Calmodulin Regulated Spectrin Associated Protein 1 mRNA is Directly Regulated by miR-126 in Primary Human Osteoblasts.

Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR-126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR-126 gain-of-function and loss-of-function experiments in hOBs followed by microarray analysis in order to identify targets of miR-126. The transcript cluster IDs were sieved by applying cut-off criteria and by selecting transcripts which were upregulated following miR-126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR-126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR-126. In this study, we show that miR-126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756-1763, 2017. © 2016 Wiley Periodicals, Inc.

Il10 and poly-dl-lactide-ɛ-caprolactone conduits in critical size nerve defect bridging-An experimental study.

INTRODUCTION: Aim of this study is to evaluate if regeneration in repair of nerve defects can be improved by combination of a poly-dl-lactide-ɛ-caprolactone conduit (PLC) with long-term release of anti-inflammatory Interleukin 10 (IL10), which is known to reduce intraneural scarring in nerve regeneration through its anti-inflammatoric properties. METHOD: Experiments were performed at 30 female Lewis rats. Conduits filled with fibrin (PLC-group n = 10) and fibrin loaded with IL10 (IL10-group n = 10) were compared to autologs nerve grafts (NG-group n = 10) in a 15 mm sciatic nerve gap lesion. Sciatic function index (SFI) and electrophysiological analyses were performed 16 weeks after surgery prior to histological evaluation. In histological analyses total nerve count, total nerve area, myelination index, and N-ratio were measured. Additionally, gastrocnemius muscle was weighed. RESULTS: SFI (NG-group:-50.68 ± 7.03%; PLC-group:-56.48  ± 2.30%; IL10-group:-56.54  ± 8.22%) and nerve conduction velocity (NG-group: 92.52  ± 4.64 m/s; PLC-group: 92.77  ± 5.07 m/s; IL10-group: 93.78  ±3.63 m/s) showed no significant differences after 16 weeks (P > 0.05). Significant higher axon count (17.592  ± 483) were observed in the NG-group compared to PLC- (6.722 ± 553) and IL10-group (6.842 ± 681) (P < 0.001). NG-group had significant highest nerve cross sections (604.214  ± ±15.217 µm2 ) as compared to PLC- (245.669  ± ±28.034 µm2 ) and IL10-group (244.698 ± 26.772 µm2 ) (P < 0.001). Comparison of myelination index showed significant higher values for NG-group (0.46  ± 0.02) than PLC- (0.64  ± 0.01) and IL10-group (0.62  ± 0.01) (P < 0.001). N-ratios in PLC-group (0.21  ± 0.01) and IL10-group (0.24 ± 0.01) were lower than in NG-group (0.51  ± 0.03) (P < 0.001). Between PLC- and IL10-group no differences were observed (P > 0.05). Gastrocnemius muscle was heavier in NG-group (0.86 ± 0.21g) as compared to PLC- (0.26 ± 0.05g) and IL-10 group (0.29 ± 0.06 g) (P < 0.05). CONCLUSION: Bridging critical nerve defects through fibrin-filled PLC conduits is possible. Although, autologs nerve graft showed superior histological results. Long-term release of IL10 in the conduit did not improve regeneration of critical nerve defects. © 2015 Wiley Periodicals, Inc. Microsurgery 36:410-416, 2016.

Growth differentiation factor 11 supports migration and sprouting of endothelial progenitor cells.

