Visualization of the pulp chamber roof and residual dentin thickness by spectral-domain optical coherence tomography in vitro.

The aim of this study was to evaluate the ability of spectral-domain optical coherence tomography (SD-OCT) to display the roof of the pulp chamber and to estimate the residual dentin thickness (RDT) of the pulp complex. The roots of 20 extracted human molars were embedded in epoxy resin, and crowns were longitudinally sectioned in the mesial-distal direction, exposing the pulp chamber. The coronal part of the crown was removed up to an RDT to the pulp chamber roof of 2 mm. Samples were imaged by SD-OCT from coronal view and by light microscopy (LM) in the sagittal plane. Using a microtome, dentin was subsequently removed in four levels from the occlusal aspect in steps of 250 µm. At each level, RDT was documented and measured by both methods. The data were compared (Spearman's rho correlation coefficient, Wilcoxon signed-rank test). Using OCT, the roof of the pulp chamber was first displayed at a maximum RDT of 1.94 mm. The minimal RDT that could be imaged by OCT was 0.06 mm. Values from both methods were strongly correlated (r, 0.83-0.95; pi ≤ 0.05) and differed significantly for large RDTs (dentin levels 1, 2; pi < 0.05) but not for small RDTs (levels 3, 4; pi ≥ 0.226). The roof of the dental pulp chamber could be already visualized by SD-OCT with a RDT of 1.94 mm. Therefore, the method could be a useful diagnostic tool during the preparation of deep dentin cavities and might help to preserve the integrity of the pulp chamber.

Protein kinase D1 mediates anchorage-dependent and -independent growth of tumor cells via the zinc finger transcription factor Snail1.

We here identify protein kinase D1 (PKD1) as a major regulator of anchorage-dependent and -independent growth of cancer cells controlled via the transcription factor Snail1. Using FRET, we demonstrate that PKD1, but not PKD2, efficiently interacts with Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11. There was no change in the nucleocytoplasmic distribution of Snail1 using wild type Snail1 and Ser-11 phosphosite mutants in different tumor cells. Regardless of its phosphorylation status or following co-expression of constitutively active PKD, Snail1 was predominantly localized to cell nuclei. We also identify a novel mechanism of PKD1-mediated regulation of Snail1 transcriptional activity in tumor cells. The interaction of the co-repressors histone deacetylases 1 and 2 as well as lysyl oxidase-like protein 3 with Snail1 was impaired when Snail1 was not phosphorylated at Ser-11, which led to reduced Snail1-associated histone deacetylase activity. Additionally, lysyl oxidase-like protein 3 expression was up-regulated by ectopic PKD1 expression, implying a synergistic regulation of Snail1-driven transcription. Ectopic expression of PKD1 also up-regulated proliferation markers such as Cyclin D1 and Ajuba. Accordingly, Snail1 and its phosphorylation at Ser-11 were required and sufficient to control PKD1-mediated anchorage-independent growth and anchorage-dependent proliferation of different tumor cells. In conclusion, our data show that PKD1 is crucial to support growth of tumor cells via Snail1.

Calcineurin (PPP3CB) regulates angiotensin II-dependent vascular remodelling by potentiating EGFR signalling in mice.

AIM: This study investigates the role of calcineurin for angiotensin II (AngII)-induced vascular remodelling with the help of a mouse model lacking the catalytic beta subunit of calcineurin (PPP3CB KO). METHODS: Wildtype (WT) and PPP3CB KO mice were treated for 4 weeks with AngII followed by assessment of blood pressure, histological evaluation of aortas and mRNA analysis of aortic genes PPP3CB-dependently regulated by AngII. Primary murine vascular smooth muscle cells (VSMCs) were used for qPCR, ELISA and Western Blot experiments as well as wound healing and cell proliferation assays. RESULTS: Upon AngII treatment, PPP3CB KO mice showed less aortic media thickening, lumen dilation and systolic blood pressure compared to WT mice. Next-generation sequencing data of aortic tissue indicated an increase in extracellular matrix components (EMCs), cell migration and cell proliferation. A PPP3CB-dependent increase in EMC was confirmed by qPCR in aorta and VSMCs. PPP3CB-dependent stimulation of VSMC migration could be verified by wound healing assays but markers of enhanced cell proliferation were only detectable in aortic tissue of WT mice but not in isolated WT or KO VSMCs. We could demonstrate in VSMCs with pharmacological inhibitors that PPP3CB leads to enhanced heparin-binding EGF-like growth factor (HB-EGF) secretion, epidermal growth factor receptor (EGFR) activation and consecutive stimulation of transforming growth factor ß(TGFß) and connective tissue growth factor (CTGF) signalling that enhances collagen expression. CONCLUSION: AngII-induced vascular remodelling involves PPP3CB, which leads to enhanced EMC production, VSMC migration and sustained increase in systolic blood pressure via HBEGF/EGFR-TGFß-CTGF signalling.

Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases.

Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies.