Respiratory defects in the CrtapKO mouse model of osteogenesis imperfecta.

Respiratory disease is a leading cause of mortality in patients with osteogenesis imperfecta (OI), a connective tissue disease that causes severely reduced bone mass and is most commonly caused by dominant mutations in type I collagen genes. Previous studies proposed that impaired respiratory function in OI patients was secondary to skeletal deformities; however, recent evidence suggests the existence of a primary lung defect. Here, we analyzed the lung phenotype of Crtap knockout (KO) mice, a mouse model of recessive OI. While we confirm changes in the lung parenchyma that are reminiscent of emphysema, we show that CrtapKO lung fibroblasts synthesize type I collagen with altered posttranslation modifications consistent with those observed in bone and skin. Unrestrained whole body plethysmography showed a significant decrease in expiratory time, resulting in an increased ratio of inspiratory time over expiratory time and a concomitant increase of the inspiratory duty cycle in CrtapKO compared with WT mice. Closed-chest measurements using the forced oscillation technique showed increased respiratory system elastance, decreased respiratory system compliance, and increased tissue damping and elasticity in CrtapKO mice compared with WT. Pressure-volume curves showed significant differences in lung volumes and in the shape of the curves between CrtapKO mice and WT mice, with and without adjustment for body weight. This is the first evidence that collagen defects in OI cause primary changes in lung parenchyma and several respiratory parameters and thus negatively impact lung function.

Krüppel-like Factor 9 (KLF9) Suppresses Hepatocellular Carcinoma (HCC)-Promoting Oxidative Stress and Inflammation in Mice Fed High-Fat Diet.

Obesity, oxidative stress, and inflammation are risk factors for hepatocellular carcinoma (HCC). We examined, in mice, the effects of Krüppel-like factor 9 (KLF9) knockout on: adiposity, hepatic and systemic oxidative stress, and hepatic expression of pro-inflammatory and NOX/DUOX family genes, in a high-fat diet (HFD) context. Male and female Klf9+/+ (wild type, WT) and Klf9-/- (knockout, KO) mice were fed HFD (beginning at age 35 days) for 12 weeks, after which liver and adipose tissues were obtained, and serum adiponectin and leptin levels, liver fat content, and markers of oxidative stress evaluated. Klf9-/- mice of either sex did not exhibit significant alterations in weight gain, adipocyte size, adipokine levels, or liver fat content when compared to WT counterparts. However, Klf9-/- mice of both sexes had increased liver weight/size (hepatomegaly). This was accompanied by increased hepatic oxidative stress as indicated by decreased GSH/GSSG ratio and increased homocysteine, 3-nitrotyrosine, 3-chlorotyrosine, and 4HNE content. Decreased GSH to GSSG ratio and a trend toward increased homocysteine levels were observed in the corresponding Klf9-/- mouse serum. Gene expression analysis showed a heightened pro-inflammatory state in livers from Klf9-/- mice. KLF9 suppresses hepatic oxidative stress and inflammation, thus identifying potential mechanisms for KLF9 suppression of HCC and perhaps cancers of other tissues.

Lesion Genotype Modifies High-Fat Diet Effects on Endometriosis Development in Mice.

Endometriosis is a chronic, estrogen-dependent gynecologic disorder that affects reproductive-aged women and to a lesser extent, post-menopausal women on hormone therapy. The condition is associated with systemic and local immune dysfunctions. While its underlying mechanisms remain poorly understood, endometriosis has a genetic component and propensity for the disease is subject to environmental, nutritional, and lifestyle influences. Previously, we showed that high-fat diet (HFD) increased ectopic lesion numbers, concurrent with systemic and peritoneal changes in inflammatory and oxidative stress status, in immunocompetent recipient mice ip administered with endometrial fragments null for Krüppel-like factor 9 gene. Herein, we determined whether HFD modifies lesion parameters, when recipient peritoneal environment is challenged with ectopic wild-type (WT) endometrial fragments, the latter simulating retrograde menstruation common in women during the menstrual period. WT endometrium-recipient mice fed HFD (45% kcal from fat) showed reduced lesion incidence, numbers, and volumes, in the absence of changes in systemic ovarian steroid hormone and insulin levels, relative to those fed the control diet (CD, 17% kcal from fat). Lesions from HFD- and CD-fed recipients demonstrated comparable gene expression for steroid hormone receptors (Esr and Pgr) and cytokines (Il-6, Il-8, and CxCL4) and similar levels of DNA oxidative biomarkers. HFD moderately altered serum (3-nitrotyrosine and methionine/homocysteine) and peritoneal (reduced glutathione/oxidized glutathione) pro-oxidative status but had no effect on peritoneal inflammatory (tumor necrosis factor α and tumor necrosis factor receptor 1) mediators. Results indicate that lesion genotype modifies dietary effects on disease establishment and/or progression and if translated, could be important for provision of nutritional guidelines to women with predisposition to, or affected by endometriosis.