BACKGROUND: Neovascularization plays an important role in tissue engineering applications. In animal models, it was demonstrated that implantation of endothelial progenitor cells (EPCs) from cord blood led to the formation of a complex functional neovasculature, whereas EPCs isolated from peripheral blood (pbEPCs) showed a limited vasculogenic potential, which may be attributed to age-related dysfunction. Growth differentiation factor 11 (GDF11) was recently identified as a rejuvenation factor, which was able to reverse age-related dysfunction of stem cells. Therefore, we hypothesized that GDF11 may improve the vasculogenesis-related phenotype of pbEPCs. MATERIALS AND METHODS: pbEPCs were isolated from adult peripheral blood. Transforming growth factor (TGF)-ß type-I receptor expression was analyzed by immunostaining. pbEPCs were treated with recombinant GDF11 for various time periods. Thereafter, phosphorylation of Smad2/Smad3, adhesion, proliferation, cell survival, migration, and in vitro sprout formation was investigated. RESULTS: pbEPCs express the TGF-ß type-I receptors ALK4 and ALK5, but not ALK7. Treatment of pbEPCs with recombinant GDF11 resulted in activation of the Smad2/Smad3 pathway and in increased migration, which was inhibited by the TGF-ß1 superfamily type-I activin receptor-like kinase inhibitor SB431542, demonstrating that the TGF-ß receptor-Smad2/Smad3 pathway is involved in GDF11 induced migration. Moreover, in vitro sprout formation was increased as well by GDF11 treatment. However, other parameters such as adherence, proliferation, and apoptosis were not affected by GDF11. CONCLUSIONS: This study provides evidence that GDF11 improves vasculogenesis-related growth parameters in pbEPCs and may represent a therapeutic option to ameliorate the angiogenic and vasculogenic properties of pbEPCs.

C3 toxin and poly-DL-lactide-ε-caprolactone conduits in the critically damaged peripheral nervous system: a combined therapeutic approach.

INTRODUCTION: Peripheral nerve regeneration over longer distances through conduits is limited. In the presented study, critical size nerve gap bridging with a poly-DL-lactide-ε-caprolactone (PLC) conduit was combined with application of C3 toxin to facilitate axonal sprouting. MATERIALS AND METHODS: The PLC filled with fibrin (n = 10) and fibrin gel loaded with 1-µg C3-C2I and 2-µg C2II (n = 10) were compared to autologous nerve grafts (n = 10) in a 15-mm sciatic nerve gap lesion model of the rat. Functional and electrophysiological analyses were performed before histological evaluation. RESULTS: Evaluation of motor function and nerve conduction velocity at 16 weeks revealed no differences between the groups. All histological parameters and muscle weight were significantly elevated in nerve graft group. No differences were observed in both PLC groups. CONCLUSIONS: The PLCs are permissive for nerve regeneration over a 15-mm defect in rats. Intraluminal application of C3 toxin did not lead to significant enhancement of nerve sprouting.

Are there differences in LDL-C target value attainment in Austrian federal states? Yes!

UNLABELLED: Low density lipoprotein (LDL-C) levels determine the cardiovascular risk. Previous studies indicated an LDL-C target attainment of around 50%, but no Austrian wide analysis on results for the federal states was available. We therefore sought to detect potential differences. DESIGN: Open-label, non-interventional, longitudinal study, registered: www.clinicaltrials.gov NCT 01381679. In all, 746 statin treated patients not at LDL-C goal received intensified therapy for 12 months. The sample was split into nine subgroups, representing the federal states of Austria.We detected an east-west gradient for baseline LDL-C. Individual target values were achieved by 37.2% (range: 26.1-57.7%). After 12 months, LDL-C < 70 mg/l was achieved by 13.5% (5.9-38.5%). Univariate ANCOVA retrieved significant differences within the states (Upper Austria and Salzburg, p = 0.001 and p = 0.0015, respectively). Furthermore, the capacity of intensified lipid lowering therapy applied in practice was as high as -42% as compared to previous standard therapy (additional LDL-C reduction after switch from baseline therapy in Vorarlberg).

Effect of home-based telemonitoring using mobile phone technology on the outcome of heart failure patients after an episode of acute decompensation: randomized controlled trial.

