José G. Pichel

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P0 in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Cochlear abnormalities in insulin-like growth factor-1 mouse mutants

Insulin-like growth factor 1 (IGF-1) modulates inner ear cell proliferation, differentiation and survival in culture. Its function in human hearing was first evidenced by a report of a boy with a homozygous deletion of the Igf-1 gene, who showed severe sensorineural deafness [Woods et al., New Engl. J. Med. 335 (1996) 1363-1367]. To better understand the in vivo role of IGF-1 during inner ear differentiation and maturation, we studied the cochleae of Igf-1 gene knockout mice by performing morphometric stereological analyses, immunohistochemistry and electron microscopy on postnatal days 5 (P5), P8 and P20. At P20, but not at P5, the volumes of the cochlea and cochlear ganglion were significantly reduced in mutant mice, although the reduction was less severe than whole body dwarfism. A significant decrease in the number and average size of auditory neurons was also evident at P20. IGF-1-deficient cochlear neurons showed increased apoptosis, along with altered expression of neurofilament 200 kDa and vimentin. The eighth nerve, the cochlear ganglion and the fibers innervating the sensory cells of the organ of Corti of the P20 mouse mutants presented increased expression of vimentin, whereas the expression of neurofilament was decreased. In addition, the myelin sheath was severely affected in ganglion neurons. In conclusion, IGF-1 deficit in mice severely affects postnatal survival, differentiation and maturation of the cochlear ganglion cells.

Developmental cooperation of leukemia inhibitory factor and insulin-like growth factor I in mice is tissue-specific and essential for lung maturation involving the transcription factors Sp3 and TTF-1

The multifunctional proteins leukemia inhibitory factor (LIF) and insulin-like growth factor I (IGF-I) are expressed in overlapping patterns during development and, therefore, may act cooperatively. We show that mice doubly deficient in LIF and IGF-I all died at birth of apparent respiratory failure. Growth retardation, muscle hypoplasia and delayed ossification in IGF-I-deficient E18.5 mice were exacerbated by the absence of LIF. The transcription factor Sp3 was decreased in the skeleton of the double null mice. Pronounced depletion of olfactory bulb neurons, in contrast, was only IGF-I-dependent. The lungs displayed reduced air space in the IGF-I-deficient embryos and neonates, phenotype exacerbated in the double nulls, which showed abnormal epithelial cells and decreased Sp3 expression. In addition, the transcription factor TTF-1 and the surfactant protein B were lower in the lung of the double null neonates than in all other genotypes. LIF and IGF-I, thus, have cooperative and distinct tissue functions during development. Their essential role in bone ossification apparently involves Sp3, and in lung maturation Sp3 together with TTF-1.

Increased leptin and white adipose tissue hypoplasia are sexually dimorphic in Lif null/Igf‐I haploinsufficient mice

We previously showed cooperation of leukemia inhibitory factor (LIF) and insulin‐like growth factor I (IGF‐I) during development. Mice doubly deficient in LIF and IGF‐I died at birth. We now analyze the possible combined influence of both factors on postnatal growth. The haploinsufficiency of the Igf‐I gene on a Lif null background caused a marked reduction in body mass index and white adipose tissue only in female mice. These animals had increased leptin, increased serum IGF‐I and apparent substitution of white adipose tissue by brown adipose tissue. The complex interrelationships between LIF and IGF‐I in regulating weight thus involve sexually dimorphic effects on adipose tissue differentiation and circulating leptin.

Involvement of Igf1r in Bronchiolar Epithelial Regeneration: Role during Repair Kinetics after Selective Club Cell Ablation

Regeneration of lung epithelium is vital for maintaining airway function and integrity. An imbalance between epithelial damage and repair is at the basis of numerous chronic lung diseases such as asthma, COPD, pulmonary fibrosis and lung cancer. IGF (Insulin-like Growth Factors) signaling has been associated with most of these respiratory pathologies, although their mechanisms of action in this tissue remain poorly understood. Expression profiles analyses of IGF system genes performed in mouse lung support their functional implication in pulmonary ontogeny. Immuno-localization revealed high expression levels of Igf1r (Insulin-like Growth Factor 1 Receptor) in lung epithelial cells, alveolar macrophages and smooth muscle. To further understand the role of Igf1r in pulmonary homeostasis, two distinct lung epithelial-specific Igf1r mutant mice were generated and studied. The lack of Igf1r disturbed airway epithelial differentiation in adult mice, and revealed enhanced proliferation and altered morphology in distal airway club cells. During recovery after naphthalene-induced club cell injury, the kinetics of terminal bronchiolar epithelium regeneration was hindered in Igf1r mutants, revealing increased proliferation and delayed differentiation of club and ciliated cells. Amid airway restoration, lungs of Igf1r deficient mice showed increased levels of Igf1, Insr, Igfbp3 and epithelial precursor markers, reduced amounts of Scgb1a1 protein, and alterations in IGF signaling mediators. These results support the role of Igf1r in controlling the kinetics of cell proliferation and differentiation during pulmonary airway epithelial regeneration after injury.