Evaluating body composition in infancy and childhood: A comparison between 4C, QMR, DXA, and ADP.

BACKGROUND: Accurate and precise methods to measure of body composition in infancy and childhood are needed. OBJECTIVES: This study evaluated differences and precision of three methods when compared with the four-compartment (4C) model for estimating fat mass (FM). METHODS: FM of children (age 14 days to 6 years of age, N = 346) was obtained using quantitative nuclear magnetic resonance (QMR, EchoMRI-AH), air-displacement plethysmography (ADP, PeaPod, less than or equal to 8 kg, BodPod age 6 years or older), and dual-energy X-ray absorptiometry (DXA, Hologic QDR). The 4C model was computed. Correlation, concordance, and Bland-Altman analyses were performed. RESULTS: In infants, PeaPod had high individual FM accuracy, whereas DXA had high group FM accuracy compared with 4C. In children, DXA had high group and individual FM accuracies compared with 4C. QMR underestimated group FM in infants and children (300 and 510 g, respectively). The instrument FM precision was best for QMR (10 g) followed by BodPod (34 g), PeaPod (38 g), and DXA (45 g). CONCLUSIONS: In infants, PeaPod was the best method to estimate individual FM whereas DXA was best to estimate group FM. In children, DXA was best to estimate individual and group FM. QMR had the highest instrument precision.

Maternal Adiposity is Associated with Fat Mass Accretion in Female but not Male Offspring During the First 2 Years of Life.

OBJECTIVE: This study investigated which antenatal and postnatal factors determine offspring adiposity during the first 2 years of life. METHODS: Participants were mother and child pairs (N = 224). Offspring percent fat mass (%FM) was obtained using quantitative nuclear magnetic resonance at 11 time points between ages 0.5 and 24 months. Independent variables included race, age, gestational weight gain, first-trimester %FM, delivery mode, gestational measures of resting energy expenditure, respiratory exchange ratio, physical activity, serum cytokines and lipids, and dietary intake for the mothers, as well as sex, birth weight and length, breastfeeding duration, and physical activity at age 2 years for the children. Linear mixed models were used to construct the best-fitted models for the entire cohort and for each sex. RESULTS: Maternal %FM (P = 0.006), high-density lipoprotein (HDL) (P < 0.001), and breastfeeding duration (P = 0.023) were positively associated with female offspring adiposity, whereas maternal dietary fiber intake (P = 0.016) had a negative association. Birth weight (P = 0.004), maternal HDL (P = 0.013), and breastfeeding duration (P = 0.015) were all positively associated with male offspring adiposity. CONCLUSIONS: Antenatal and postnatal factors differentially impact male and female offspring adiposity during the first 2 years of life.

The Osteocyte Transcriptome Is Extensively Dysregulated in Mouse Models of Osteogenesis Imperfecta.