BACKGROUND: Telemonitoring of patients with chronic heart failure (CHF) is an emerging concept to detect early warning signs of impending acute decompensation in order to prevent hospitalization. OBJECTIVE: The goal of the MOBIle TELemonitoring in Heart Failure Patients Study (MOBITEL) was to evaluate the impact of home-based telemonitoring using Internet and mobile phone technology on the outcome of heart failure patients after an episode of acute decompensation. METHODS: Patients were randomly allocated to pharmacological treatment (control group) or to pharmacological treatment with telemedical surveillance for 6 months (tele group). Patients randomized into the tele group were equipped with mobile phone-based patient terminals for data acquisition and data transmission to the monitoring center. Study physicians had continuous access to the data via a secure Web portal. If transmitted values went outside individually adjustable borders, study physicians were sent an email alert. Primary endpoint was hospitalization for worsening CHF or death from cardiovascular cause. RESULTS: The study was stopped after randomization of 120 patients (85 male, 35 female); median age was 66 years (IQR 62-72). The control group comprised 54 patients (39 male, 15 female) with a median age of 67 years (IQR 61-72), and the tele group included 54 patients (40 male, 14 female) with a median age of 65 years (IQR 62-72). There was no significant difference between groups with regard to baseline characteristics. Twelve tele group patients were unable to begin data transmission due to the inability of these patients to properly operate the mobile phone ("never beginners"). Four patients did not finish the study due to personal reasons. Intention-to-treat analysis at study end indicated that 18 control group patients (33%) reached the primary endpoint (1 death, 17 hospitalizations), compared with 11 tele group patients (17%, 0 deaths, 11 hospitalizations; relative risk reduction 50%, 95% CI 3-74%, P = .06). Per-protocol analysis revealed that 15% of tele group patients (0 deaths, 8 hospitalizations) reached the primary endpoint (relative risk reduction 54%, 95% CI 7-79%, P= .04). NYHA class improved by one class in tele group patients only (P< .001). Tele group patients who were hospitalized for worsening heart failure during the study had a significantly shorter length of stay (median 6.5 days, IQR 5.5-8.3) compared with control group patients (median 10.0 days, IQR 7.0-13.0; P= .04). The event rate of never beginners was not higher than the event rate of control group patients. CONCLUSIONS: Telemonitoring using mobile phones as patient terminals has the potential to reduce frequency and duration of heart failure hospitalizations. Providing elderly patients with an adequate user interface for daily data acquisition remains a challenging component of such a concept.

In vivo evaluation of an electrospun gelatin nonwoven mat for regeneration of epithelial tissues.

One major objective in epithelial tissue engineering is to identify a suitable biomaterial that supports epithelial tissue formation. Therefore, the purpose of this study is to elucidate a novel electrospun gelatin nonwoven mat (NWM) for epithelial tissue engineering purposes in vivo. This NWM was seeded with either human gingival keratinocytes (GK, in coculture with gingival fibroblast) or human skin epithelial keratinocytes (EK, in coculture with skin dermal fibroblasts). These constructs were ex vivo cultured for 4 days before subcutaneous implantation into athymic nude mice. After 7 days, the constructs were explanted and investigated by immunohistology. Our results show that GK form a stratified epithelium on the surface of the NWM, mostly independent of a fibroblastic counterpart. Like native mucosa, the regenerated epithelium showed expression of epidermal growth factor receptor, cytokeratin-14 and -1, and involucrin. Only the expression of the basement membrane constituent laminin 5 was more pronounced in cocultures. Comparing GK and skin EK, we found that skin EK form a less developed epithelial tissue. Furthermore, the NWM allows not only for epithelial tissue formation by GK, but also for infiltration of human fibroblasts and mouse immune cells, thus representing a biomaterial with potential regenerative capacity for oral mucosa tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1605-1614, 2019.

The effect of the K+ agonist nicorandil on peripheral vascular resistance.

