The Yes-associated protein YAP is a downstream effector of the Hippo pathway of cell cycle control which plays important roles in tumorigenesis. Hippo-mediated phosphorylation YAP, mainly at S127, inactivates YAP function. In this study, we define a mechanism for positive regulation of YAP activity that is critical for its oncogenic function. Specifically, we found that YAP is phosphorylated in vitro and in vivo by the cell cycle kinase CDK1 at T119, S289, and S367 during G2/M phase of the cell cycle. We also found that ectopic expression of a phosphomimetic YAP mutant (YAP3D, harboring T119D/S289D/S367D) was sufficient to induce mitotic defects in immortalized epithelial cells, including centrosome amplification, multipolar spindles and chromosome missegregation. Finally, we documented that mitotic phosphorylation of YAP was sufficient to promote cell migration and invasion in a manner essential for neoplastic cell transformation. In support of our findings, CDK1 inhibitors largely suppressed cell motility mediated by activated YAP-S127A but not the phosphomimetic mutant YAP3D. Collectively, our results reveal a previously unrecognized mechanism for controlling the activity of YAP that is crucial for its oncogenic function mediated by mitotic dysregulation.

Periodontitis is characterized by the progressive destruction of tooth-supporting alveolar bone, which is mainly caused by chronic inflammation in response to persistent bacterial insult. It has recently become clear that the pathogenesis of periodontitis is associated with a high ratio of proinflammatory M1 (classically activated) macrophages to anti-inflammatory M2 (alternatively activated). To decrease the inflammatory activity, we locally delivered the C-C motif chemokine ligand 2 (CCL2) using controlled-release microparticles (MPs). CCL2 is known to promote chemotaxis of M0 or M2 phenotype macrophages to the inflamed site and induce M2 phenotype polarization locally. Our in vitro data showed that CCL2 increased the number of M2 phenotype macrophages, decreased TNF-α secretion, and enhanced chemotaxis of RAW264.7 cells toward CCL2 MPs. Moreover, we induced periodontal disease in 2 animal models through inoculation of Porphyromonas gingivalis and ligature around the murine molar. Micro–computed tomography analysis showed significant reduction of alveolar bone loss in the CCL2 MP treatment group when compared with a blank MP group and a no-treatment periodontitis group in both models. Immunohistologic analysis showed a significant increase in the M2 phenotype subset and a decrease in the M1 phenotype subset in the CCL2 MP group of the P. gingivalis–induced model. Also, in both models, tartrate-resistant acidic phosphatase staining showed significantly fewer numbers of osteoclasts in the CCL2 MP group in alveolar bone area. Moreover, quantitative polymerase chain reaction results showed a significant increase in IL-1RA (interleukin 1 receptor antagonist) mRNA expression and a decrease in RANKL (receptor activator of nuclear factor kappa-Β ligand) mRNA expression in the CCL2 MP group in the ligature model. In summary, manipulation of endogenous M2 phenotype macrophages with CCL2 MPs decreased the M1 phenotype:M2 phenotype ratio and prevented alveolar bone loss in mouse periodontitis models. The delivery of CCL2 MPs provides a novel approach to treat periodontal disease.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling. The obesity epidemic has produced great interest in the relationship between small visceral adipose tissue depots producing localized inflammatory conditions, which may link metabolism, innate immunity, and vascular remodeling. This study used novel micro computed tomography (microCT) three-dimensional modeling to investigate the degree of remodeling of the lung vasculature and differential proteomics to determine small visceral adipose dysfunction in rats with severe PAH. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) with subsequent hypoxia exposure for 3 weeks (SU/hyp). At 12 weeks after hypoxia, microCT analysis showed a decrease in the ratio of vascular to total tissue volume within the SU/hyp group (mean ± standard deviation: 0.27 ± 0.066; P = 0.02) with increased vascular separation (0.37 ± 0.062 mm; P = 0.02) when compared with the control (0.34 ± 0.084 and 0.30 ± 0.072 mm). Differential proteomics detected an up-regulation of complement protein 3 (C3; SU/hyp ∶ control ratio = 2.86) and the adipose tissue-specific fatty acid binding protein-4 (FABP4, 2.66) in the heart adipose of the SU/hyp. Significant remodeling of the lung vasculature validates the efficacy of the SU/hyp rat for modeling human PAH. The upregulation of C3 and FABP4 within the heart adipose implicates small visceral adipose dysfunction. C3 has been associated with vascular stiffness, and FABP4 suppresses peroxisome proliferator-activated receptor, which is a major regulator of adipose function and known to be downregulated in PAH. These findings reveal that small visceral adipose tissue within the SU/hyp model provides mechanistic links for vascular remodeling and adipose dysfunction in the pathophysiology of PAH.

