Imipramine and olanzapine block apoE4-catalyzed polymerization of Aß and show evidence of improving Alzheimer's disease cognition.

BACKGROUND: The apolipoprotein E (APOE) ε4 allele confers the strongest risk for late-onset Alzheimer's disease (AD) besides age itself, but the mechanisms underlying this risk are debated. One hypothesis supported by evidence from multiple labs is that apoE4 binds to the amyloid-ß (Aß) peptide and catalyzes its polymerization into neurotoxic oligomers and fibrils. Inhibiting this early step in the amyloid cascade may thereby reduce or prevent neurodegeneration and AD. METHODS: Using a design of experiments (DOE) approach, we developed a high-throughput assay to identify inhibitors of apoE4-catalyzed polymerization of Aß into oligomers and fibrils. We used it to screen the NIH Clinical Collection of small molecule drugs tested previously in human clinical trials. We then evaluated the efficacy and cytotoxicity of the hit compounds in primary neuron models of apoE4-induced Aß and phosphorylated tau aggregation. Finally, we performed retrospective analyses of the National Alzheimer's Coordinating Center (NACC) clinical dataset, using Cox regression and Cox proportional hazards models to determine if the use of two FDA-approved hit compounds was associated with better cognitive scores (Mini-Mental State Exam), or improved AD clinical diagnosis, when compared with other medications of the same clinical indication. RESULTS: Our high-throughput screen identified eight blood-brain barrier (BBB)-permeable hit compounds that reduced apoE4-catalyzed Aß oligomer and fibril formation in a dose-dependent manner. Five hit compounds were non-toxic toward cultured neurons and also reduced apoE4-promoted Aß and tau neuropathology in a dose-dependent manner. Three of the five compounds were determined to be specific inhibitors of apoE4, whereas the other two compounds were Aß or tau aggregation inhibitors. When prescribed to AD patients for their normal clinical indications, two of the apoE4 inhibitors, imipramine and olanzapine, but not other (non-hit) antipsychotic or antidepressant medications, were associated with improvements in cognition and clinical diagnosis, especially among APOE4 carriers. CONCLUSIONS: The critical test of any proposed AD mechanism is whether it leads to effective treatments. Our high-throughput screen identified two promising FDA-approved drugs, imipramine and olanzapine, which have no structural, functional, or clinical similarities other than their shared ability to inhibit apoE4-catalyzed Aß polymerization, thus identifying this mechanism as an essential contribution of apoE4 to AD.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice.

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12-14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 µg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

Astrogliosis and episodic memory in late life: higher GFAP is related to worse memory and white matter microstructure in healthy aging and Alzheimer's disease.

Astrocytes play a formative role in memory consolidation during physiological conditions; when dysregulated, astrocytes release glial fibrillary acidic protein (GFAP), which has been linked with negative memory outcomes in animal studies. We examined the association between blood GFAP, memory, and white matter (WM) integrity, accounting for blood markers of AD pathology (i.e., Aß42) and neurodegeneration (i.e., total tau; neurofilament light chain) in 114 older adults (asymptomatic, n = 69; MCI/AD dementia, n = 45). Higher levels of GFAP were associated with lower memory scores (p < 0.0001), such that for 1 SD increase in mean GFAP values, the memory composite score decreased on average by 0.49 (Standard error = 0.071). These results remained significant after controlling for diagnostic status and AD-related blood biomarkers. Higher GFAP was also related to lower WM integrity in regions vulnerable to AD pathology; however, WM integrity did not account for the association between GFAP and memory. Study findings suggest that higher blood levels of a marker of astrogliosis may reflect impoverished memory functions and white matter health, independent of markers of amyloid or neurodegeneration.

Trisomy 21 causes changes in the circulating proteome indicative of chronic autoinflammation.

Trisomy 21 (T21) causes Down syndrome (DS), but the mechanisms by which T21 produces the different disease spectrum observed in people with DS are unknown. We recently identified an activated interferon response associated with T21 in human cells of different origins, consistent with overexpression of the four interferon receptors encoded on chromosome 21, and proposed that DS could be understood partially as an interferonopathy. However, the impact of T21 on systemic signaling cascades in living individuals with DS is undefined. To address this knowledge gap, we employed proteomics approaches to analyze blood samples from 263 individuals, 165 of them with DS, leading to the identification of dozens of proteins that are consistently deregulated by T21. Most prominent among these proteins are numerous factors involved in immune control, the complement cascade, and growth factor signaling. Importantly, people with DS display higher levels of many pro-inflammatory cytokines (e.g. IL-6, MCP-1, IL-22, TNF-α) and pronounced complement consumption, resembling changes seen in type I interferonopathies and other autoinflammatory conditions. Therefore, these results are consistent with the hypothesis that increased interferon signaling caused by T21 leads to chronic immune dysregulation, and justify investigations to define the therapeutic value of immune-modulatory strategies in DS.

Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.

Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD.

Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death.

Cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine) is a non-proteinogenic thioether containing amino acid. In mammals, cystathionine is formed as an intermediate of the transsulfuration pathway by the condensation of serine and homocysteine (Hcy) in a reaction catalyzed by cystathionine ß-synthase (CBS). Cystathionine is subsequently converted to cysteine plus ammonia and α-ketobutyrate by the action of cystathionine γ-lyase (CGL). Pathogenic mutations in CBS result in CBS-deficient homocystinuria (HCU) which, if untreated, results in mental retardation, thromboembolic complications and connective tissue disorders. Currently there is no known function for cystathionine other than serving as an intermediate in transsulfuration and to date, the possible contribution of the abolition of cystathionine synthesis to pathogenesis in HCU has not been investigated. Using both mouse and cell-culture models, we have found that cystathionine is capable of blocking the induction of hepatic steatosis and kidney injury, acute tubular necrosis, and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Northern and Western blotting analysis indicate that the protective effects of cystathionine occur without any obvious alteration of the induction of the unfolded protein response. Our data constitute the first experimental evidence that the abolition of cystathionine synthesis may contribute to the pathology of HCU and that this compound has therapeutic potential for disease states where ER stress is implicated as a primary initiating pathogenic factor.

Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia.

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.

Moderate postnatal hyperoxia accelerates lung growth and attenuates pulmonary hypertension in infant rats after exposure to intra-amniotic endotoxin.

To determine the separate and interactive effects of fetal inflammation and neonatal hyperoxia on the developing lung, we hypothesized that: 1) antenatal endotoxin (ETX) causes sustained abnormalities of infant lung structure; and 2) postnatal hyperoxia augments the adverse effects of antenatal ETX on infant lung growth. Escherichia coli ETX or saline (SA) was injected into amniotic sacs in pregnant Sprague-Dawley rats at 20 days of gestation. Pups were delivered 2 days later and raised in room air (RA) or moderate hyperoxia (O2, 80% O2 at Denver's altitude, ∼65% O2 at sea level) from birth through 14 days of age. Heart and lung tissues were harvested for measurements. Intra-amniotic ETX caused right ventricular hypertrophy (RVH) and decreased lung vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein contents at birth. In ETX-exposed rats (ETX-RA), alveolarization and vessel density were decreased, pulmonary vascular wall thickness percentage was increased, and RVH was persistent throughout the study period compared with controls (SA-RA). After antenatal ETX, moderate hyperoxia increased lung VEGF and VEGFR-2 protein contents in ETX-O2 rats and improved their alveolar and vascular structure and RVH compared with ETX-RA rats. In contrast, severe hyperoxia (≥95% O2 at Denver's altitude) further reduced lung vessel density after intra-amniotic ETX exposure. We conclude that intra-amniotic ETX induces fetal pulmonary hypertension and causes persistent abnormalities of lung structure with sustained pulmonary hypertension in infant rats. Moreover, moderate postnatal hyperoxia after antenatal ETX restores lung growth and prevents pulmonary hypertension during infancy.

Bone marrow-derived angiogenic cells restore lung alveolar and vascular structure after neonatal hyperoxia in infant mice.

Neonatal hyperoxia impairs vascular and alveolar growth in mice and decreases endothelial progenitor cells. To determine the role of bone marrow-derived cells in restoration of neonatal lung structure after injury, we studied a novel bone marrow myeloid progenitor cell population from Tie2-green fluorescent protein (GFP) transgenic mice (bone marrow-derived angiogenic cells; BMDAC). We hypothesized that treatment with BMDAC would restore normal lung structure in infant mice during recovery from neonatal hyperoxia. Neonatal mice (1-day-old) were exposed to 80% oxygen for 10 days. BMDACs (1 x 10(5)), embryonic endothelial progenitor cells, mouse embryonic fibroblasts (control), or saline were then injected into the pulmonary circulation. At 21 days of age, saline-treated mice had enlarged alveoli, reduced septation, and a reduction in vascular density. In contrast, mice treated with BMDAC had complete restoration of lung structure that was indistinguishable from room air controls. BMDAC comprised 12% of distal lung cells localized to pulmonary vessels or alveolar type II (AT2) cells and persist (8.8%) for 8 wk postinjection. Coculture of AT2 cells or lung endothelial cells (luEC) with BMDAC augmented AT2 and luEC cell growth in vitro. We conclude that treatment with BMDAC after neonatal hyperoxia restores lung structure in this model of bronchopulmonary dysplasia.