BACKGROUND: The vasoactive effect of nicorandil on coronary arteries is well known. Nicorandil exerts its vasodilatory effect through a dual mechanism of action: involving on the one hand cyclic guanosine monophosphate (c GMP) as a nitrovasodilatator, and on the other hand, acting as a potassium channel opener. OBJECTIVE: To address the question if nicorandil works in peripheral arteries, its effect on peripheral vascular resistance was evaluated in isolated perfused guinea pig hind limbs. METHODS: A catheter was inserted via the distal aorta and common iliac artery. Perfusion pressure was monitored under constant perfusion with Tyrode's solution, therefore changes in perfusion pressure represent changes in vascular resistance. After stabilization precontraction of the peripheral vascular bed was achieved with noradrenaline 3 microM and nicorandil was added in concentrations of 1, 10 and 100 microM. The effect of nicorandil (1, 10 and 100 microM) was tested in the presence of L-NAME and glybenclamide. RESULTS: A significant reduction of vascular peripheral resistance was already achieved at a concentration of 1 microM nicorandil (30.3+/-6.1%, mean S.E.M., p < 0.001). At a concentration of 100 microM nicorandil the reduction of peripheral vascular resistance was 94.4+/-16.4%. Peripheral vascular resistance was less but nearly comparable reduced by nicorandil (100 microM) if the endothelial NO effect was inhibited by L-NAME (58.6+/-18.6%) or if the ATP-dependent potassium channels were blocked by glybenclamide (56.4+/-14.6%). CONCLUSIONS: In peripheral arteries the nitrovasodilator effect of nicorandil is nearly comparable to the potassium agonistic effect, and the concentration, which is necessary to reduce peripheral vascular resistance significantly, is comparable with dosages necessary for reduction of coronary resistance.

Medical applications of near-eye display devices: an exploratory study.

INTRODUCTION: Near-eye display devices (such as Google Glass) may improve the efficiency and effectiveness of clinical care by giving clinicians information (such as the patient's vital signs) continuously within their field of vision during various procedures. We describe the use of Glass during a radiological intervention in three patients. Other possible applications (including tele-mentoring and the supervision of trainees) are discussed and a classification proposed. METHODS: An app was developed to facilitate the use of Glass, so vital physical signs (pulse and blood pressure) could be projected on the near-eye display, via an intranet to protect sensitive data. The device was then used during radiological interventions (percutaneous transluminal angioplasty) in three patients, and assessed by the interventionalists who were interviewed before and after each procedure. RESULTS: The interventionalists reported that Google Glass improved concentration on the task in hand by reducing head and neck movements (which would be needed to view several remote monitors). However, heat generation by the device and low battery capacity are shortcomings for which solutions must be developed, and data protection is mandatory. CONCLUSION: Google Glass may have a number of clinical applications and can quicken interventions where vital signs or other visual data need to be monitored by the operator.

Platelet-derived growth factor receptor signaling is not involved in osteogenic differentiation of human mesenchymal stem cells.

Platelet-derived growth factor (PDGF) receptor signaling plays an important role in the regulation of proliferation and migration of skeletal cells such as osteoblasts or mesenchymal stem cells (MSCs). However, involvement of these receptors in the process of osteoblastic differentiation of MSCs is still a matter of debate. The aim of our study was to examine the role of PDGF receptor signaling in osteogenic differentiation of human MSCs. For this purpose, we performed PDGF receptor stimulation as well as inhibition experiments. Inhibition experiments were carried out with Tyrphostin AG1296, a potent and specific inhibitor of PDGF receptor activity. As expected, Tyrphostin AG1296 treatment caused a concentration-dependent decrease in fetal calf serum and PDGF-BB-induced proliferation of MSCs and effectively inhibited PDGF-BB-induced phosphorylation of extracellular-regulated kinase 1/2. However, PDGF receptor inhibition had no significant effect on osteoblastic differentiation of MSCs, as evaluated histochemically by von Kossa, Alizarin-Red, and osteocalcin stainings. Moreover, mineralized matrix production, as assayed by quantitative Ca(2+)-measurements, was also not modulated by Tyrphostin AG1296 treatment. These results were noticeable irrespective of whether MSCs were grown under nonosteogenic or osteogenic differentiation conditions. Similarly, PDGF-BB treatment of MSCs in receptor stimulation experiments also failed to modulate mineralization. However, expression of alkaline phosphatase was suppressed by Tyrphostin AG1296 treatment at later stages of osteogenesis but not in the early stages, as assessed by enzyme activity and mRNA expression assays. Expression of other osteogenic marker genes such as osteocalcin, runt-related transcription factor 2, osteopontin, collagen type I, and bone sialoprotein was almost unaffected in our perturbation studies. From these experiments, we conclude that PDGF receptor signaling sustains proliferation without affecting osteogenic differentiation of MSCs.