Dentin sialophosphoprotein (DSPP) is one of the major non-collagenous proteins present in dentin, cementum and alveolar bone; it is also transiently expressed by ameloblasts. In humans many mutations have been found in DSPP and are associated with two autosomal-dominant genetic diseases — dentinogenesis imperfecta II (DGI-II) and dentin dysplasia (DD). Both disorders result in the development of hypomineralized and mechanically compromised teeth. The erupted mature molars of Dspp–/– mice have a severe hypomineralized dentin phenotype. Since dentin and enamel formations are interdependent, we decided to investigate the process of enamel onset mineralization in young Dspp–/– animals. We focused our analysis on the constantly erupting mouse incisor, to capture all of the stages of odontogenesis in one tooth, and the unerupted first molars. Using high-resolution microCT, we revealed that the onset of enamel matrix deposition occurs closer to the cervical loop and both secretion and maturation of enamel are accelerated in Dspp–/– incisors compared to the Dspp+/– control. Importantly, these differences did not translate into major phenotypic differences in mature enamel in terms of the structural organization, mineral density or hardness. The only observable difference was the reduction in thickness of the outer enamel layer, while the total enamel thickness remained unchanged. We also observed a compromised dentin-enamel junction, leading to delamination between the dentin and enamel layers. The odontoblast processes were widened and lacked branching near the DEJ. Finally, for the first time we demonstrate expression of Dspp mRNA in secretory ameloblasts. In summary, our data show that DSPP is important for normal mineralization of both dentin and enamel.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by significant vascular remodeling within the lung. Clinical computed tomography (CT) scans are routinely used to aid in PAH diagnosis. Animal models, including the Sugen-hypoxic rat model (SU/hyp), of PAH closely mimic human PAH development. We have previously used micro-computed tomography (microCT) to find extensive right lung vascular remodeling in the SU/hyp. We hypothesized that the individual right lung lobes may not contribute equally to overall lung vascular remodeling. Sprague-Dawley rats were subjected to a subcutaneous injection of vascular endothelial growth factor receptor blocker (Sugen 5416) and subsequently exposed to chronic hypoxic conditions (10% O2) for three weeks. Following perfusion of the lung vasculature with an opaque resin (Microfil), the right lung lobes were microCT-imaged with a 10-µm voxel resolution and 3D morphometry analysis was performed separately on each lobe. As expected, we found a significantly lower ratio of vascular volume to total lobe volume in the SU/hyp compared with the control, but only in the distal lobes (inferior: 0.23 [0.21–0.30] versus 0.35 [0.27–0.43], P = 0.02; accessory: 0.27 [0.25–0.33] versus 0.37 [0.29–0.43], P = 0.06). Overall, we observed significantly fewer continuous blood vessels and reduced vascular density while having greater vascular lumen diameters in the distal lobes of both groups (P < 0.05). In addition, the vascular separation within the SU/hyp lobes and the vascular surface area to volume ratio were significantly greater in the SU/hyp lobes compared with controls (P < 0.03). Results for the examined parameters support the overall extensive vascular remodeling in the SU/hyp model and suggest this may be lobe-dependent.

No abstract

Magnesium (Mg) and its alloys are candidate materials for resorbable implantable devices, such as orthopedic devices or cardiovascular stents. Mg has a number advantages, including mechanical properties, light weight, its osteogenic effects and the fact that its degradation products are nontoxic and naturally present in the body. However, production of H gas during the corrosion reaction can cause formation of gas pockets at the implantation site, posing a barrier to clinical applications of Mg. It is therefore desirable to develop methods to control corrosion rate and gas pocket formation around the implants. Here we evaluate the potential of self-assembled multilayer alkylsilane (AS) coatings to control Mg device corrosion and formation of gas pockets in vivo and to assess effects of the AS coatings on the surrounding tissues in a subcutaneous mouse model over a 6 weeks' period. The coating significantly slowed down corrosion and gas pocket formation as evidenced by smaller gas pockets around the AS coated implants (ANOVA; p = 0.013) and decrease in the weight loss values (t test; p = 0.07). Importantly, the microCT and profilometry analyses demonstrated that the coating inhibited the pitting corrosion. Specifically, the roughness of the coated samples was ∼30% lower than uncoated specimen (p = 0.02). Histological assessment of the tissues under the implant revealed no inflammation or foreign body reaction. Overall, our results demonstrate the feasibility of use of the seld assembled AS coatings for reduction of gas pocket formation around the resorbable Mg devices. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.

<p>PDF file 210K, Identification of Phosphorylation Sites on YAP.</p>