Inhaled Nitric Oxide Reverses Vascular and Respiratory Effects of ET-1 and PAF in Pigs

In anaesthetized paralysed, mechanically ventilated pigs, the vascular and respiratory effects of 80 ppm nitric oxide (NO) inhaled for 6 min were evaluated. To evoke different levels of smooth muscle contraction ET-1 or PAF, mediators involved in pulmonary disorders, were used. In control conditions, inhaled NO caused selective pulmonary vasodilatation without affecting respiratory resistances. This pulmonary vascular activity influenced the distensibility of the respiratory system and decreased inspiratory work. ET-1 administration significantly increased pulmonary arterial pressure and modestly changed mechanical properties of the respiratory system, while PAF caused potent vasoconstriction and bronchoconstriction associated with a marked change in volume-pressure relationship. In both cases, the changes in vascular and mechanical properties of the respiratory system increased inspiratory work. The vascular and respiratory activities of inhaled NO were correlated with preconstriction levels. The data show that the combination of vascular and respiratory effects improves pulmonary function, suggesting that inhalation of NO is a possible therapeutic approach for obstructive and inflammatory pulmonary diseases

Dental and craniofacial defects in the Crtap−/− mouse model of osteogenesis imperfecta type VII

BackgroundInactivating mutations in the gene for cartilage‐associated protein (CRTAP) cause osteogenesis imperfecta type VII in humans, with a phenotype that can include craniofacial defects. Dental and craniofacial manifestations have not been a focus of case reports to date. We analyzed the craniofacial and dental phenotype of Crtap−/− mice by skull measurements, micro‐computed tomography (micro‐CT), histology, and immunohistochemistry.ResultsCrtap−/− mice exhibited a brachycephalic skull shape with fusion of the nasofrontal suture and facial bones, resulting in mid‐face retrusion and a class III dental malocclusion. Loss of CRTAP also resulted in decreased dentin volume and decreased cellular cementum volume, though acellular cementum thickness was increased. Periodontal dysfunction was revealed by decreased alveolar bone volume and mineral density, increased periodontal ligament (PDL) space, ectopic calcification within the PDL, bone‐tooth ankylosis, altered immunostaining of extracellular matrix proteins in bone and PDL, increased pSMAD5, and more numerous osteoclasts on alveolar bone surfaces.ConclusionsCrtap−/− mice serve as a useful model of the dental and craniofacial abnormalities seen in individuals with osteogenesis imperfecta type VII.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155878/1/dvdy166.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155878/2/dvdy166_am.pd

A Rab33b missense mouse model for Smith-McCort dysplasia shows bone resorption defects and altered protein glycosylation

Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either RAB33B or DYM genes. These genes codes for proteins that are located at the Golgi apparatus and have a role in intracellular vesicle trafficking. We generated mice that carry a Rab33b disease-causing variant, c.136A>C (p.Lys46Gln), which is identical to that of members from a consanguineous family diagnosed with SMC. In male mice at 4 months of age, the Rab33b variant caused a mild increase in trabecular bone thickness in the spine and femur and in femoral mid-shaft cortical thickness with a concomitant reduction of the femoral medullary area, suggesting a bone resorption defect. In spite of the increase in trabecular and cortical thickness, bone histomorphometry showed a 4-fold increase in osteoclast parameters in homozygous Rab33b mice suggesting a putative impairment in osteoclast function, while dynamic parameters of bone formation were similar in mutant versus control mice. Femur biomechanical tests showed an increased in yield load and a progressive elevation, from WT to heterozygote to homozygous mutants, of bone intrinsic properties. These findings suggest an overall impact on bone material properties which may be caused by disturbed protein glycosylation in cells contributing to skeletal formation, supported by the altered and variable pattern of lectin staining in murine and human tissue cultured cells and in liver and bone murine tissues. The mouse model only reproduced some of the features of the human disease and was sex-specific, manifesting in male but not female mice. Our data reveal a potential novel role of RAB33B in osteoclast function and protein glycosylation and their dysregulation in SMC and lay the foundation for future studies

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. Keywords: Osteocyte, RANKL, Osteogenesis imperfecta, Bone fragilit

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

Loss of Sc65 results in dermal tears, abnormal collagen fibrils and skin fragility.

