Increased epithelial cell proliferation in very premature baboons with chronic lung disease

Coordinated proliferation of lung cells is required for normal lung growth and differentiation. Chronic injury to developing lung may disrupt normal patterns of cell proliferation. To examine patterns of cell proliferation in injured developing lungs, we investigated premature baboons delivered at 125 days gestation (approximately 67% of term) and treated with oxygen and ventilation for 6, 14, or 21 days (PRN). Each PRN treatment group contained 3 or 4 animals. During normal in utero lung development, the proportion of proliferating lung cells declined as measured by the cell-cycle marker Ki67. In the PRN group, the proportion of proliferating lung cells was 2.5-8.5-fold greater than in corresponding gestational controls. By 14 days of treatment, the proportion of cells that expressed pro-surfactant protein B (proSP-B) was ~2.5-fold greater than in gestational controls. In the PRN group, 41% of proliferating cells expressed proSP-B compared with 5.8% in the gestational controls. By 21 days of treatment, proliferation of proSP-B-expressing epithelial cells declined substantially, but the proportion of proliferating non-proSP-B-expressing cells increased approximately sevenfold. These data show that the development of chronic lung disease is associated with major alterations in normal patterns of lung-cell proliferation.     

Induced p21Cip1 in premature baboons with CLD: implications for alveolar hypoplasia

Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells. The percentage of cells expressing p21 increased threefold in all PRN-treated animals, but different cell populations expressed it during disease progression. Between 2 and 6 days of treatment, p21 was detected in 30-40% of pro-SP-B cells. In contrast, only 12% of pro-SP-B cells expressed p21 by 14 and 21 days of treatment, by which time p21 was also detected in mesenchymal cells. Even though increased epithelial and mesenchymal cell proliferation occurs during disease progression, those cells expressing p21 did not also express the proliferative marker Ki67. Thus two populations of epithelial and mesenchymal cells can be identified that are either expressing Ki67 and proliferating or expressing p21 and not proliferating. These data suggest that p21 may play a role in disorganized proliferation and alveolar hypoplasia seen in newborn chronic lung disease.     

Angiogenic factors in women ten years after severe very early onset preeclampsia

Background

Women with a history of mainly severe and early onset preeclampsia have an increased risk of future cardiovascular disease. During these complicated pregnancies increased levels of anti-angiogenic factors can be found. We hypothesize that women with a history of severe very early onset preeclampsia still have increased levels of these biomarkers years after this pregnancy, resulting in increased risk for cardiovascular disease.

Methods

Twenty women with severe early onset preeclampsia before 24 weeks'' gestation, who delivered between 1993–2003 in a tertiary referral centre and twenty matched controls with uncomplicated pregnancies and healthy term infants, were addressed for participation in the study. Venous plasma samples were analyzed for basic fibroblast growth factor (bFGF), placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), vascular endothelial growth factor (VEGF), E- and P-selectin, soluble intercellular adhesion molecule-3 (sICAM-3) and thrombomodulin by ELISA.

Results

Sixteen case subjects and 18 control subjects consented participation. The median time interval index pregnancy to study was 9.4 and 9.7 years for cases and controls, respectively. Median levels for cases-controls (p-value) were not different; bFGF: 17.43–11.11 pg/mL (0.33), sFlt-1: 102.98–101.92 pg/ml (0.84), PLGF: 3.57–4.20 pg/mL (0.38), VEGF: 64.05–45.72 pg/mL (0.73), E-selectin: 5.11–4.68 ng/mL (0.20), P-selectin: 85.35–71.69 ng/mL (0.69), sICAM-3: 0.42–0.63 ng/mL (0.41) and Thrombomodulin: 0.92–0.93 ng/mL (0.59).

Conclusion

There were no differences in angiogenic biomarkers between women with a history of severe early onset preeclampsia versus uncomplicated pregnancy almost 10 years later, suggesting that these angiogenic factors will not contribute to the early detection of women at risk for future cardiovascular disease.     