Osteocytes are long-lived, highly interconnected, terminally differentiated osteoblasts that reside within mineralized bone matrix. They constitute about 95% of adult bone cells and play important functions including in the regulation of bone remodeling, phosphate homeostasis, and mechanical stimuli sensing and response. However, the role of osteocytes in the pathogenesis of congenital diseases of abnormal bone matrix is poorly understood. This study characterized in vivo transcriptional changes in osteocytes from CrtapKO and oim/oim mouse models of osteogenesis imperfecta (OI) compared with wild-type (WT) control mice. To do this, RNA was extracted from osteocyte-enriched cortical femurs and tibias, sequenced and subsequently analyzed to identify differentially expressed transcripts. These models were chosen because they mimic two types of OI with different genetic mutations that result in distinct type I collagen defects. A large number of transcripts were dysregulated in either model of OI, but 281 of them were similarly up- or downregulated in both compared with WT controls. Conversely, very few transcripts were differentially expressed between the CrtapKO and oim/oim mice, indicating that distinct alterations in type I collagen can lead to shared pathogenic processes and similar phenotypic outcomes. Bioinformatics analyses identified several critical hubs of dysregulation that were enriched in annotation terms such as development and differentiation, ECM and collagen fibril organization, cell adhesion, signaling, regulatory processes, pattern binding, chemotaxis, and cell projections. The data further indicated alterations in important signaling pathways such as WNT and TGF-ß but also highlighted new candidate genes to pursue in future studies. Overall, our study suggested that the osteocyte transcriptome is broadly dysregulated in OI with potential long-term consequences at the cellular level, which deserve further investigations. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Loss of RANKL in osteocytes dramatically increases cancellous bone mass in the osteogenesis imperfecta mouse (oim).

Osteogenesis imperfecta (OI) is characterized by osteopenia and bone fragility, and OI patients during growth often exhibit high bone turnover with the net result of low bone mass. Recent evidence shows that osteocytes significantly affect bone remodeling under physiological and pathological conditions through production of osteoclastogenic cytokines. The receptor activator of nuclear factor kappa-B ligand (RANKL) produced by osteocytes for example, is a critical mediator of bone loss caused by ovariectomy, low-calcium diet, unloading and glucocorticoid treatment. Because OI bone has increased density of osteocytes and these cells are embedded in matrix with abnormal type I collagen, we hypothesized that osteocyte-derived RANKL contributes to the OI bone phenotype. In this study, the conditional loss of RANKL in osteocytes in oim/oim mice (oim-RANKL-cKO) resulted in dramatically increased cancellous bone mass in both the femur and lumbar spine compared to oim/oim mice. Bone cortical thickness increased significantly only in spine but ultimate bone strength in the long bone and spine was minimally improved in oim-RANKL-cKO mice compared to oim/oim mice. Furthermore, unlike previous findings, we report that oim/oim mice do not exhibit high bone turnover suggesting that their low bone mass is likely due to defective bone formation and not increased bone resorption. The loss of osteocyte-derived RANKL further diminished parameters of formation in oim-RANKL-cKO. Our results indicate that osteocytes contribute significantly to the low bone mass observed in OI and the effect of loss of RANKL from these cells is similar to its systemic inhibition.

Expression characterization and functional implication of the collagen-modifying Leprecan proteins in mouse gonadal tissue and mature sperm.

The Leprecan protein family which includes the prolyl 3-hydroxylase enzymes (P3H1, P3H2, and P3H3), the closely related cartilage-associated protein (CRTAP), and SC65 (Synaptonemal complex 65, aka P3H4, LEPREL4), is involved in the post-translational modification of fibrillar collagens. Mutations in CRTAP, P3H1 and P3H2 cause human genetic diseases. We recently showed that SC65 forms a stable complex in the endoplasmic reticulum with P3H3 and lysyl hydroxylase 1 and that loss of this complex leads to defective collagen lysyl hydroxylation and causes low bone mass and skin fragility. Interestingly, SC65 was initially described as a synaptonemal complex-associated protein, suggesting a potential additional role in germline cells. In the present study, we describe the expression of SC65, CRTAP and other Leprecan proteins in postnatal mouse reproductive organs. We detect SC65 expression in peritubular cells of testis up to 4 weeks of age but not in cells within seminiferous tubules, while its expression is maintained in ovarian follicles until adulthood. Similar to bone and skin, SC65 and P3H3 are also tightly co-expressed in testis and ovary. Moreover, we show that CRTAP, a protein normally involved in collagen prolyl 3-hydroxylation, is highly expressed in follicles and stroma of the ovary and in testes interstitial cells at 4 weeks of age, germline cells and mature sperm. Importantly, CrtapKO mice have a mild but significant increase in morphologically abnormal mature sperm (17% increase compared to WT). These data suggest a role for the Leprecans in the post-translational modification of collagens expressed in the stroma of the reproductive organs. While we could not confirm that SC65 is part of the synaptonemal complex, the expression of CRTAP in the seminiferous tubules and in mature sperm suggest a role in the testis germ cell lineage and sperm morphogenesis.