<p>a) H&E stained sections of WT and <i>Sc65KO</i> skin. Note the decreased density of collagen, the frayed dermis indicated by arrows and the reduced thickness of the muscle layer in the <i>Sc65-null</i> samples. b) Serial skin sections were stained with Sirius red. <i>Sc65-null</i> skin exhibits fewer large collagen fibers (red staining) and greater number of smaller collagen fibers stained in green compared to WT counterparts. c) Electron micrographs of 7 month-old mouse skin biopsy from WT and <i>Sc65KO</i> mice. Collagen fibrils, shown in cross-section, from <i>Sc65-null</i> skin tended to be smaller and have a decreased range of fibril diameter compared to WT fibrils. Loss of Sc65 also resulted in the presence of collagen fibrils with irregular profile and several large “cauliflower-like” fibrils (red arrow) which indicate abnormal fibrillogenesis (scale bar represents 500nm). d) Distribution of collagen fibril diameter in WT and <i>Sc65KO</i> mouse skin as measured from electron microscopy images. Measurements were collected from three different mice/genotype and >200 fibril/mouse. e) Skin EMs from <i>Sc65KO</i> mice also exhibited significantly more empty space among collagen fibrils compared to WT mice indicating a less densely packed collagen (*p = 0.01). Five electron micrograph images of non-overlapping areas were quantified from each mouse. f-h) Skin samples from WT and <i>Sc65KO</i> mice were subjected to a biomechanical skin loading test to measure tensile strength. Skin that lacks SC65 expression ruptured at a significantly lower peak load compared to WT skin indicating significant skin fragility (*p<0.01).</p

Schematic representation of a fibrillar collagen molecule in the ER.

<p>The long uninterrupted triple-helical domain is shown here already folded and without N- and C-propeptides. Depicted in different colors are some of the prolyl 3-hydroxylase enzymes (P3Hs), lysyl hydroxylase enzymes (LHs) and cyclophilin B (purple) with some of their known substrate residues. Our previous work identified the prolyl 3-hydroxylation complex that modifies P986; our current work suggests the existence of a new complex, possibly including CYPB, responsible for the hydroxylation of K87 and K930. Evidence indicates that CRTAP and SC65 act as unique orchestrators of essential molecular complexes for collagen post-translational modification. (LH2 is likely to also act within a protein complex but direct interactions have not yet been published).</p

<i>Sc65KO</i> mouse generation and confirmation of bone loss phenotype.

<p>a) Strategy for the creation of the <i>Sc65-null</i> allele. The schematic diagram shows the <i>Sc65</i> wild-type, targeted, floxed and excised allele. <i>Sc65</i> coding regions are in light blue while non-coding regions are in dark blue. Also note the proximity to the <i>Fkbp10</i> gene which is transcribed in the opposite orientation. b) PCR genotyping of <i>Sc65KO</i> mice (upper panel) and Western blot confirmation of SC65 protein (arrow) loss in multiple <i>Sc65KO</i> tissues from 3 day-old mice compared to WT controls (lower panel—Cal = calvaria, Kid = Kidney). c) Immunohistochemistry detection of SC65 in adult femur section from a WT mouse showing specific intracellular staining in bone forming cells (osteoblasts) aligned on the surface of a bone trabecula. <i>Sc65</i> expression is lost in a similar section from a <i>Sc65KO</i> mouse. Scale bars = 100μM (10x) or 20μM (63x). d) MicroCT analysis of long bones from 6 month-old WT and <i>Sc65KO</i> male mice (n = 9). Both femurs and tibias from <i>Sc65KO</i> mice exhibited decreased trabecular bone volume/tissue volume (BV/TV), connectivity density (Conn.D) and cortical thickness (Ct.Th) compared to WT controls (*p<0.05).</p

Increased electrophoretic mobility and altered cross-linking of type I collagen from <i>Sc65-null</i> skin.

<p>a) SDS-6%PAGE of type I collagen extracted from skin and decalcified bone of <i>Sc65KO</i> and WT mice shows increased mobility of α-chains and reduced ratio of cross-linked β to γ components in the <i>Sc65KO</i> skin extracts. An acetic acid extract from skin of the original Sc65-null mouse¹⁹ (1 mo.) created by gene-trap insertion is compared with that from the new <i>Sc65KO</i> (6 mo.) and their respective WT controls. Total heat denatured extracts of skin and bone collagens from new <i>Sc65KO</i> mice are shown on the right for comparison. Bone collagen from <i>Sc65KO</i> mice does not show the differences from WT in β/γ intensities evident for skin collagen. The strong (lower) γ band in SC65 skin extracts was identified as γ _112. Both original and new <i>Sc65KO</i> mice showed the same altered pattern of chain intensities from WT most pronounced in the acetic acid extracts of skin with an apparent increase in γ₁₁₂ at the expense of β_12. b) Densitometric analysis of collagen bands on SDS-PAGE. Densitometry was performed on bands 1–8 (counted from top to bottom) of acetic acid extracts from 1mo and 6mo skin samples of both original and new <i>Sc65KO</i> mice using NIH imageJ software. Values are means ± SD, n = 6; *p<0.01.</p