Role of lung injury in the pathogenesis of hyaline membrane disease in premature baboons

To test the hypothesis that hyaline membrane disease (HMD) has a multifactorial etiology in which barotrauma plays a major role, we compared the immediate institution of high-frequency oscillatory ventilation (HFOV; 15 Hz, n = 5) with positive-pressure ventilation with positive end-expiratory pressure (PPV; n = 7) in premature baboons (140-days gestation) with HMD. Measurements of ventilation settings and physiological parameters were obtained and arterial-to-alveolar O2 (PaO2-to-PAO2) ratio and oxygenation index [(PaO2/PAO2)-to-mean airway pressure ratio (IO2)] were calculated. At death (24 h), static pressure-volume (PV) curves were performed, and phospholipids (PL) and platelet-activating factor (PAF) were measured in lung lavage fluid. Morphological inflation patterns were analyzed using a panel of standards. By design, mean airway pressure was initially higher (19 vs. 13 cmH2O) in the HFOV animals. PaO2-to-PAO2 ratio and IO2 progressively deteriorated in the PPV animals and then stabilized at significantly lower levels than with HFOV. PV curves from HFOV animals had significant increases in lung volume at maximum distending pressure, deflation volume at 10 cmH2O, and hysteresis area compared with PPV, which showed no hysteresis. Seven of seven PPV and only one of five HFOV animals had morphological findings of HMD. PL amount and composition in both groups were consistent with immaturity, even though the quantity was significantly greater in the PPV group. PAF was present (greater than or equal to 0.10 pmol) in six of seven PPV and in the only HFOV animal with HMD. We conclude that HFOV protected PL-deficient premature baboons from changes in gas exchange, lung mechanics, and morphology typical of HMD in this model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiogenic and anti-angiogenic factors in adrenal tumours

It appears that neoangiogenesis is an important factor in tumour invasion and the formation of metastases in several human cancers, and studies on pro-angiogenic and antiangiogenic factors are therefore of considerable interest to researchers. In this review we present pro-angiogenic and anti-angiogenic factors and other growth factors and their role in the formation of new blood vessels in adrenal tumours. Assessment of the angiogenic status of adrenal tumours and their vascular pattern may be useful for discriminating benign from malignant lesions and knowledge of their angiogenesis may be essential to the drawing up of promising treatment strategies for patients with malignant tumours.     

Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury

Capillary leakage and alveolar edema are hallmarks of acute lung injury (ALI). Neutrophils and serum macromolecules enter alveoli, promoting inflammation. Vascular endothelial growth factor (VEGF) causes plasma leakage in extrapulmonary vessels. Angiopoietin (Ang)-1 and -4 stabilize vessels, attenuating capillary leakage. We hypothesized that VEGF and Ang-1 and -4 modulate vessel leakage in the lung, contributing to the pathogenesis of ALI. We examined a murine model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 and 129/J mice were studied at baseline and 24, 48, and 96 h after single or multiple doses of aerosolized LPS. Both strains exhibited time- and dose-dependent increases in inflammation and a deterioration of lung mechanics. Bronchoalveolar lavage (BAL) protein levels increased significantly, suggesting capillary leakage. Increased BAL neutrophil and total protein content correlated with time-dependent increased tissue VEGF and decreased Ang-1 and -4 levels, with peak VEGF and minimum Ang-1 and -4 expression after 96 h of LPS challenge. These data suggest that changes in the balance between VEGF and Ang-1 and -4 after LPS exposure may modulate neutrophil influx, protein leakage, and alveolar flooding during early ALI.     

Maternal heme oxygenase 1 regulates placental vasculature development via angiogenic factors in mice

The placental vasculature is critical for nutrient, gas, and waste exchange between the maternal and fetal systems. Its development depends on the proper expression and interaction of angiogenesis and associated growth factors. Heme oxygenase (HMOX), the enzyme for heme degradation, plays a role in angiogenesis and is highly expressed in the placenta. To evaluate the role of maternal HMOX1, the inducible HMOX isozyme, on placental vasculature formation, mice with a partial deficiency in Hmox1 (Hmox1(+/-)) were used. Three-dimensional images of placental vasculatures as well as spiral arteries from Hmox1(+/+) or Hmox1(+/-) placentas were created by vascular corrosion casting technique and imaged by micro-computerized tomography (microCT). The structures and morphologies of fetomaternal interfaces were observed by histological staining and the ultrastructure of uterine natural killer (uNK) cells, a major regulator in spiral artery remodeling, was analyzed by transmission electron microscopy. A group of growth factors and angiogenic factors from the decidua/mesometrial lymphoid aggregate of pregnancy (MLAp) as well as labyrinth regions were quantified using an angiogenesis PCR array kit and compared between Hmox1(+/+) or Hmox1(+/-) placentas. In conclusion, a partial deficiency of maternal Hmox1 resulted in the malformation of fetomaternal interface, insufficiency of spiral artery remodeling, and alteration of uNK cell differentiation and maturation. These changes were independent of the fetal genotype, but relied on the maternal HMOX1 level, which determined the balance of expression levels of pro- and antiangiogenic factors in the decidua/MLAp region. These results implied that Hmox1 polymorphisms among the human population might contribute to some unexplained cases of pregnancy disorders, such as fetal growth retardation and preeclampsia.     

