Interplay between vascular endothelial growth factor-A and extracellular matrix in angiogenesis: molecular and cellular mechanisms

The induction of angiogenesis by stimulation of physiological vessel growth using pro- angiogenic growth factors is currently under intense investigation in medical research. It is well accepted, that angiogenesis is a rate-limiting step in skin regeneration, as it ensures supply of novel tissue with nutrients and oxygen. Chronic wounds are characterized by a lack of angiogenesis and thus represent a major target for the induction of angiogenesis by therapeutic means. For the delivery of VEGF-A165 to chronic wounds and the induction of effective angiogenesis, in general it is proposed that first, the stabilization of the growth factor against protease activities, and second, the tight control of its release are beneficial. The aim of this study was to optimize wound angiogenesis in non-healing wounds in response to recombinant VEGF-A165 by improving bioavailability and potency of the growth factor. To stabilize the growth factor in the chronic wound environment, recently a mutated form of VEGF-A165 (VEGFmut) resistant to plasmin cleavage was generated in our group. This mutant was used in this study for the analysis of protein delivery. For this purpose, a hybrid protein composed of VEGFmut and a factor XIIIa transglutaminase substrate sequence (TG) was generated. This sequence allows covalent incorporation of the recombinant protein a into fibrin matrix. Furthermore, a second strategy to increase the angiogenic response was investigated which aimed at the stimulation of synergistic signaling downstream of VEGFR-2 and integrin αvβ3. To this end, bi-functional proteins consisting of the fibronectin type III domain 10 (FNIII10) and VEGFmut denoted as FLV were generated for the concomitant activation of both receptors. The recombinant proteins were expressed in E. coli. FNIII10 and TG-FNIII10 were purified as GST-fusion proteins, whereas VEGF-containing constructs were produced in bacterial inclusion bodies, refolded, dimerized, and purified by heparin affinity chromatography. However, purification of the bi-functional proteins was challenging, as they tended to precipitate and dimerization was ineffective. To overcome these problems, in a subsequent approach, FNIII10, TG-FNIII10, VEGFmut, TG-VEGFmut, FLV and TG-FLV were expressed in eucaryotic cells (HEK293-EBNA) and purified by a C-terminal poly-histidine tag. The biological activity of these proteins was confirmed in various in vitro assays. First, HUVECs were shown to attach to TG-FNIII10 in a concentration dependent manner, and this attachment was reduced by integrin αvβ3 function-blocking antibodies. Second, recombinant proteins fused to a TG sequence were covalently incorporated into fibrin gels by the activity of factor XIIIa, and were retained by up to 90 % after two days of washing, whereas their soluble counterparts were released in a burst release during the first 8 hours. Third, the biological activity of the VEGF-variants was shown in vitro by their ability to induce VEGFR-2 phosphorylation in HUVECs by western blot analysis and ELISA. Intriguingly, the bi-functional protein FLV failed to induce detectable synergistic signaling on the receptor level when it was added to HUVECs. In contrast, when HUVECs were seeded on microscopy slides coated with the recombinant proteins, FLV promoted attachment and spreading to a higher degree than FNIII10 or VEGFmut alone. Together, these findings indicate that the immobilized proteins show different potential in the induction of cellular responses. The potency of these proteins to induce angiogenesis was also assessed in vivo using wound healing as a model. Fibrin gels containing 0.468 µM (corresponding to 20 µg/mL effective VEGF-concentration) of either VEGFmut, TG-VEGFmut, TG-FLV or no recombinant protein were applied to full-thickness punch biopsy wounds created on the back of db/db mice, which are used as a model of impaired wound healing. When compared to fibrin treatment only, wound closure was accelerated upon treatment with various VEGF-proteins. More important, both TG-VEGF and TG-FLV proved to be significantly more potent in inducing blood vessel growth into the wound area, when compared to soluble VEGFmut. Differences between the two TG- isoforms were observed in the maturity of neovessels as indicated by the recruitment of pericytes: pericyte recruitment was more efficient in fibrin/TG-VEGF treated wounds than in fibrin/TG-FLV treated wounds at day 10. Collectively, the findings of this study support a critical role for the interplay between VEGF-A and extracellular matrix during wound angiogenesis, and suggest that protein engineering provides a novel molecular approach to use these interactions for therapeutic angiogenesis

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway

First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors

Purpose: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. Patients and Methods: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon–Wed–Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3β levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. Results: Fifty-one patients were treated on study. The safety of 5–300 mg of AT13148 was studied. Further, the doses of 120–180–240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean Cmax and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. Conclusions: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer

Altered neural dynamics in people who report spontaneous out of body experiences

It has been suggested that individual differences in cortical excitability leading to disruption of the timing and integration of sensory information processing may explain why some people have out of body experiences (OBE) in the absence of any known pathological or psychiatric condition. Here we recorded EEG from people who either had, or had not experienced an OBE in order to investigate the neural dynamics of OBE in the non-clinical population. A screening questionnaire was completed by 551 people, 24% of whom reported having at least one OBE. Participants who were free of any psychiatric or neurological diagnoses, including migraines, were invited to take part in subsequent EEG recording. EEG data were obtained from 19 people who had had an OBE and 20 who had not. Amplitude of the visual P1 ERP deflection and consistency of alpha-band phase locking were significantly reduced in the participants who had had an OBE. We did not find any group differences in resting state power or in visually induced gamma oscillations. These results provide support for the claim that cortical differences, particularly with respect to the timing of visual information processing, may give rise to OBE in clinically healthy individuals. To our knowledge, this study is the first to compare EEG variables obtained from people who have, and have not, had an OBE

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Comparative characterization of bacterial immune stimuli

The immune system responds to invading pathogens with a broad spectrum of inflammatory reactions. The pathogens are recognized by different immune cells via the pathogen recognition receptors. These pathogen recognition receptors detect conserved structures of pathogens, the pathogen-associated molecular patterns. The pathogen-associated molecular patterns are structures of the bacterial cell wall or intracellular molecules. Eminent structures from Gram-negative bacteria are lipopolysaccharide, peptidoglycan and bacterial DNA, while conserved structures from Gram-positive bacteria are lipoteichoic acid, peptidoglycan as well as bacterial DNA. Naturally occurring breakdown products of peptidoglycan are called muropeptides. One prominent muropeptide is muramyl dipeptide, which is known as minimal principle for adjuvant activity.In this work a comparative characterization of whole Staphylococcus aureus, lipoteichoic acid, bacterial DNA, peptidoglycan and muropeptides have been carried out. This work shows that lipoteichoic acid is the main immunostimulatory principle of Staphylococcus aureus. Muramyl dipeptide and other muropeptides synergize in cytokine induction with lipopolysaccharide and bacterial DNA. The structural requirements and the mechanism of synergy of lipopolysaccharide and bacterial DNA with muramyl dipeptide have been characterized. These findings add to our understanding of bacterial immune recognition by the innate immune system