Inhaled nitric oxide improves lung structure and pulmonary hypertension in a model of bleomycin-induced bronchopulmonary dysplasia in neonatal rats.

Whether inhaled nitric oxide (iNO) prevents the development of bronchopulmonary dysplasia (BPD) in premature infants is controversial. In adult rats, bleomycin (Bleo) induces lung fibrosis and pulmonary hypertension, but the effects of Bleo on the developing lung and iNO treatment on Bleo-induced neonatal lung injury are uncertain. Therefore, we sought to determine whether early and prolonged iNO therapy attenuates changes of pulmonary vascular and alveolar structure in a model of BPD induced by Bleo treatment of neonatal rats. Sprague-Dawley rat pups were treated with Bleo (1 mg/kg ip daily) or vehicle (controls) from day 2 to 10, followed by recovery from day 11 to 19. Treatment groups received early (days 2-10), late (days 11-19), or prolonged iNO therapy (10 ppm; days 2-19). We found that compared with controls, Bleo increased right ventricular hypertrophy (RVH), and pulmonary arterial wall thickness, and reduced vessel density alveolarization. In each iNO treatment group, iNO decreased RVH (P < 0.01) and wall thickness (P < 0.01) and restored vessel density after Bleo (P < 0.05). iNO therapy improved alveolarization for each treatment group after Bleo; however, the values remained abnormal compared with controls. Prolonged iNO treatment had greater effects on lung structure after bleomycin than late treatment alone. We conclude that Bleo induces lung structural changes that mimic BPD in neonatal rats, and that early and prolonged iNO therapy prevents right ventricle hypertrophy and pulmonary vascular remodeling and partially improves lung structure.

Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants.

Exposure of preterm infants to hyperoxia impairs vascular growth, contributing to the development of bronchopulmonary dysplasia and retinopathy of prematurity. Disruption of vascular endothelial growth factor (VEGF)-nitric oxide (NO) signaling impairs vascular growth. Endothelial progenitor cells (EPCs) may play an important role in vascular growth. Endothelial colony-forming cells (ECFCs), a type of EPC, from human preterm cord blood are more susceptible to hyperoxia-induced growth impairment than term ECFCs. Therefore, we hypothesized that hyperoxia disrupts VEGF-NO signaling and impairs growth in preterm ECFCs and that exogenous VEGF or NO preserves growth in hyperoxia. Growth kinetics of preterm cord blood-derived ECFCs (gestational ages, 27-34 wk) were assessed in room air (RA) and hyperoxia (40-50% oxygen) with or without VEGF, NO, or N(omega)-nitro-l-arginine. VEGF, VEGF receptor-2 (VEGFR-2), and endothelial NO synthase (eNOS) protein expression and NO production were compared. Compared with RA controls, hyperoxia significantly decreased growth, VEGFR-2 and eNOS expression, and NO production. VEGF treatment restored growth in hyperoxia to values measured in RA controls and significantly increased eNOS expression in hyperoxia. NO treatment also increased growth in hyperoxia. N(omega)-nitro-l-arginine treatment inhibited VEGF-augmented growth in RA and hyperoxia. We conclude that hyperoxia decreases growth and disrupts VEGF-NO signaling in human preterm ECFCs. VEGF treatment restores growth in hyperoxia by increasing NO production. NO treatment also increases growth during hyperoxia. Exogenous VEGF or NO may protect preterm ECFCs from the adverse effects of hyperoxia and preservation of ECFC function may improve outcomes of preterm infants.

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro.

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.

Intrauterine pulmonary hypertension impairs angiogenesis in vitro: role of vascular endothelial growth factor nitric oxide signaling.