Notch signaling in ocular vasculature development and diseases

Ocular angiogenesis, characterized by the formation of new blood vessels in the avascular area in eyes, is a highly coordinated process involved in retinal vasculature formation and several ocular diseases such as age-related macular degeneration, proliferative diabetic retinopathy and retinopathy of prematurity. This process is orchestrated by complicated cellular interactions and vascular growth factors, during which endothelial cells acquire heterogeneous phenotypes and distinct cellular destinations. To date, while the vascular endothelial growth factor has been identified as the most critical angiogenic agent with a remarkable therapeutic value, the Notch signaling pathway appears to be a similarly important regulator in several angiogenic steps. Recent progress has highlighted the involvement, mechanisms and therapeutic potential of Notch signaling in retinal vasculature development and pathological angiogenesis-related eye disorders, which may cause irreversible blindness.     

Maternal care and development of stress responses in baboons

The ability to mount a successful response to threats is critical for an organism's survival. A key element of the stress response is its nonspecificity toward the stress source, with similar endocrine and behavioral changes expected under a variety of stressors. In this project we utilized an experimental design that accounts for multiple sources of variation to further understand the nature of stress responsivity and its relationship to the early rearing environment. A sample of baboons (n=73) was observed during the early phase of life in their social group, and then tested as juveniles in a challenging situation. Maternal cortisol levels were measured during the peripartum period. The challenging situation (individuals were isolated for a few minutes in a single cage) was designed to be a moderate source of psychological stress. Patterns in individual differences during the stress test were "mapped" by means of multidimensional scaling (MDS). After the observation was made, the subject was sedated and a blood sample was taken to measure cortisol levels. Our results indicate that when juvenile baboons are confronted with a source of psychological stress, they show a multidimensional behavioral response, probably mediated by the activation and synergic interaction among different neurohormonal systems that, ultimately, act on the hypothalamus-pituitary-adrenal (HPA) axis. Different components of the multidimensional, or nonspecific, behavioral response are associated with the quality and quantity of interactions with their mothers during early life. Juveniles whose mothers displayed higher levels of positive interaction were characterized by vigilant but less active reactions to the stress test, whereas juveniles of mothers that displayed high levels of stress-related behaviors had higher cortisol and locomotion levels.     

Induction of early alveolar injury by inhaled asbestos and silica

Inhaled asbestos fibers and silica crystals are known to cause interstitial fibrotic lung disease in animals and humans. The initial cellular events and biochemical mechanisms that lead to development of disease are poorly understood. In ongoing studies reviewed here it has been shown that inhaled particulates small enough to pass through the conducting airways are deposited initially at the bifurcations of alveolar ducts. Within hours after brief exposure, alveolar epithelial cells phagocytose inhaled particles that subsequently are translocated to interstitial matrix and fibroblasts. Within 48 h after exposure, inhaled asbestos on alveolar surfaces activates a complement-dependent chemotactic factor for macrophages that accumulate at duct bifurcations. Epithelial cells, macrophages, fibroblasts, and the interstitial matrix are significantly altered by brief (1- 5-h) exposure to chrysotile asbestos. The basic mechanisms that mediate these initial events remain to be defined.     

