Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system

Smart matrices are required in bone tissueengineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bioartificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery

Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit alpha(2)delta-1 with thrombospondin-4

The alpha(2)delta-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of alpha(2)delta-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/alpha(2)delta-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of alpha(2)delta-1 (alpha(2)delta-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing alpha(2)delta-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal a2d-1 and suggest that gabapentin does not directly target TSP/alpha(2)delta-1 interaction to alleviate neuropathic pain

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-Twinkle(Epi) mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-Twinkle(Epi) mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts

Angiotensin II type 1 receptor localizes at the blood-bile barrier in humans and pigs

Animal models and clinical studies suggest an influence of angiotensin II (AngII) on the pathogenesis of liver diseases via the renin-angiotensin system. AngII application increases portal blood pressure, reduces bile flow, and increases permeability of liver tight junctions. Establishing the subcellular localization of angiotensin II receptor type 1 (AT1R), the main AngII receptor, helps to understand the effects of AngII on the liver. We localized AT1R in situ in human and porcine liver and porcine gallbladder by immunohistochemistry. In order to do so, we characterized commercial anti-AT1R antibodies regarding their capability to recognize heterologous human AT1R in immunocytochemistry and on western blots, and to detect AT1R using overlap studies and AT1R-specific blocking peptides. In hepatocytes and canals of Hering, AT1R displayed a tram-track-like distribution, while in cholangiocytes AT1R appeared in a honeycomb-like pattern; i.e., in liver epithelia, AT1R showed an equivalent distribution to that in the apical junctional network, which seals bile canaliculi and bile ducts along the blood-bile barrier. In intrahepatic blood vessels, AT1R was most prominent in the tunica media. We confirmed AT1R localization in situ to the plasma membrane domain, particularly between tight and adherens junctions in both human and porcine hepatocytes, cholangiocytes, and gallbladder epithelial cells using different anti-AT1R antibodies. Localization of AT1R at the junctional complex could explain previously reported AngII effects and predestines AT1R as a transmitter of tight junction permeability

Vital staining of blood vessels and bile ducts with carboxyfluorescein diacetate succinimidyl ester: a novel tool for isolation of cholangiocytes

Background and aim: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes. Methods: Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively. Results: Upon histomorphological analysis of cryoand paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size. Conclusions: Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE

When and why was the phrenicoabdominal branch of the left phrenic nerve placed into the esophageal hiatus in German textbooks of C anatomy? An anatomical study on 400 specimens reevaluating its course through the diaphragm

Background: The phrenicoabdominal branch of the left phrenic nerve passes between muscle fiber bundles within the costal part of the diaphragm near the pericardium. In most German textbooks of anatomy, however, its passage is described to be found in the esophageal hiatus. The aim of this study was to reevaluate its topography relative to the diaphragm in a multicentric study and to identify the initiation of this description. Methods: In this multicentric study, the most dorsomedial branch of the left phrenic nerve was identified as the phrenicoabdominal branch in 400 embalmed anatomic specimens of Caucasian origin. The distance between its passage and the apex of the pericardium, the left border of the esophageal hiatus, and the inner aspect of the left sixth rib was measured on the cranial aspect of the diaphragm. Textbooks on human anatomy published in German language between 1700 and 2018 were reviewed for their description of the passage of the left phrenicoabdominal branch through the diaphragm. Results: The first statement on the passage of the left phrenicoabdominal branch through the esophageal hiatus was given in 1791 by Sommering. Since then, in German textbooks of anatomy, a duality in the description of the passage of the left phrenicoabdominal branch persists. In none of the individuals examined in this study, the left phrenicoabdominal branch passed through the esophageal hiatus. In 99.5% of all cases, it pierced the costal part of the diaphragm dorsal to or at the same level as the apex of the pericardium. The mean distances (standard deviations) were 3.4 (+/- 1.5) cm to the apex of the pericardium, 5.8 (+/- 2.2) cm to the esophageal hiatus, and 5.5 (+/- 1.6) cm to the inner aspect of the left sixth rib. Conclusion: The findings on the position of the left phrenicoabdominal branch relative to the diaphragm help to improve topographical knowledge and prevent inadvertent nerve injury during surgical interventions on or near the diaphragm. Further to this, these results may form a substantial basis to adopt the correct description of the passage of the left phrenicoabdominal branch to anatomical textbook knowledge. (C) 2019 Elsevier GmbH. All rights reserved

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Calc1(-/-) compared with Cold1(+/+) skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KO-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cold1(-/-) than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure

Role of guanylate binding protein-1 in vascular defects associated with chronic inflammatory diseases

Rheumatic autoimmune disorders are characterized by a sustained pro-inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein-1 (GBP-1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro-inflammatory cytokines. In addition, GBP-1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP-1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP-1 in EPC. Interestingly, stable expression of GBP-1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up-regulation of both Flk-1 and von Willebrand factor expression. In addition, GBP-1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP-1 inhibited vessel-directed migration of EPC at the tissue level using the rat arterio-venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP-1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation

Transglutaminase mediated asprosin oligomerization allows its tissue storage as fibers

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived from profibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the tissue distribution of asprosin. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. This oligomerization is facilitated by transglutaminase activity and requires an intact fibrillin fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in oligomerized form which may regulate its cellular bioavailability in tissues