RATIONALE: Mechanisms that impair angiogenesis in neonatal persistent pulmonary hypertension (PPHN) are poorly understood. OBJECTIVES: To determine if PPHN alters fetal pulmonary artery endothelial cell (PAEC) phenotype and impairs growth and angiogenesis in vitro, and if altered vascular endothelial growth factor-nitric oxide (VEGF-NO) signaling contributes to this abnormal phenotype. METHODS: Proximal PAECs were harvested from fetal sheep that had undergone partial ligation of the ductus arteriosus in utero (PPHN) and age-matched control animals. Growth and tube formation +/- VEGF and NO stimulation and inhibition were studied in normal and PPHN PAECs. Western blot analysis was performed for VEGF, VEGF receptor-2 (VEGF-R2), and endothelial NO synthase (eNOS) protein content. NO production with VEGF administration was measured in normal and PPHN PAECs. MEASUREMENTS AND MAIN RESULTS: PPHN PAECs demonstrate decreased growth and tube formation in vitro. VEGF and eNOS protein expression were decreased in PPHN PAECs, whereas VEGF-R2 protein expression was not different. VEGF and NO increased PPHN PAEC growth and tube formation to values achieved in normal PAECs. VEGF inhibition decreased growth and tube formation in normal and PPHN PAECs. NOS inhibition decreased growth in normal and PPHN PAECs, but tube formation was only reduced in normal PAECs. NO reversed the inhibitory effects of VEGF-R2 inhibition on tube formation in normal and PPHN PAECs. VEGF increased NO production in normal and PPHN PAECs. CONCLUSIONS: PPHN in utero causes sustained impairment of PAEC phenotype in vitro, with reduced PAEC growth and tube formation and down-regulation of VEGF and eNOS protein. VEGF and NO enhanced growth and tube formation of PPHN PAECs.

Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition.

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.

Hyperoxia reduces bone marrow, circulating, and lung endothelial progenitor cells in the developing lung: implications for the pathogenesis of bronchopulmonary dysplasia.

Hyperoxia disrupts vascular and alveolar growth of the developing lung and contributes to the development of bronchopulmonary dysplasia (BPD). Endothelial progenitor cells (EPC) have been implicated in repair of the vasculature, but their role in lung vascular development is unknown. Since disruption of vascular growth impairs lung structure, we hypothesized that neonatal hyperoxia impairs EPC mobilization and homing to the lung, contributing to abnormalities in lung structure. Neonatal mice (1-day-old) were exposed to 80% O(2) at Denver's altitude (= 65% at sea level) or room air for 10 days. Adult mice were also exposed for comparison. Blood, lung, and bone marrow were harvested after hyperoxia. Hyperoxia decreased pulmonary vascular density by 72% in neonatal but not adult mice. In contrast to the adult, hyperoxia simplified distal lung structure neonatal mice. Moderate hyperoxia reduced EPCs (CD45-/Sca-1+/CD133+/VEGFR-2+) in the blood (55%; P < 0.03), bone marrow (48%; P < 0.01), and lungs (66%; P < 0.01) of neonatal mice. EPCs increased in bone marrow (2.5-fold; P < 0.01) and lungs (2-fold; P < 0.03) of hyperoxia-exposed adult mice. VEGF, nitric oxide (NO), and erythropoietin (Epo) contribute to mobilization and homing of EPCs. Lung VEGF, VEGF receptor-2, endothelial NO synthase, and Epo receptor expression were reduced by hyperoxia in neonatal but not adult mice. We conclude that moderate hyperoxia decreases vessel density, impairs lung structure, and reduces EPCs in the circulation, bone marrow, and lung of neonatal mice but increases EPCs in adults. This developmental difference may contribute to the increased susceptibility of the developing lung to hyperoxia and may contribute to impaired lung vascular and alveolar growth in BPD.

Recombinant human VEGF treatment transiently increases lung edema but enhances lung structure after neonatal hyperoxia.

Recent studies suggest that VEGF may worsen pulmonary edema during acute lung injury (ALI), but, paradoxically, impaired VEGF signaling contributes to decreased lung growth during recovery from ALI due to neonatal hyperoxia. To examine the diverse roles of VEGF in the pathogenesis of and recovery from hyperoxia-induced ALI, we hypothesized that exogenous recombinant human VEGF (rhVEGF) treatment during early neonatal hyperoxic lung injury may increase pulmonary edema but would improve late lung structure during recovery. Sprague-Dawley rat pups were placed in a hyperoxia chamber (inspired O(2) fraction 0.9) for postnatal days 2-14. Pups were randomized to daily intramuscular injections of rhVEGF(165) (20 microg/kg) or saline (controls). On postnatal day 14, rats were placed in room air for a 7-day recovery period. At postnatal days 3, 14, and 21, rats were killed for studies, which included body weight and wet-to-dry lung weight ratio, morphometric analysis [including radial alveolar counts (RAC), mean linear intercepts (MLI), and vessel density], and lung endothelial NO synthase (eNOS) protein content by Western blot analysis. Compared with room air controls, hyperoxia increased pulmonary edema by histology and wet-to-dry lung weight ratios at postnatal day 3, which resolved by day 14. Although treatment with rhVEGF did not increase edema in control rats, rhVEGF increased wet-to-dry weight ratios in hyperoxia-exposed rats at postnatal days 3 and 14 (P < 0.01). Compared with room air controls, hyperoxia decreased RAC and increased MLI at postnatal days 14 and 21. Treatment with VEGF resulted in increased RAC by 181% and decreased MLI by 55% on postnatal day 14 in the hyperoxia group (P < 0.01). On postnatal day 21, RAC was increased by 176% and MLI was decreased by 58% in the hyperoxia group treated with VEGF. rhVEGF treatment during hyperoxia increased eNOS protein on postnatal day 3 by threefold (P < 0.05). We conclude that rhVEGF treatment during hyperoxia-induced ALI transiently increases pulmonary edema but improves lung structure during late recovery. We speculate that VEGF has diverse roles in hyperoxia-induced neonatal lung injury, contributing to lung edema during the acute stage of ALI but promoting repair of the lung during recovery.

BAY 41-2272, a direct activator of soluble guanylate cyclase, reduces right ventricular hypertrophy and prevents pulmonary vascular remodeling during chronic hypoxia in neonatal rats.

Exposure to hypoxia during the first weeks of life in newborn rats decreases vascular growth and alveolarization and causes pulmonary hypertension (PH). BAY 41-2272 is a novel direct activator of soluble guanylate cyclase independent of nitric oxide, effective as an acute pulmonary vasodilator in an animal model of persistent pulmonary hypertension of the newborn, but whether prolonged BAY 41-2272 therapy is effective in the setting of chronic PH is unknown. We hypothesize that BAY 41-2272 would prevent PH induced by chronic exposure to neonatal hypoxia. At 2 days of age, newborn rats were randomly exposed to hypoxia (FiO2, 0.12) or room air, and received daily intramuscular treatment with BAY 41-2272 (1 mg/kg) or saline. After 2 weeks, rats were killed for assessment of right ventricular hypertrophy (RVH), wall thickness of small pulmonary arteries, vessels density, radial alveolar counts and mean linear intercepts. In comparison with control, hypoxia increased RVH and artery wall thickness, reduced vessels density, decreased radial alveolar counts and increased mean linear intercepts. In comparison with hypoxic controls, prolonged BAY 41-2272 treatment during chronic hypoxia reduced RVH (0.67 +/- 0.03 vs. 0.52 +/- 0.05; p < 0.05), and attenuated artery wall thickness (48.2 +/- 2.8% vs. 35.7 +/- 4.1 microm; p < 0.01). However, BAY 41-2272 did not change vessels density, radial alveolar counts or mean linear intercepts. We conclude that BAY 41-2272 prevents the vascular structural effects of PH and reduces RVH but does not protect from hypoxia-induced inhibition of alveolarization and vessel growth. We speculate that BAY 41-2272 may provide a new therapy for chronic PH.

Inhaled NO restores lung structure in eNOS-deficient mice recovering from neonatal hypoxia.