Regulation of factors controlling angiogenesis in liver development: a role for PEDF in the formation and maintenance of normal vasculature

PEDF and VEGF are important inhibitors and promoters of angiogenesis, and the ratio between the two is an important indicator in many neovascular diseases. In mouse liver PEDF and VEGF(165) were co-expressed at very early stages of liver development and their expression increased as liver embryogenesis progressed, suggesting that PEDF and VEGF are both crucial to vasculogenesis as well. VEGF(189) only appears at the P0 stage in liver organogenesis and is maintained at high levels thereafter. PEDF and the two VEGF isoforms are synthesized by fresh and cultured hepatocytes. Expression of VEGF(121) and overexpression of VEGF(165) were only seen in HepG2, a well-characterized hepatocellular carcinoma line. The results suggest that hepatic vascular architecture is under the control of both PEDF and VEGF, and that VEGF(165) and VEGF(189) have distinct functions in normal vascular development of the liver. The VEGF isoforms 121 and 189 may be key regulators of increased vascularity and progression of hepatocellular carcinoma, one of the most common malignant tumors, and may be of prognostic significance for this tumor.     

Angiogenic factors as potential drug target: Efficacy and limitations of anti-angiogenic therapy

Formation of new blood vessels (angiogenesis) has been demonstrated to be a basic prerequisite for sustainable growth and proliferation of tumor. Several growth factors, cytokines, small peptides and enzymes support tumor growth either independently or in synergy. Decoding the crucial mechanisms of angiogenesis in physiological and pathological state has remained a subject of intense interest during the past three decades. Currently, the most widely preferred approach for arresting tumor angiogenesis is the blockade of vascular endothelial growth factor (VEGF) pathway; however, the clinical usage of this modality is still limited by several factors such as adverse effects, toxicity, acquired drug resistance, and non-availability of valid biomarkers. Nevertheless, angiogenesis, being a normal physiological process imposes limitations in maneuvering it as therapeutic target for tumor angiogenesis. The present review offers an updated relevant literature describing the role of well-characterized angiogenic factors, such as VEGF, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), placenta growth factor (PLGF), hepatocyte growth factor/scatter factor (HGF/SF) and angiopoetins (ANGs) in regulating tumor angiogenesis. We have also attempted to discuss tumor angiogenesis with a perspective of ‘an attractive target with emerging challenges’, along with the limitations and present status of anti-angiogenic therapy in the current state-of-the-art.     

Unbuffered osmium staining of cell organelles: alterations induced by cell injury

We have studied the localization of osmium reduction products to investigate the functional state of organelles as well as organelle interrelationships during cell injury. In normal hepatocytes osmium deposits of variable intensity are seen in nuclear envelope, endoplasmic reticulum. Golgi cisternae and vesicles and lysosomes. Buffering of osmium with s- collidine (pH 7.4) prevents the deposition of osmium. Reversible (30 min) and irreversible (60 min) ischemia without reflow causes no change in the pattern of osmium deposition. Irreversible ischemia followed by reflow causes decreased staining of endoplasmic reticulum (ER) and redistribution of the osmium deposits through the cytoplasm. Reversibly injured pancreatic acinar cells in cultured explants manifest a similar loss of osmium staining in the endoplasmic reticulum cisternae. The administration of antimicrotubule drugs induces an accentuation of osmium staining in localized cisternal elements of hepatocytes. These heavily stained cisternae appear to give rise to the bounding membranes of drug-induced autophagic vacuoles. Cytoplasmic organelles sequestered inside the autophagic vacuoles acquire intense staining when they begin to undergo degradation. In homogenized liver tissue all the subcellular organelles show osmium deposits. The deposits are preferentially localized along the organelle membranes. In particular the dense deposits in the ER lumen are not seen in the subcellular fractions. Phospholipase A2 (3 units/mg protein) enhances the deposition of osmium in the lumen of microsomal vesicles, whereas the presence of detergent has no such effect. Addition of EDTA to the homogenizing medium enhances the ultrastructural preservation of the subcellular fractions but has little effect on the deposition of osmium. OsO4 deposition occurs at acid pH and the intensity and pattern of the stain can be modified in vivo and in vitro. Osmium tetroxide deposition is induced at sites of membrane transformation (autophagic vacuoles) and degradation (lysosomes). Calcium influx and phospholipase activation (ischemia, tissue homogenization, phospholipase addition) enhance osmium deposition and/or influence the localization of the staining pattern.     

The hepatic vasculature and its response to hepatic injury: a working hypothesis

The hepatic circulation is unique in that high volumes of low pressure blood flow are supplied through a dual venous and arterial circulation. This vascular supply is modulated both by the gastrointestinal vascular bed and an intrahepatic microcirculation. This complex vascular system is influenced by pathologic processes within the liver. Alterations in the hepatic circulation reflect hepatic metabolic adaptation and injury. It seems reasonable to assume that in some circumstances hepatic circulatory alterations are inappropriate, exaggerated or inadequate and contribute to the initiation or perpetuation of hepatic injury. This paper attempts to focus on evidence derived from studies of the normal and abnormal hepatic circulation that provide insights into hepatic circulatory responses and their role in the initiation and perpetuation of hepatic injury. A possible relationship of these vascular changes to pathologic processes within the liver is proposed. Ultimately, precise measurement and understanding of hepatic vasculature changes may allow appropriate intervention to offset injury or stimulate maximum effective repair.     

Telomerase in alveolar epithelial development and repair

Telomerase expression and activity were examined in the developing lung and in the adult lung during repair after injury. Both whole lung tissue and primary cultures of type 2 alveolar epithelial cells (AEC2) isolated from fetal and adult rodents were analyzed for 1) telomerase expression by immunohistochemistry and 2) telomerase activity with a telomerase repeat amplification protocol. We found that telomerase was expressed in a temporally regulated manner in fetal lung through the late stages of gestation, with peak expression just before birth. Expression persisted for a brief period in neonates, then decreased to nearly undetectable levels by postnatal day 9. Telomerase expression and activity were reinduced in normally quiescent adult lung by in vivo treatment with hyperoxia. In populations of AEC2 isolated from both developing and repairing lungs, telomerase expression and activity showed a strong correlation with the proliferation marker proliferating cell nuclear antigen. It has been suggested that telomerase expression and activity are hallmarks of stem or progenitor cells. Our observations suggest that a telomerase-positive subpopulation is present within the general AEC2 population. Telomerase may act as a marker for the proliferative status of this subpopulation.     

Prediction of mortality in very premature infants: a systematic review of prediction models

Context

Being born very preterm is associated with elevated risk for neonatal mortality. The aim of this review is to give an overview of prediction models for mortality in very premature infants, assess their quality, identify important predictor variables, and provide recommendations for development of future models.

Methods

Studies were included which reported the predictive performance of a model for mortality in a very preterm or very low birth weight population, and classified as development, validation, or impact studies. For each development study, we recorded the population, variables, aim, predictive performance of the model, and the number of times each model had been validated. Reporting quality criteria and minimum methodological criteria were established and assessed for development studies.

Results

We identified 41 development studies and 18 validation studies. In addition to gestational age and birth weight, eight variables frequently predicted survival: being of average size for gestational age, female gender, non-white ethnicity, absence of serious congenital malformations, use of antenatal steroids, higher 5-minute Apgar score, normal temperature on admission, and better respiratory status. Twelve studies met our methodological criteria, three of which have been externally validated. Low reporting scores were seen in reporting of performance measures, internal and external validation, and handling of missing data.

Conclusions

Multivariate models can predict mortality better than birth weight or gestational age alone in very preterm infants. There are validated prediction models for classification and case-mix adjustment. Additional research is needed in validation and impact studies of existing models, and in prediction of mortality in the clinically important subgroup of infants where age and weight alone give only an equivocal prognosis.     

Lysolecithin acyltransferase and alveolar phosphatidylcholine palmitate in experimental acute alveolar injury in the dog lung

Lysolecithin acyltransferase (EC 2.3.1. 23) activities in lung homogenates and in subcellular fractions, and fatty acid composition of phosphatidylcholine (PC) in lung lavage were studied in dogs with acute alveolar injury induced by N-nitroso-N-methylurethane. The specific activity in the microsomal fraction was 10 and 3 times higher than those of homogenate and mitochondrial fractions, respectively. Both the lysolecithin acyltransferase activities and the proportions of palmitate in alveolar lavage PC increased during the early phase of injury (days 2–4), and decreased during peak injury (days 6–8). Such correlation was not found during the recovery period (day 15). During recovery, specific and total activities of the enzyme were nearly 2- and 3-fold, respectively, those of controls. Nevertheless, the palmitate proportions in PC were normal, indicating that the increased enzyme activity in vitro was not reflected in increased PC palmitate during recovery. This finding indicates that the enzyme activity per cell was normal during recovery and suggests that the increase in specific and total activities is due to massive regeneration of type II cells and that the enzyme is localized mainly in these cells. The decrease in the proportion of palmitate in lavage PC during peak injury may lead to abnormality of surfactant function.