Aggravation of pre-existing atrioventricular block, Wenckebach type, provoked by application of X-ray contrast medium.

BACKGROUND: Significant bradycardia followed by cardiac arrest related to single bolus administration of X-ray contrast medium into a peripheral artery has not, to our knowledge, been described in the literature. METHODS AND RESULTS: While performing a percutaneous transluminal angioplasty of the left superficial femoral artery in a 68-year old patient with a pre-existing atrioventricular (AV) block, Wenckebach type, he developed an AV block III after a single bolus injection of intra-arterial X-ray contrast medium. CONCLUSION: We believe that application of contrast medium causes a transitory ischemia in the obstructed vessel and therefore elevation of endogenous adenosine. In the case of a previously damaged AV node this elevation of endogenous adenosine may be responsible for the development of a short period of third-degree AV block.

The influence of magnesium on the electrophysiological effects of erythromycin in the isolated Guinea pig heart.

There have been several case reports that erythromycin can prolong the QT interval, resulting in torsades de pointes. As magnesium is well established in the treatment of torsades de pointes, we aimed to investigate how this trace element influences the electrophysiological effects of erythromycin. The effects of erythromycin and magnesium on cardiac conduction and refractoriness were evaluated in isolated guinea pig hearts perfused by the method of Langendorff. Erythromycin was given in concentrations of 10, 30 and 100 microM. In the magnesium group MgSO4 was elevated to 3.4 mM. Magnesium at a concentration of 3.4 mM did not affect cardiac conduction and refractoriness. Erythromycin prolongs the QT interval in a dose-dependent manner. The prolongation of the QT interval by erythromycin was diminished by the elevated magnesium concentration (3.4 mM) at high rates (pacing cycle length 180 vs. 220 ms). The most pronounced effect of erythromycin (100 microM) was the prolongation of the atrial effective refractory period. This effect was nearly abolished by the elevated magnesium concentration (129.0 +/- 8.3% vs. 41.9 +/- 10.6%, P < 0.01, n = 6, x +/- SEM). In summary, magnesium minimizes the effects of erythromycin on the repolarization period in the ventricle and on the effective refractory period of the atrium.

The outback catheter: a new device for true lumen re-entry after dissection during recanalization of arterial occlusions.

PURPOSE: To report the initial experience with a new catheter system (The Outback catheter) designed to allow fluoroscopically controlled re-entry of the true arterial lumen after subintimal guidewire passage during recanalization procedures of arterial occlusions. METHODS: The catheter was used in 10 patients with intermittent claudication caused by chronic segmental occlusions of the superficial femoral or popliteal arteries. In all patients, conventional guidewire recanalization had failed. RESULTS: In 8 patients, successful true lumen re-entry was achieved with the Outback catheter. Percutaneous transluminal angioplasty was successfully performed in these patients without complications. Two technical failures occurred in heavily calcified arteries. CONCLUSION: The Outback catheter was safe and effective when used in complicated recanalization procedures in the superficial femoral and popliteal artery and the tibial trunk.

Comparison of the Effect of Prostaglandin E(1), Prostacycline and Adenosine on Peripheral Vascular Resistance.