We have previously shown that neonatal mice deficient in endothelial nitric oxide synthase (eNOS-/-) are more susceptible to hypoxic inhibition of alveolar and vascular growth. Although eNOS is downregulated, the role of nitric oxide (NO) during recovery after neonatal lung injury is poorly understood. We hypothesized that lung vascular and alveolar growth would remain impaired in eNOS-/- mice during recovery in room air and that NO therapy would augment compensatory lung growth in the eNOS-/- mice during recovery. Mice (1 day old) from heterozygous (eNOS+/-) parents were placed in hypobaric hypoxia (Fi(O2) = 0.16). After 10 days, pups were to recovered in room air (HR group) or inhaled NO (10 parts/million; HiNO group) until 3 wk of age, when lung tissue was collected. Morphometric analysis revealed that the eNOS-/- mice in the HR group had persistently abnormal lung structure compared with eNOS-sufficient (eNOS+/+) mice (increased mean linear intercept and reduced radial alveolar counts, nodal point density, and vessel density). Lung morphology of the eNOS+/- was not different from eNOS+/+. Inhaled NO after neonatal hypoxia stimulated compensatory lung growth in eNOS-/- mice that completely restored normal lung structure. eNOS+/- mice (HR group) had a 2.5-fold increase in lung vascular endothelial growth factor (VEGFR)-2 protein compared with eNOS+/+ (P < 0.05). eNOS-/- mice (HiNO group) had a 66% increase in lung VEGFR-2 protein compared with eNOS-/- (HR group; P < 0.01). We conclude that deficiency of eNOS leads to a persistent failure of lung growth during recovery from neonatal hypoxia and that, after hypoxia, inhaled NO stimulates alveolar and vascular growth in eNOS-/- mice.

Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats.

Exposure of newborn rats to hyperoxia impairs alveolarization and vessel growth, causing abnormal lung structure that persists during infancy. Recent studies have shown that impaired angiogenesis due to inhibition of vascular endothelial growth factor (VEGF) signaling decreases alveolar and vessel growth in the developing lung, and that nitric oxide (NO) mediates VEGF-dependent angiogenesis. The purpose of this study was to determine whether hyperoxia causes sustained reduction of lung VEGF, VEGF receptor, or endothelial NO synthase (eNOS) expression during recovery, and whether inhaled NO improves lung structure in infant rats after neonatal exposure to hyperoxia. Newborn rat pups were randomized to hyperoxia [fraction of inspired oxygen (Fio(2)), 1.00] or room air exposure for 6 d, and then placed in room air with or without inhaled NO (10 ppm) for 2 wk. Rats were then killed for studies, which included measurements of body weight, lung weight, right ventricular hypertrophy (RVH), morphometric analysis of alveolarization (by mean linear intercept (MLI), radial alveolar counts (RAC), and vascular volume (Vv), and immunostaining and Western blot analysis. In comparison with controls, neonatal hyperoxia reduced body weight, increased MLI, and reduced RAC in infant rats. Lung VEGF, VEGFR-2, and eNOS protein expression were reduced after hyperoxia. Inhaled NO treatment after hyperoxia increased body weight and improved distal lung growth, as demonstrated by increased RAC and Vv and decreased MLI. We conclude that neonatal hyperoxia reduced lung VEGF expression, which persisted during recovery in room air, and that inhaled NO restored distal lung growth in infant rats after neonatal hyperoxia.

Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats.

VEGF signaling inhibition decreases alveolar and vessel growth in the developing lung, suggesting that impaired VEGF signaling may contribute to decreased lung growth in bronchopulmonary dysplasia (BPD). Whether VEGF treatment improves lung structure in experimental models of BPD is unknown. The objective was to determine whether VEGF treatment enhances alveolarization in infant rats after hyperoxia. Two-day-old Sprague-Dawley rats were placed into hyperoxia or room air (RA) for 12 days. At 14 days, rats received daily treatment with rhVEGF-165 or saline. On day 22, rats were killed. Tissue was collected. Morphometrics was assessed by radial alveolar counts (RAC), mean linear intercepts (MLI), and skeletonization. Compared with RA controls, hyperoxia decreased RAC (6.1 +/- 0.4 vs. 11.3 +/- 0.4, P < 0.0001), increased MLI (59.2 +/- 1.8 vs. 44.0 +/- 0.8, P < 0.0001), decreased nodal point density (447 +/- 14 vs. 503 +/- 12, P < 0.0004), and decreased vessel density (11.7 +/- 0.3 vs. 18.9 +/- 0.3, P < 0.001), which persisted despite RA recovery. Compared with hyperoxic controls, rhVEGF treatment after hyperoxia increased RAC (11.8 +/- 0.5, P < 0.0001), decreased MLI (42.2 +/- 1.2, P < 0.0001), increased nodal point density (502 +/- 7, P < 0.0005), and increased vessel density (23.2 +/- 0.4, P < 0.001). Exposure of neonatal rats to hyperoxia impairs alveolarization and vessel density, which persists despite RA recovery. rhVEGF treatment during recovery enhanced vessel growth and alveolarization. We speculate that lung structure abnormalities after hyperoxia may be partly due to impaired VEGF signaling.