which would allow for serial assessments of long-term neurodevelopmental impairment. METHODS: In 12 newborn piglets, we produced hypoxia by 8% oxygen breathing for 5-91 minutes. We combined ischemia by reversible bilateral common carotid artery occlusion for varying times. Outcome measures were clinical neurological evaluation, magnetic resonance spectroscopy studies and brain histology. RESULTS: Those animals which received intravenous sedation and no mechanical ventilation showed poor tolerance to hypoxia-ischemia and died early in the course of the experiments. The use of inhalation anesthesia during surgical procedures and mechanical ventilation during hypoxia-ischemia was associated with long-term survival. Seven of eight animals that survived > or = 48 hr showed clinical neurological abnormalities, that later resolved. Magnetic resonance spectroscopy measurements did not change significantly following hypoxia-ischemia. None of the animals had histopathological brain lesions. CONCLUSION: When subjected to acute hypoxia-ischemia, piglets were likely to survive only if they were given such supportive measures as anesthesia and mechanical ventilation. Even with hypoxic-ischemic injury sufficient to produce acute signs of neurological dysfunctions, longterm, stable survival with no evident brain histopathological abnormalities was possible.

Elevated immunoreactive endothelin-1 levels in newborn rabbit lungs after meconium aspiration.

OBJECTIVE: Our prime objective was to study the production of big endothelin-1 (Big ET-1) and its conversion to ET-1 in the lungs of newborn rabbits exposed to meconium. Our second objective was to study the effect of captopril on endothelin expression. DESIGN: Prospective, comparative study. SETTING: Research laboratory of the Michael Reese Hospital and the University of Illinois, Chicago. SUBJECTS: Two-wk-old rabbit pups. INTERVENTIONS: Rabbit pups were instilled with meconium or saline into the lungs. Another group, pretreated with captopril, was also instilled with either meconium or saline. MEASUREMENTS AND MAIN RESULTS: After meconium or saline instillation, lung lavage was performed. Big ET-1 and ET-1 were measured in lung lavage fluid by using a commercially available enzyme-linked immunosorbent assay kits in all groups. Also, lungs were studied by histochemistry analysis for a morphologic evaluation of meconium-induced damage. In the lavage fluid of saline-instilled pups, ET-1 remained low and no increase in Big ET-1 levels was observed. In meconium-instilled animals, bioactive ET-1 levels were significantly higher, with a peak at 8 hrs after instillation. The conversion ratio of Big ET-1 to ET-1 in the meconium group increased from 2.19 at the initial period to 7.19 at 8 hrs after meconium instillation. CONCLUSIONS: Our conclusion is that aspiration of meconium causes lung injury in the newborn and that this injury is associated with a significant increase in ET peptide production in the lungs. We also showed that ET production is inhibited by pretreatment of rabbits with captopril before meconium-induced injury. ET-1 and its conversion from ET-1 in response to meconium may play important roles in increasing pulmonary vascular resistance and lung cell death, even in the absence of hypoxia. In general, we conclude, that ET-1 levels are significantly elevated in meconium-instilled rabbits compared with saline-instilled ones, and both can be significantly inhibited by pretreatment with captopril. Whether ET-1 contributes directly to the pathophysiology of or is simply a marker of meconium aspiration syndrome remains speculative.

Cytokine expression in meconium-induced lungs.

OBJECTIVE: In this article the authors present relationship between meconium exposure and inflammatory cytokine release in newborn lungs. METHODS: The authors used forty 2-week-old rabbit pups for the study. One-half of the group were instilled with meconium and the other half with saline. Rabbits were sacrificed at 0, 2, 4, 8, and 24 hrs after installation and lung lavage was obtained and was examined for cytokine mRNA expression using RT-PCR and for cytokine proteins using ELISA technique. The data were collected in each of the study group. RESULTS: Meconium instillation caused significant expression of inflammatory cytokines TNFalpha, IL-6, and IL-8 (p < 0.05) with a peak at 8 hrs after meconium instillation. Levels of IL-10 were insignificant (p > 0.05). Also, we found significant increase in necrotic cells and neutrophils (p < 0.05), compared to the control, saline instilled rabbit lungs. CONCLUSION: The present studies demonstrates that meconium induces inflammatory response and cytokines gene and protein expression in the lungs.