Prostacycline, prostaglandin E(1), and adenosine are highly effective vasodilators. These three drugs are widely used in the treatment of peripheral arterial occlusive disease. The aim of the study was to compare the vasodilatory potency of these substances in the isolated perfused guinea pig hind limb. After equilibration with Tyrode's solution and precontraction with noradrenaline 3µM, prostaglandin E(1) and adenosine were administered at dosages of 0.1, 0.3, and 1µM, whereas prostacycline was administered at a dosage of 0.01, 0.03 and 0.1µM. 0.01µM prostacycline, 0.1 µM prostaglandin E(1), and 0.1µM adenosine were the lowest dosages at which a significant vasodilation could be reached for each substance. The reduction of peripheral vascular resistance at comparable dosages of 0.1µM was 11.0 +/- 2.6% (x +/- SEM, n = 5) for adenosine, 12.0 +/- 1.0% (n = 5) for prostaglandin E(1), but 28.0 +/- 9.3% (n = 5) for prostacycline (p < 0.05 versus adenosine and prostaglandin E(1)). Even at a dosage of 0.01 µM prostacycline, a comparable reduction in peripheral vascular resistance (16.0 +/- 2.8%) could be reached, compared to a ten-fold higher dosage of prostaglandin E(1) and adenosine. At the highest concentration of 1 µM, the vasodilatory effect of adenosine was significantly less expressed, compared to that of prostaglandin E(1) (18.0 +/- 3.4% versus 33.0 +/- 4.7%). In summary, prostacycline, at a ten-fold lower concentration, showed comparable vasodilatory effects to adenosine and prostaglandin E(1). The rank order at the vasodilatory potency is prostacycline > prostaglandin E(1) > adenosine.

The effect of caffeine on peripheral vascular resistance in isolated perfused guinea pig hind limbs.

BACKGROUND: The role of caffeine in cardiovascular disease is controversial. Most of its pharmacologic actions are attributed to its role as an adenosine antagonist. Adenosine is one of the most important endogenous vasodilatative substances and is released under ischemic conditions, for example, in the skeletal muscle of patients with peripheral arterial occlusive disease. We aimed to investigate the influence of caffeine on peripheral vascular resistance and on the beneficial vasodilatory effect of adenosine in isolated perfused guinea pig hind limbs. MATERIALS AND METHODS: (1) Caffeine was administered at 0.5, 5, and 50 micromol/L under normoxic conditions. (2) The vasculature of the perfused guinea pig hind limb was precontracted with noradrenaline (3 micromol/L), followed by adenosine (10 micromol/L) under normoxic conditions. When vascular resistance (VR) had reached a steady state, caffeine was administered additionally at dosages of 0.5, 5, and 50 micromol/L. (3) This protocol was repeated using iloprost 0.1 micromol/L instead of adenosine as vasodilatory substance. (4) Under hypoxia, caffeine was again administered at the above dosages. (5) Under hypoxia, experiments with adenosine A2-receptor antagonists (alloxazine 10 micromol/L and ZM 241385 100 nmol/L) were done. RESULTS: Under normoxic conditions, 0.5 and 5 micromol/L caffeine had nearly no effect on vascular resistance compared with baseline conditions. A slight, but statistically not significant decrease in VR was achieved with 50 micromol/L caffeine. In the presence of noradrenaline, the vasodilatory effect of adenosine was reduced by 7.6 +/- 1.6% after the addition of 0.5 micromol/L caffeine, and by 37.3 +/- 3.8% at a dosage of 5 micromol/L caffeine. A dosage of 50 micromol/L caffeine completely abolished the vasodilatative effect of adenosine. In the presence of iloprost, only a slight but statistically insignificant inhibitory influence (0.9%) of caffeine at a dosage of 50 micromol/L could be seen. Hypoxia significantly reduced VR. Caffeine at 0.5 micromol/L diminished this effect by about 53.2 +/- 4.6% and abolished it at 5 and 50 micromol/L. The hypoxia-induced adenosine-mediated vasodilatation seems to be an adenosine A2A-receptor-mediated effect. CONCLUSIONS: The observed effect of hypoxia-induced vasodilatation in peripheral arteries may be the result of the vasodilatory effect of elevated endogenous adenosine during hypoxia. For patients with peripheral arterial disease, drinking of caffeine-containing beverages may reduce the beneficial vasodilatory effect of elevated endogenous adenosine levels.