IGF1R deficiency attenuates acute inflammatory response in a bleomycin-induced lung injury mouse model

IGF1R (Insulin-like Growth Factor 1 Receptor) is a tyrosine kinase with pleiotropic cellular functions. IGF activity maintains human lung homeostasis and is implicated in pulmonary diseases such as cancer, ARDS, COPD, asthma and fibrosis. Here we report that lung transcriptome analysis in mice with a postnatally-induced Igf1r gene deletion showed differentially expressed genes with potentially protective roles related to epigenetics, redox and oxidative stress. After bleomycin-induced lung injury, IGF1R-deficient mice demonstrated improved survival within a week. Three days post injury, IGF1R-deficient lungs displayed changes in expression of IGF system-related genes and reduced vascular fragility and permeability. Mutant lungs presented reduced inflamed area, down-regulation of pro-inflammatory markers and up-regulation of resolution indicators. Decreased inflammatory cell presence in BALF was reflected in diminished lung infiltration mainly affecting neutrophils, also corroborated by reduced neutrophil numbers in bone marrow, as well as reduced lymphocyte and alveolar macrophage counts. Additionally, increased SFTPC expression together with hindered HIF1A expression and augmented levels of Gpx8 indicate that IGF1R deficiency protects against alveolar damage. These findings identify IGF1R as an important player in murine acute lung inflammation, suggesting that targeting IGF1R may counteract the inflammatory component of many lung diseases.

Prominent release of lipoxygenase generated mediators in a murine house dust mite-induced asthma model

The profile of activation of lipid mediator (LM) pathways in asthmatic airway inflammation remains unclear. This experimental study quantified metabolite levels of ω3-, ω6- and ω9-derived polyunsaturated fatty acids in bronchoalveolar lavage fluid (BALF) after 4-weeks of repeated house dust mite (HDM) exposure in a murine (C57BL/6) asthma model. The challenge induced airway hyperresponsiveness, pulmonary eosinophil infiltration, but with low and unchanged mast cell numbers. Of the 112 screened LMs, 26 were increased between 2 to >25-fold in BALF with HDM treatment (p < 0.05, false discovery rate = 5%). While cysteinyl-leukotrienes were the most abundant metabolites at baseline, their levels did not increase after HDM treatment, whereas elevation of PGD2, LTB4 and multiple 12/15-lipoxygenase products, such as 5,15-DiHETE, 15-HEDE and 15-HEPE were observed. We conclude that this model has identified a global lipoxygenase activation signature, not linked to mast cells, but with aspects that mimic chronic allergic airway inflammation in asthma.

Characterization of the acute inflammatory profile and resolution of airway inflammation after Igf1r-gene targeting in a murine model of HDM-induced asthma

Asthma is a chronic inflammatory disease characterized by bronchial hyperresponsiveness, mucus overproduction and airway remodeling. Notably, we have recently demonstrated that insulin-like growth factor 1 receptor (IGF1R) deficiency in mice attenuates airway hyperresponsiveness and mucus secretion after chronic house dust mite (HDM) exposure. On this basis, inbred C57BL/6 and Igf1r-deficient mice were given HDM extract to study the acute inflammatory profile and implication of Igf1r in acute asthma pathobiology. Additionally, Igf1r-deficiency was therapeutically induced in mice to evaluate the resolution of HDM-induced inflammation. Acute HDM exposure in inbred C57BL/6 mice led to a progressive increase in inflammation, airway remodeling and associated molecular indicators. Preventively-induced Igf1r-deficiency showed reduced neutrophil and eosinophil numbers in BALF and bone marrow, a significant reduction of airway remodeling and decreased levels of related markers. In addition, therapeutic targeting of Igf1r promoted the resolution of HDM-induced-inflammation. Our results demonstrate for the first time that Igf1r is important in acute asthma pathobiology and resolution of HDM-induced inflammation. Thus, IGF1R is suggested to be a promising candidate for future therapeutic approaches for the treatment and prevention of asthma.